Hello everyone and thank you very much for being here today for yet another CNCF webinar about how we at RML are leveraging chat GPT to create custom security controls. You may be asking yourselves, what are security controls? So security controls define rules that restrict access to resources, they verify the correctness of configuration settings and they detect suspicious activity. There are two CNCF projects that can be used to create policies. These projects are OPA, the Open Policy Agent and Kaverno. While they share some similarities, there are also some significant differences between them. Let's go over a few. The policy language for instance, OPA uses a language called Rego, which is declarative. It's a rule-based language that allows developers to write policies using a set of predefined rules and Kaverno on the other hand uses YAML-based policies that can be easily understood and written by Kubernetes users without a deep knowledge of programming languages. So while Kaverno is easily accessible to anybody that ever wrote YAML, OPA with Rego requires a learning curve. In terms of policy enforcement, OPA is a standalone policy engine that can be integrated with other Kubernetes tools such as Kubernetes submission controllers. It's used to enforce policies. Kaverno is a native Kubernetes submission controller, which means it integrates directly into Kubernetes API server and enforces policies in real time. The policy scope, OPA policies can be used to enforce policies across multiple clusters while Kaverno policies are scoped to a single Kubernetes cluster. And in terms of policy updates, for instance, in OPA, policies can be updated on the fly without requiring a restart to the policy agent. In Kaverno, policies need to be applied as a Kubernetes resource and they may require a restart of the Kubernetes API server. And lastly, in terms of management, OPA provides a comprehensive policy management API which enables users to manage policies programmatically. Kaverno, on the other hand, provides Kubernetes native policy management experience, where policies are managed as Kubernetes resources. In summary, while OPA and Kaverno are both policy engines that enable policy-based governance of Kubernetes clusters, they differ in their policy language, the enforcement, the scope, the updates, and the management. The choice between the two will probably depend on the specific use case and it is possible to use them both in conjunction to achieve a comprehensive policy-based governance. At RMO, we have the largest library of security controls available. There are over 200 at your disposal and they are all written in Rego. From time to time, RMO platform and Cubescape users want to write their own custom policies and controls to meet their specific needs and security policies. And to do so, they need to know how to use OPA and Rego, which is insintrivial. So this is where AI comes in. RMO Labs has harnessed GPT-3 to help users create their own custom controls without the need to know how to use OPA and Rego. All you need to do is to write what they want to check in natural language. And RMO with GPT-3 will generate the exact control written in Rego with a description and also the suggested remediation. Once the custom control is generated, the user can download it and run the control in the CLI as part of improving their Kubernetes security posture. So how do we do it? Let's take a look at RMO platform. All you need to do in order to use this feature is just head over to the settings page, to the control section, and click on Create Custom Control. Here you type what you want the control to test in plain English and it's that simple. That's it. You can also provide an example object to test the test should run on. For instance, you can put a deployment manifest or a secret manifest or whatever. And if you know exactly what you're looking for and what you're testing in this control, you can also provide the description and the necessary remediation, but you don't have to do that because RMO does that for you. Here I'm going to give it a name. It's going to be pod test. And what I want this control to test is I want this control to fail. If a pod, for instance, has a CPU resource request with a value higher than 300. Now after the control is created, you can use it right away. You can download the control to your machine and you can use Cubescape Scan Command in the CLI in order to test the control against your environment or your manifest files. Now I want to show you RMO platform in general and how the context is being connected. The first thing you see when you start using the RMO platform is the dashboard. It's a single view of everything you should know about your Kubernetes environments from CI CD and clusters and it's all aggregated into one single view that helps you focus on what really matters and what's really important from a security compliance perspective. What we see here are the different clusters that I've scanned in the past and it's prioritized by the risk score calculated based on the different framework we use. And you understand which cluster you should start working on first, right? We also show you configuration risks trend over time so you can identify drift changes in your configuration so maybe you notice something changed and there's maybe a spike in the graph so you need to pay attention to it and you need to take action about it. Also when it comes to vulnerabilities you can see right here in the lower graph and we also show you what are the top five failed controls that we ran against your environment and what are the top five CVEs that we found on your clusters. This is actionable items from the dashboard screen. Now this is usually the first view that a typical user sees after the onboarding and this is where you should start your Kubernetes security journey first thing in the morning. The solution of RMO platform is focusing on two main areas. One is happening inside your cluster and also on the outside of your cluster. So let's talk about compliance. What you see here are different clusters that I've scanned and you can see which provider the cluster is on and any other information that we provide. We support managed and unmanaged Kubernetes clusters like AKS, EKS, GKE or any unmanaged Kubernetes type that you are using. And if I want to, when I dive in to one of these different cluster scans reports we see the different controls that the system tested and which of them failed meaning I need to pay attention to. And before I dive in I would like to explain how we manage the controls that we just talked about according to the different frameworks. So let's navigate again to the settings page and under the framework section you can see the different frameworks that we have. We have out of the box frameworks like MITRE, NSA, CAS and there is also an option to customize and build your own framework and cherry pick the controls that are relevant to your security needs and to your compliance requirements. Each framework consists of many types of controls meaning tests that are being tested against your environment. And currently we support more than 200 controls that are Kubernetes specific that you'll be able to run inside your environments. Some of the controls can be configured according to specific needs. So let's say I look for application credentials and configuration files which is of course not something that is recommended. It's not recommended to save secrets in your configuration files. And I can decide on which secret the control should look for. I can add or remove different types of values and this is also true for other controls that may be configurable in the platform. And this provides a high level of flexibility and customization. Let's navigate back to the platform and now I will navigate to the compliance section. You can run scans manually or according to a schedule interval using ChromeJobs. Focusing on the control that we just looked at the application credentials, when I click on it I see which resource failed against this control. I can also exclude findings that are approved by me because you know some manifest files have those configurations by design and some of them are being excluded for you in order to reduce all the noise.  If you click on the fix button the platform will offer a remediation including specific information regarding what the issue is, where it is found and how you can fix it in order for you to fix it. I can also share the issue with my teammates. I can send that via Slack channel or I can even open a Jira ticket straight from here using the collaboration feature. And let's move to the vulnerability section. The platform scans for images that run inside the cluster and we can see vulnerabilities that were found. So we can filter or sort according to certain criteria like show me only the RCE vulnerabilities which are considered more likely a risky vulnerability or only the ones that have effects meaning I can do something about them or let's combine them both with the severity filter and this reduces the noise and filters everything that you know you need your attention to go to. From there I'll dive into one of the results, one of the scans and you see which vulnerability, which component, which version, how severe it is and which version I should upgrade this component to in order to fix this vulnerability. I can also use the exception if I don't want to be alert about it anymore and I can also look for a specific vulnerability in case I have that information and see where it is found in my images on my images in my cluster. The next section I want to talk about is the RBAC visualizer. It's considered by many to be one of the most complex things to manage in Kubernetes. So the platform offers an interactive visualizer to understand all your resources, your role bindings, your roles, cluster roles, who can access what, verbs, everything. We made it even easier to query using some built-in queries so you get useful information and important information and insights with no trouble. For example, show me all the cluster admins that I have in my cluster. And by the way, the C35 means that it's also misconfiguration covered as one of the controls. So we actually get context between the misconfiguration and the RBAC and this show how different section of the platform provides context when combined. And now I see all the cluster admins in my cluster. I can also have a reverse look and see which other roles this user is related to or which subjects are related to searching cluster roles depending on the need that I want. And I can go top down, I can go bottom up. Another example is show me all the unassigned roles, which is not a good practice to leave unassigned roles in your environment. And you can easily take action based on the information that you see here. I can also use the investigate tool where I can search for specific roles or users or services counts and so on and investigate from there. I can ask to see all the related resources or roles. And using the who can tool, I can further investigate and question and ask who can get or list for instance secrets in my environment. And you see there you go, it's actionable items. Everything we just played with is in the context of your clusters. But the platform also provides the same information regarding misconfiguration vulnerabilities outside the cluster. We can connect the ARML platform to different types of repository providers that you work with. This means that I can also scan for private or public repos before they are being pulled into my clusters. I can scan for image container registries before pulling them into my cluster. So this help prevents vulnerabilities from getting to my clusters and hardening my Kubernetes cluster. Back to the custom controls, there are many potential use cases for using chat GPT within the context of Kubernetes. Kubernetes can be challenging to navigate and fully utilize due to its complex nature and the need for a substantial understanding and set of skills. Any help that AI based tools like chat GPT can provide to make this test easier is welcomed. From writing a new Yano file to deploying a new cluster to securing it. In the case of the implementation I just showed you, we envision a custom framework made up of custom controls which will grow as you add controls to it. Our roadmap includes adding the ability to create custom frameworks that will host and save all the custom controls and enable the users to run them directly for ARML platform, thus making it easier for users to access and utilize their custom controls as well as streamline the process of adding new ones. This new type of custom framework will provide a more flexible and user friendly experience for our users. And in terms of upstream Cubescape, users will be able to contribute new controls to the Cubescape Paragl library and give back to the community that way. Once merged, these controls will be available for selection and use by all Cubescape and ARML platform users, thus making you part of our Cubescape community. And that's what I wanted to talk to you about today. I hope this has been informative for you and you know a little more about custom controls and ARML platform. I'll see you in the next webinar. Thank you for watching.


In this module, we would be studying Sharia treatment of credit and credit card. Not only in this module, actually this would be our focus in the next 10 or 11 module. It is an important topic hence we would be spending some considerable time on understanding the treatment of credit slash debt slash loan in Islam and its implications for the offering of Islamic credit cards. What is credit? Let's go back to the basics and try to understand the nature of credit.  I am not talking about that credit. I am talking about the credit which we use in financial markets. In its simplest form credit is a debt, loan or receivable. This is basically when I buy something on a deferred payment basis then this could be called a credit sale. We have studied that credit sale is permissible in Islamic law. Actually that is an example of credit sale and this is acceptable in an Islamic banking and finance framework. There are quite a number of products which are based on this principle. While trade can take place, trade of commodities, trade of goods and services, it can take place on the basis of credit sale. Money can only be given on credit or IE can be lent but without charging extra which is deemed as prohibited interest. This is a very clear difference of treatment of credit sale and money on credit. We would look into these differences with the help of some diagrams. Let us see, there is a party A and there is a party B and they want to exchange a commodity X for a price P. We have time dimension here. This is time dimension and this is time dimension. If party B gives commodity X to party A at T naught meaning spot now and party A gives the price at the same time to party B that would be considered as a spot sale. And a lot of sales actually are spot sales. Whenever I go to a shop, to a store, to a selling place, I actually do these spot sales. This is okay. Now if party A gives the commodity X to party B at T naught and party B actually pays the price at a future date T1, we call it a credit sale. And as I mentioned previously, that would be bad module. Then we have another possibility whereby party A actually delivers the commodity later while receiving the price from party B now. So party B is paying the price now to party A and party B delivers the commodity at T1. This is a depiction of Bacellum which we have already studied. This is the case of Baistisna. The price is paid by party B i.e. the buyer in installments at T naught, T1, T2. So party B is paying the price now after some time with the progression of the project. And the commodity is delivered by the seller on a future date when it has been made on order. So that is the case of Baistisna. When it comes to money, if money is exchanged dollars for dollars, if they are exchanged on spot. So party A is giving dollars now and party B is giving the dollars at the same time. That would be the spot exchange of dollars. Thus dollars abhi die or thus dollars abhi le li. You might be thinking that I am crazy. What kind of an exchange is this one? Actually this happens many a times without realizing that this is what we are doing. Do you have any example in mind? Many a times people exchange Nai note with Purane note. Same price in some cases with different quantities and we will come to that one. Now if this party is delivering dollars now and party B is paying the same amount of dollars in future. That is called as loan. Now the situation changes when we have two different currencies. If party A gives dollars to party B now and party B actually gives rupees to party A at the same time. That is called spot currency exchange. What happens when party A gives dollars now in exchange for party B delivering rupees later on? This is prohibited in Islam. The previous example there is acceptance or there is provision for acceptance in an Islamic legal framework. But if I receive ten dollars today in exchange for an amount in rupees after one week, after one month. That would not be considered acceptable in an Islamic economic or Islamic financial or Islamic legal framework. Now this remains prohibited even when I receive dollars now in lump sum and I am required to pay rupees in easy installments over a certain time period. That is not acceptable either. And this information, general information is crucial to our understanding of the treatment of credit and credit cards in Islamic banking and finance. And this is something we would be covering in the next few modules.


Hello. Let's find out whether you, the audience, are smarter than the fish that I work with. I'm going to give you a task. You won't have much time to solve it, so try to do it as fast as you can. Ready? Count how many fish you see on this slide. Go! Okay, stop. You're out of time already. Now, the answer was 15, but if you didn't get it in that time, I'm afraid you are no smarter than a fish. They can do it in a split second. Now, brain is no doubt the most fascinating organ that ever evolved on this planet, yet we know so little about it. How can a fish, a size of a pinky finger, outsmart us? It's a mystery, and I want to know the answer. I want to know why some animals are smarter than others. So here's what I do. I design IQ tests for fish. I build special mazes and arenas so that I can measure their memory, learning, navigation skills, even their abilities to control emotions. After watching thousands and thousands of fish swim around the tank, I can give each fish a score and then I look for patterns. Are there differences between males and females, young and old? Does inbreeding or pollution affect intelligence? I compare fish performance to other animals, and I try to find biological explanations for the results that I see. There is one pattern in particular that I'd like to share with you today. Intelligence is an adaptation to problem solving. When animals face certain challenges in their lives, we expect that over time they might evolve intelligence to deal with these problems. Okay, sure. But what happens, say, if males and females, for some reason, face different challenges? Well, in that case, we expect that males and females will evolve different intelligence independently. For example, our male fish right here happen to be quite aggressive. They do a lot of fighting. And I found that their ability to control emotions is actually quite poor compared to that of the females who provide parental care and avoid impulsive actions. But sometimes we find no differences between the sexes at all. Let's suppose there is a predator nearby. As a fish, you want to quickly find the largest group of other fish and swim away to safety. You see a bunch of your fish friends over here, some fish buddies over there. Which group do you join? Well, you've got to count them first to find the largest group. This is why fish are so good at counting because for them it is the matter of life and death. But we humans face different challenges in our lives, like attempting to communicate effectively in under three minutes. Intelligence is an adaptation to problem solving. Being smart doesn't mean that you can count very fast. It means you're well adapted to your environment. So as you can see, my little fish friends might not look very impressive, but they helped me answer some of the most interesting questions in the field of animal intelligence. Thank you.


In today's video, we're talking about how to kick a soccer ball or how to kick a football whatever you call this beautiful game I want to help you have more consistent shots now You're searching for this video. I'm assuming because you're not having consistency with your shots Sometimes going straight sometimes going over there sometimes going over there Okay, but you want to have more consistency you want to hit that net more often So I want to help you with that so I got three things that I want you to think about and the first thing It's kind of a more advanced topic, but that is I got to come a bit closer I want you to talk about the physics of ball striking Okay, and what I mean by that is in order for this ball to go straight I have to hit it straight Okay, the ball is going to go exactly where I tell it to go So there should never be a time when you had a shot and you're like, oh, I did everything right But it went over there. No, you obviously didn't do everything right This ball is only going to do what you tell it to do. Okay, so what I mean by that is if I hit it straight in the middle It's going to go straight if I hit it on this side of the ball It's going to spin off and go that way if I hit it on this side of the ball. It's going to spin off and Go this way. Okay, so this ball is just going to do what you tell it now I want you think just think about like say for example, you're playing pool Okay, you're playing pool. You're trying to hit the ball This ball and make it go into that pocket Well, you have to hit it on that side of the ball in order to make it go there Well, this is the exact same thing or say for example, you're playing golf Okay, if you hit the ball here, it's going to hook and go over there if you hit it here It's going to slice and go over there. Okay, if you hit it here, it's going to go straight down the middle Okay, so think about the physics of shooting the physics of ball striking So the reason I want you to think about this number one is just it's going to help you right away You're gonna say, okay, I want to go straight. Well, I got to hit it straight in the middle or I want it to go To the bottom corner. I can hit it a bit more on here. I want to go to that top corner I can hit it more here. Okay, you're gonna understand that it's gonna help you But you can correct your bad shots a lot better.  So unless you really focus on holding it straight the shots gonna want to go that way, right? It's gonna want to go to that far corner like that. Okay, so personally I hate missing the net that way I would much rather miss the net that way So instead I know when I shoot with my right the tendency is to hit the ball and it's gonna pull that way Okay, so what I will do is I'll aim a bit more on this side and that will correct it And that will make sure that the ball goes straight. Let's see Whoa Okay, and like I said, I would much rather miss Going that way then pull it that way. I absolutely hate to do that So if that's a problem you have then maybe think about that that'll help you out right away Okay so next thing We're talking about the physics is your foot technique. Okay, so I'm assuming we're talking laces strike and guys I have a video on my channel. It's called. I think it's called six Crucial shooting techniques every player must know and I'll put a link in the description of this video But that video will teach you how to shoot in different ways It will teach you how to curve the ball how to chip a ball how to scoop a ball How to drive a ball how to ping a ball how to hit it with your laces It will teach you all these different ways to shoot. Okay, so go and watch that video But today just talking about laces. Okay foot technique. So if you can see my foot technique, I hope you can see My toes are pointed down in the ground. My foot is flexed. My ankle is firm. Okay. I'm not like this. I'm not loose like that Okay, it's strong. It's flexed now What you want to think about when you're hitting the ball is keeping that foot Flexed and strong through the ball. Okay, so a lot of us we have like a weak foot a weak ankle And we're hitting a hard ball and we're having a really weak shot and we're wondering why it's so weak Well, you have to make it strong. You have a hard foot. You're going to have a hard shot Okay, but just make sure when you hit the ball when you hit the ball Your ankle doesn't go weak. Okay. A lot of us we're flexing and then we hit and then we're weak You want that flexed strong foot through the ball? Okay, and that's going to help you with your shooting. Okay. So think about that strong flexed foot through the ball Okay Boom with both feet, especially with your weaker foot your weaker foot. It's going to feel weird. It's going to feel unnatural Okay, but make sure that you hit the ball nice and strong Especially with your weaker foot Keep those toes flexed strong ankle lock through the ball Through the ball. Okay. The final thing I want you to think about is Your range of motion. Okay. And what I mean by that is When I'm shooting the ball, it should be a nice natural swinging motion Like that. Okay. If you were to compare Compare your strong foot to your weak foot A lot of us are we're shooting with our strong foot. It's a nice backswing nice fall through nice Nice natural swinging motion. Okay. This is where you're going to generate a lot of power Naturally without trying hard. It's this nice flowing motion like that. The thing is when we're doing Nice flowing motion like that when we're shooting with our weaker foot or our left it looks something like this.  It's like so heavy and weak through the ball. There's no range of motion Okay, so you want a nice backswing nice fall through with both feet backswing fall through Back swing fall through. Okay, so think about those things think about Where you want to hit the ball the specific point where you want to hit the ball think about your flex foot Think about nice natural backswing Okay, and then Think about very important You should always add use your body positioning Okay, if you want to hit the target more often and you saw that shot. I just hit right there It's nothing special, but it's on net. I tell my kids my students all the time I do not care how hard you can hit the ball if you can hit it hard. That's great But what's more important is if you can hit the net every time or at least most of the time Okay, so if you want to make sure you hit the net You don't sky it up over there get your body over the ball so important Whenever you're shooting think about, okay, I gotta stay over the ball Especially if it's like a one-timer ball is coming to you. It's coming straight at you If you are up like this at all It's very hard to control that ball. Make sure you get over it And then try to make good contact focus on the ball Okay, so coming back to the physics. It's impossible for me To hit the exact spot on the ball I want if i'm not focusing on the ball Watch the ball until your foot makes contact And you hit the ball, okay And the same things apply with both feet, right? And that's right down the middle, but even that's fine A shot on target is a good shot. Okay, and realistically that shot Is harder for a keeper to save than a shot that's right here or right here Okay, hard and low is the way to go Remember that Hard and low Is the way to go. Okay, so if you want to improve your shooting think about those things, but most importantly you have to practice Okay, you have to practice that shooting technique But like I said go to my channel watch that video six crucial shooting techniques that every player should know Thanks for watching this video guys. Please like give me a thumbs up Please comment below And if you have any other tips to add to those three things I know there's lots of other things out there if there's something that helps you specifically Please comment below and share that with other people watching this video Please share this video on social media share with your friends share with your teammates And thank you for watching this video on how to kick a soccer ball or how to kick a football


differences there. There'll be flexibility that we will have basically based on the fact that they're going to be custom in some manner. Also, the job cost system will typically have higher value, higher dollar values for the products as opposed to a process cost system because and that leads through the customization. If we're going to customize it, then it's going to take more time and it's going to cost more as opposed to if it was all just being the same thing. Our goal there is to lower costs and have it be typically cheaper. So if we can contrast this to a process cost system, the idea of a process cost system is we're going to apply the costs to the process. And the reason we can do that is because all the things that we make are going to be in essence the same. So that's going to be our route again that we want to think about as opposed to something being customized. Here everything it is what it is. You get what you get. It's all going to be the same. So we don't need to apply the costs in the same way. We need to apply the costs all evenly to the units. So what we need to do then we can do that by using a process cost system, assigning costs as they go through the process rather than two particular units of inventory particular jobs. So in this I would think of something like taffy if we're making taffy or caramel candies or something like that. They're all the same. We're not going to vary the caramel candies. They're all the same. So we're producing our inventory. Other things like oil is going to be like that for processing oil. It's all the same at the end of the process. So our goal here is to make everything the same and lower costs. So we're going to have then repetitive procedures. Everything is going to be the same. And our goal here again is just to make everything the same even mechanized if possible have the whole machine make the taffy and go through just the process of making the taffy. And once we automate the system because they're all the same, then that should be lowering the cost. We're going to have a homogeneous product products or service as opposed to heterogeneous. So they're all the same. And again, this is probably the one that here you want to focus in on or the fact that they're not custom. So we don't have custom products. They're all going to be the same in nature. And therefore everything else pretty much derives from that. We're gonna have high production value. That's the point of us making everything the same. We're going to say, Yeah, there is no variance. You want carmels with different kind of things in them or whatnot. We don't do that. We just make the carmels. They're all the same. And we have a pretty low cost because of that. The more we try to customize the carmels, then the more it's going to be, you know, difficult for us to automate the process. Then we're gonna have more customization. So if we keep them all the same, we can have high production volume and lower cost, low production flexibility. Again, that follows from them all being pretty much the same and not being customized. So we're gonna we're not going to vary what we produce. We're going to say that these are all we're all going to produce the same thing for every every unit is going to be the same. So if we look at these together, then the job costs and the process cost system. Remember that the job cost system has the custom order as opposed to not non custom orders or just repetitive procedures on the process cost system. So this side's going to be custom. This kind of side is not custom. And that means that we're going to have everything be repeated. It's just it is going to be what it is. And then we're going to have heterogeneous production for the for the job costs versus the process cost homogeneous. And again, this this kind of falls out from the fact that these are custom and these are non custom. So these are going to be all different products and services through that customization. These will all be the same. The job cost system, low production volume, again, because they're all different in nature, and a high production volume for the process cost system, because they're all the same, we can then scale up and be able to produce a lot more than the job cost system, high product flexibility, we can vary that's the point that's the point of having them be custom. On the process cost system, we have low product flexibility. And again, that's the that's the point because that's what leads us to be able to produce more at that time.


The thesis of this research project is it is the intention of this research to explore the prevalence of what the mainstream might consider deviant sexual behaviors, especially BDSM and the associated culture. The research objectives are to define sexual, deviant behavior, define BDSM, determine the prevalence of BDSM in America, to ascertain the thoughts and feelings of those who engage in the lifestyle, and to objectively observe the culture. The research methods will be surveys, interviews, and direct observation. The sample groups will be reviewed by the federal public, and those within the BDSM community.


Hi everyone, Joe for jaspyscasebrakes.com with 20 coming at you with 2021 Leaf Lumber Kings baseball two box random hit break number one. So we're going to out of a fresh case we're going to pop a select two boxes pop them open and bust out eight hits and big things that these folks are getting into the action and we'll type in the hits here and we'll randomize your names, randomize the hits and match you up with something. So let's bust open this fresh case here. There it is leaf lumber Kings. So we're going to roll we're going to select a die we're going to select that middle one right there and we'll go one two three four on the dice roll if I roll a five or a six we'll just roll again four one two three four and the count is actually too fast they're counting they're counting down the time the honest is at the free throw line but they got to do some one alligators when you guys do those counts when you're like before when you're playing like flag or touch football with your buddies and you go and you're counting down before you can blitz do you go one alligator do you go one one thousand one Mississippi what's I went alligators I go out I still do alligators alright so here's an example of what you can get out of here some pretty crazy stuff let's see what we got Sean D goes Mississippi Jeff Jeff goes alligators yeah I go alligators too all right there's Daryl strawberry 21 out of 25 Adam Wolf goes Mississippi's so when we'll type these cards in in the order that I that I pull them and we've got six out of nine Roberto Clemente couple pieces of his lumber that's pretty cool you've never heard alligators I grew up in Southern California I think alligators happen Jeff Jeff did alligators Jeff where are you from again and we've got four out of five this is pretty cool a piece of Duke Snyder's lumber Harman Killers lumber and Eddie Matthews bat rack three that's pretty awesome what about thousand anyone do thousands one one thousand two one thousand three one thousand four one thousand five one thousand maybe it's a West Coast thing Jeff and Jersey in auto Fred Lynn five out of nine bat in auto that is piece of his lumber lumber Kings there's our first four and we've got another two box break of this in the store right now if you want to run this back right is that why we say alligators out here but we'll have to do a survey based on region how people count down count things down or count things off Albert Bell 15 out of 25 four out of nine Frank Thomas 1500 RBI Club couple pieces of his lumber right there no one mentioned thousand sides we did that we did alligators or thousands I feel like sometimes Mississippi's three out of 25 nice the franchise Tony Fernandez Roberto Alomar Joe Carter Vernon Wells pretty good blue jays there pieces of their lumber nice that's pretty awesome yeah I think these are all game used to franchise for and we've got super teams one out of six wow that's awesome Montreal Rusty Strubb Tim Reigns Larry Walker Vladimir Guerrero senior Andre Dawson Gary Carter Anders Galarraga and Pedro Martinez it's a good group of players I think this this rusty guy is the only one that I'm not familiar with we hope you enjoy this piece of history I hope so too all right now let's we're gonna pause the video actually when we come back I'll have the hits all typed in and then we'll run that randomizer BRB all right welcome back everybody so I got all the hits typed in right here there's one two three four five six seven eight with the eight people right here you're guaranteed is something a random something so let's roll it and randomize both lists three and a three six the hard way your names first one two three four five and six and final time got Daniel down to John three and a three six times for the hits good luck one two three four five and six and final time after six we got the Fred Lynn relic auto and the triple bat relic featuring Snyder Kilibrew and Eddie Matthews let's match you up with your guys all right Daniel with the Fred Lynn bat and autograph 5 out of 9 Robert with the Albert Bell bat relic 15 out of 25 Michael May with the Daryl Strawberry piece of his lumber 21 out of 25 Robert with the with a few hits right here in a row the franchise for featuring the Blue Jays super teams Montreal and the Roberto Clemente bat relic six out of 9 Eric with the Frank Thomas bat relic 4 out of 9 and John LeFarge with the Snyder Kilibrew Matthews bat relic 4 out of 5 all aboard the Big Hit Express whoop whoop some pretty nice stuff we got two more boxes in the store ladies and gentlemen I'm Joe for Jasper's case breaks dot com that was leaf lumber Kings baseball 2021 edition thanks for watching thanks for getting in on the action and I'll see you next time for the next break bye bye


와! All right. Welcome back. Okay. Welcome back. Good to see everybody today. We're going to be going a little bit into more detail about the function of protein. So we looked at the different shapes of proteins, but we didn't really talk about function yet. The one thing I did mention is that proteins are dynamic. So they're flexing around. And today you'll see just a beautiful example of how proteins slice and dice and do all kinds of amazing motions to carry out their function. So today we'll both be talking about proteins, we'll come to that in a second, about a protein that doesn't catalyze a chemical reaction. So you couldn't really say it's an enzyme. An enzyme is a protein that carries out a chemical reaction. Its purpose is to bind something and to vary in a physiological way, bind and release that object at the appropriate time. So perhaps against my better judgment, I thought we would come back to this confusing topic that you guys uncovered. So looking at the Leninger textbook on this topic, one is not less confused. And the primary reason is that these definitions are not standardized. They're sort of, you know it when you see it, sort of definitions. So I thought perhaps instead of giving you very precise molecular distances that define each of these types of things, that I'd give you some examples. But these are for the most part arbitrary. And so we talked about structural motifs and super secondary structure. And here are some examples of that. You can see they're small. They contain limited number of strands for example with the sheets. But here we have one, this zinc finger. And it's on the line of purpose because throughout literature you'll see it described as a motif as well as a domain. Okay, why? Because it's a definition, it's arbitrary, it's not standardized how we differentiate this. Here is a real life protein. The domains are in different colors. Do all of them have a flexible linker between them? So for example, here is one domain, this red helical domain. No, it has a flexible linker within the domain. Now these guys are more globular like, right? So here's a domain, helical domain. Here's a G domain. And here is an EH domain. Those are somewhat distant. But what happened here is someone just said that's the domain and that's the domain, right? One important thing, a tradition for the majority of domains, the amino acids in those domains are contiguous in the primary sequence. And so we have, you'll see this throughout your careers as biomedical researchers, this representation of the primary sequence of a protein. Remember, and these are the actual amino acid numbers here. And so remember we start from the N-terminus going to the C-terminus. So for example, this protein has 543 amino acids. And we have these colored boxes. And each of those boxes is one of these domains. Domains can be defined as a compact structure, a unit of folding, a unit of function such as this domain binds to something, or a unit of evolution. In general in nature, you'll see that different proteins take different combinations of those domains using the function of each of those domains in different ways to accomplish a certain type of chemical transformation. So I don't know that this clarifies anything for you. Structural biologists need to define these standards. So for example, if you gave a thousand structures to a thousand structural biologists and you said what is the average number of domains in these proteins? I bet you would get a nice L-shaped curve in terms of the average number. Okay, so let's look at some more examples. We have a little bit more time today. I had mentioned in the last lecture a particular domain called an SH2 domain. Its structure is here. It binds a phosphotyrosine. So an amino acid tyrosine that in the para position where the hydroxy group is, we've substituted in a phosphate. In that particular substrate of this domain binds in this groove. We have another domain that binds phosphotyrosine called a PTB domain. And so these are some examples of domains. Now we can take these domains and combine them in all sorts of interesting ways. So again, remember these depictions are always from the end to the C-terminus left to right. And so we have a variety of different signaling molecules that have different combinations of domains. And those give those signaling molecules the unique properties necessary for them to communicate signals from outside of the cell down in generally down into transcription. So this is just an overview but I thought it would be useful to highlight that this is a common way in which domains are represented in the primary sequence. But it's not necessarily always the case that domains are contiguous in the primary sequence. It's almost, it's usually the case, such as for these domains here, okay? I'm not gonna ask for any more questions. Oh, so Art, there is a question, however. Does motif have to have contiguous sequence of amino acids like them? Again, there is no definition. Here are some examples of motifs and you can see for these particular examples, they are contiguous, so that's how they define them. Unfortunately, it's a world of gray, not black and white. Okay, it's a little bit of review. So today we're gonna be talking about protein function, some of the most amazing proteins and some of the earliest characterized proteins out there. So we're gonna be looking at the oxygen binding myoglobin and hemoglobin and closely related hemoglobin protein. We're gonna be looking at how their flexibility contributes to their function. We'll look at all kinds of modes of regulation, which will be an ongoing theme in the class. And then we'll give another quick example after that of another protein that binds things. So this is myoglobin. Myoglobin is found in muscles. There are large amounts of myoglobin and shampoo. This was the first protein structure ever obtained. So in terms of human beings, this is the first protein ever seen by a human being. And you can imagine, up to this point it was all theoretical, line is pawling with pieces of paper and winding things up and they saw this and look at this beautiful structure. So this one is all alpha helical. They noticed something red. Well, it probably wasn't red in the X-rays, but this was something that was not attached to the protein covalently. So it's non-covalently attached thing and we'll come back to what that is. And so here it's a pretty simple structure. You just have a combination of helices. You could refer to it as a fold. So this is the globin fold. This protein has one amino acid in addition to how the globin fold is defined. That's arbitrary, right? They're all sort of stuck together. So here's the structure. There's different ways to represent the structure with these cartoon representation. If you have arrows, you're going from the end of the sea terminus, a space filling, or where you're actually showing the atoms. And so that sort of doesn't tell you much. Okay, so this is myoglobin. Here is it, sure, or not? So I'm going to just play it manually. Nice. All right. Today the proteins will not be rotating. I know your heart broken. Okay, so here's another picture of myoglobin since it's not rotating, it adds nothing. So here it's the so-called this non-covalent group. It's adding a function. So amino acids alone in myoglobin are insufficient for its function as an oxygen storage protein. And so these are prosthetic groups. So you could also call it a cofactor.  And when you add an iron, it becomes a heme molecule. And you can see the irons are typically octahedral in configuration. They're desired substituents for an octahedral configuration. So the iron is chelated by these four nitrogen atoms. And then you see some wavy line. And what's this? What's this wavy line thing hanging down from lecture one? Histidine. So histidine is showing up today. And then we have an oxygen atom here. Okay. And so this is the prosthetic group. Stably associated with the protein. And now we can look at it in a different way. You can see that there's a plane defined by the heme or the porphyrin ring around the iron atom. And you can see the fifth bond is to this histidine prosthetic group. And then we have an oxygen. And it's important that the protein provides this fifth bond to the histidine group because that helps to prevent the iron from becoming oxidized. So to function in its role of binding oxygen it needs to be iron two plus, okay? And so let's look at this in some other ways. One thing that can happen with irons is yes, they can bind to oxygen. When they do bind to oxygen it's in this bent configuration in relation to the porphyrin ring here. But other molecules combined to iron such as this carbon monoxide. And you can imagine, well that's probably not good. Probably myoglobin or we'll see hemoglobin need to differentiate the binding of this two atom molecule with this two atom molecule. But what a subtle thing for the protein to have to do to discriminate a two atom molecule that's either linear or bent to the side. So that's where we have a second histidine group provided by the myoglobin protein. So these are just hanging out in space but they're actually attached, they're all attached together through myoglobin. This histidine is referred to as the distal histidine and it's providing this exquisite selectivity to block or slow down the binding of carbon monoxide but to facilitate the binding of oxygen through a hydrogen bond, okay? So if we didn't have this histidine, carbon monoxide would bind very tightly. As it turns out, carbon monoxide binds tighter to just plain heme in solution than oxygen. So what is this subtle little difference doing? Well, free heme binds carbon dioxide 25,000 times more tightly than oxygen. The purpose of this myoglobin molecule is not to start carbon monoxide. That's stuff we need for metabolism, we need oxygen. But when you put this heme group in myoglobin, you put that histidine group in the way for carbon monoxide to bind that decreases the binding affinity of the heme 100-fold for carbon monoxide. It's still preferred. So in the atmosphere of Earth, we had a one-to-one mixture of carbon monoxide and oxygen. The gross majority of myoglobin molecules will be filled with carbon monoxide. But fortunately, the atmosphere is very high in oxygen, low in carbon monoxide. And that's reflected in the blood of organisms. So about 1% of myoglobin is normally coordinated with carbon monoxide. And so you can do some calculations. Come out and come back in. My screen's checked. So this binding ratio, binding affinity ratio of myoglobin were 25,000 to 200. Most of myoglobin would be coordinated with carbon monoxide. So this discrimination, there's still, every one of you currently has some carbon monoxide in your myoglobin and in your hemeoglobin. But luckily, you have a lot more oxygen. So it's just a minor binding partner. So where does this carbon monoxide come from? Any ideas? Yeah? Could be. So there's other non-biological sources as well, right? Partial oxidation. So you would not want to be in a sealed environment with an automobile because they produce carbon monoxide. So yeah, carbon monoxide is in the environment from the partial combustion of fuels. Okay, so today we're going to introduce some of the concepts that are gonna be critical in the fourth lecture. Thinking about the equilibrium between proteins and their substrate. So here we talk about a ligand. A ligand is simply something that binds reversibly to a protein. So in our case in myoglobin, the ligand is an oxygen molecule. And so we can describe this equilibrium by equilibrium constant, an association constant and a dissociation constant. Remember, from probably right when you started at Brown or maybe even in high school, you define this as the concentration of products over reactants. Remember that? And so for the association constant, the product is PL, concentration of that divided by concentration of P, concentration of L. In KD, the opposite direction is just the inverse one over K. So so far, might ring some bells. But now I'm gonna throw something new at you. Theta. And this is defined as the fractional occupancy of a protein for its ligand. And so it's defined as the number of occupied sites, binding sites, ligand binding sites on a protein, divided by the total number of sites. You can calculate that by if you know the concentration of the protein that's bound to a ligand compared to the total amount of protein, okay? What's made? So this is just the definition. So to make it crystal clear, we have the red circle method of showing this. And so we have here four red circles. These are proteins. Only one of the four is bound to a ligand. So theta would be 0.25 for this particular case. If half of the binding sites were bound to a ligand, theta would be 0.5. Okay, what's been so far? Not too bad so far. So all we're gonna do now is combine these two equations. We have an equation for the KA and the equation for the fractional occupancy. And if we substitute PL into here and rearrange things, theta is equal to this equation. The concentration of ligand divided by the concentration of ligand plus a constant plus one over KA. So we've just done simple arithmetic rearrangement of these equations and a substitution. And you might recognize this equation. It's a hyperbolic relationship. So the equation of a hyperbola back from maybe even junior high school is X over X plus a constant, okay? So that has this kind of shape to it. So at very low concentrations of ligand, there's a linear relationship between the fractional occupancy and the concentration of the ligand. In other words, this is sort of linear, right? So if looking at the equation, you can see this as well. If L is very small, L would be much smaller than one over KA. So theta would just equal a linear relationship there. And the same thing up here. If L is much larger, the line approaches one. So L divided by L plus something insignificant is one. Does it make sense so far? So we have a hyperbolic relationship between the fractional occupancy of a protein and its ligand concentration. So let's poke around at this equation. So here's the equation again. What happens if half the binding sites are occupied? Remember I said theta would be 0.5 if half the binding sites are occupied. So if you put 0.5 for theta and do some rearrangements, look at how miraculously it simplifies. And so at a theta of 0.5, the concentration of ligand equals KD. So the dissociation constant has units of concentration. Its value is the concentration of ligand necessary to fill half the binding sites.  It'll become very useful. But for now, that gives you sort of a physical feeling of what is this KD? How can I calculate KD? Well I just keep increasing ligand concentration, test some analytical measurement of the number of proteins bind to the ligand. I have hyperbola, it has some kind of upper level, half of that you track down on the curve and you can find the KD directly from a binding curve because it would be the concentration of ligand necessary to fill half those binding sites. So we can calculate KD. But now in this lecture, our ligand is a gaseous molecule, right? So it's an oxygen molecule. And so you don't usually think of moles of oxygen, right? You think of a partial pressure of oxygen. And so we can define a P50, a partial pressure of oxygen that is necessary to fill half those binding sites, okay? And that's just a definition. So this P50 is the pressure, is KD. So in this case, KD does not have units of concentration, it has units of pressure. You see so far? And it's rambling on, if you wanna interrupt, that's fine. Okay. Okay, so let's think about what does this mean? So the P50, the partial pressure of oxygen necessary to fill half the myoglobin oxygen binding sites is 0.26 kilopascals. So what does that mean in terms of magnitude? The environment of us, we're at sea level, Brown University is right by the sea. The atmospheric pressure here is about 101 kilopascals. I think more of atmosphere is one atmosphere, but you can also say pressure is kilopascals. But oxygen only comprises 21% of the atmosphere. The majority is nitrogen, of course. And so if you calculate 21% of this number, you come to a partial pressure of oxygen of 21 kilopascals, right? But the concentration of oxygen necessary to fill half the myoglobin binding sites is only 0.26 atmospheres, which is 1% of the amount of oxygen that's available to us. So what does that tell you about the, in general terms, the binding affinity of myoglobin for oxygen? Is it low, medium, high? High, right, from the perspective of the environment that we're in. So all of our myoglobin molecules are full with oxygen, right, unless we're running around and we're using that oxygen very rapidly. So it tells you something. So the function of myoglobin is to store... Yeah, all the movies don't work today. The function of myoglobin is to store oxygen. So a battery needs to have a large capacity, it has a large affinity for electrons, right? In this case it has a large, this oxygen battery has a large affinity for oxygen. Okay, so the role, the shape of this binding curve is made in the function of the protein to store oxygen in myoglobin, okay? So myoglobin is a storage molecule for oxygen, but we don't need to just store oxygen, we also need to transport it. I'm breathing in oxygen into my lungs, and that oxygen doesn't just get exhaled again, it needs to get transported in my body to my tissues. So we need another type of protein to transport that oxygen to my tissues. Myoglobin might not be very good. It would be great if it were in my lungs, which it's not to my muscles, but if it were in my lungs, it'd be great at picking up oxygen, but it wouldn't be good on the delivery side of things. It wouldn't wanna let go of that oxygen unless there was an intense demand for that oxygen, right, unless the pressure of oxygen was extremely low in my tissues, so I'm running around, they might release it from my muscles. So hemoglobin fits this role of transport. Hemoglobin is a hetero-tetrameric protein, so it has four polypeptides in its structure. Would this be tertiary or a quaternary structure? Quaternary structure. Each subunit would be a tertiary structure, but all together, this would be the quaternary structure. What is the alpha two, beta two means? That means that there's two subunits that are very similar. So the alpha subunits, there's two of those. They're primary sequences identical, and then we have two beta subunits whose primary sequence differs from the alpha subunit, but they're also identical to each other. But even though their primary sequences are different, the fold is the same. They have the so-called myoglobin fold, right? But there are some differences between them, and those differences are important, as you'll see in a moment, in the interfaces between the subunits that are tied to the function of hemoglobin. So hemoglobin is found in your erythrocytes. They're just chock full of hemoglobin. They're even red, right? So you can see that each of these subunits has a heme prosthetic group, and each of those potentially combined to oxygen. So whereas a single myoglobin protein could bind just one oxygen, hemoglobin combined up to four oxygens. And you can see it has a donut shape. There's a hole in the middle. So today we're gonna put something in that hole, and that's gonna help to regulate this protein. Okay? So far okay? There's left, maybe. All right, so here's that myoglobin fold. So do you see how the tertiary structure of a myoglobin is similar to one of the subunits of hemoglobin? It's all alpha helical. Remember these swirls, these are alpha helices. They orient those helices pretty similarly to the heme prosthetic group. Although they're primary sequences of myoglobin and this beta subunit hemoglobin are very different. They're structurally similar. They have the same fold, okay? But only 27 of the amino acids are the same, as it turns out, so that's pretty cool. So the action, a lot of the understanding of hemoglobin is gonna come at the interface, the noncovalent interfaces between the subunits here, between the alpha and the beta subunits. And there's going to be manipulation of the amino acid and the polypeptide at those interfaces to regulate this protein, to help it to be an even better oxygen transport molecule. And so this slide is often very confusing to people. So I'm gonna tell you what these letters mean in a little bit right now, it's just gonna confuse you. So we have two different states of this protein. What does that mean? That means that there are two crystal structures out there that people were able to determine. One with oxygen bound, the other with oxygen not bound. We do not have a crystal structure in between these states, but we're able to see that when oxygen is bound, it's in this configuration, hemoglobin's in the R state, and when oxygen is not bound, it's in the T state. Look at the dramatic shift in all of these alpha helices. So when you transition from the oxygen not bound to the oxygen bound form, the whole thing is flexing. Hard to find an amino acid that's not moved here. Some, as you'll see of these amino acids, are pointing down in the donut hole, like you could see it here. See this histidine, HC3, so this is a histidine, the C terminus of one of the hemoglobin polypeptides. It rotates way the heck up here. So it's in one state and then it comes up. That's perhaps the most dramatic motion that we can notice between these two states. So we have, again, we have the heme groups, and oxygen is gonna bind at those heme groups. And so here's where the word T and R are defined. T stands for tense and R stands for relax. And so it's tense, because do you see the contortion of this porphyrin ring? It's like, instead of being planar, it's a little bit flexed back. And the iron atom is pulled back, sort of like you would do with a fling shot, right? So this is tense, and when you're thinking about the orientation of this iron atom, you would guess iron's probably gonna wanna sort of sit in the middle of that plane. But in the tense state, it's pulled back, and in the relaxed state, it's pushed forward. But confusingly, the binding affinity is in the inverse way of thinking of things. So the binding affinity for oxygen is higher when it's in the relaxed state. So although I've told you that these are tense and relaxed, you feel free to forget that, because it's just going to confuse you. But you do need to remember that in this configuration, in the R state, the binding affinity of the iron atom is higher for oxygen, okay? Just because of the way things are contorted. Does that make sense? Do you see the flex and the puckering in that ring? Now, it's not much of a flex. It's about 0.4 angstroms. Like, so it's just a little boop, right? And you can see, you can see it here a little bit. So we see a little bit of a movement, 0.6 angstroms. This iron atom is boop. But what is the iron atom attached to? Yes, question. I think we should go through the lecture and then I think it'll become obvious. There's a lot of forces that's playing here. There's a lot of, yeah. If I tried to review the lecture, it might not be the best way, but I think if I don't answer your question, please ask it again. Okay, so we have a heme atom or an iron atom and an oxygen bind. The oxygen pulls that iron atom a grand total of 0.6 angstroms. But what's attached to the oxygen or the iron atom? A histidine, right? That's that thing on the back that's preventing it from being oxidized. Well, if you tug on the iron atom, you're tugging on the histidine. What's the histidine attached to? The histidine is attached to the alpha helix. If you tug on the histidine, you're gonna shift the alpha helix. What's the alpha helix attached to? The other alpha helix is, so if you pull on one alpha helix, the rest of them shift. And even between the subunits, you'll have a change in the types of the chemical nature of the surfaces between the subunits as you distort one of the proteins. So sort of head or foot bone, leg bone, knee bone sort of song could be talking about this situation. Okay, so we've made a tiny little tug and all of a sudden helices are moving all over. Some of these amino acids are whipping way or very dramatically magnifying this motion. So does hemoglobin have both the distal and proximal prosthetic groups bound to oxygen? Like why? Only one side of the iron atom binds to oxygen. So when we say distal and proximal, we're describing the histidine residues. So we can go back to that slide real quick. It's a good question. Define somewhere. Anybody see it? Here. So when we say proximal histidine, that's the one that oxygen is not buying on its side. The proximal histidine blocks oxygen from binding to iron. Oxygen does not bind to the histidine. It binds to the iron. The histidine, the proximal histidine blocks oxygen from binding on that side of the iron atom. Remember, there's six coordination points on iron. And this distal histidine enhances the selectivity of both myoglobin and hemoglobin for binding oxygen. It's a good question. Okay. All right, so here's another, yeah. So in this movie, you would see this circle coming out just slightly. And you would see the histidine tracking back and forth with this. And that's shifting. And you would also see this particular amino acid. The alpha helix just shifts a little bit, but this amino acid that's in the donut hole is whipping around between this T and this R state. And also, you would see, I'll put these movies on the webpage afterwards so you can see it for yourself. You would see when comparing the transition between the T and R state, you would pretty much see all of these alpha helices moving in relation to each other. So there's dramatic motions from a little .5 angstrom tug on an iron atom. But then a question comes up, well, what influences the binding affinity of an iron atom in a heme group when oxygen binds in one of these subunits? What happens to the binding affinity to oxygen in the other subunits? Well, so what are we doing? If we bind one oxygen in one subunit that pulls on that alpha helix and all of the fact that an oxygen is bound is being communicated to the other subunits through large motions of the protein. So the subunit is self-changes shape and the changes shape of that one subunit is transmitted to the other subunit at the interface between them. And so you pull on one iron atom and the other subunits are changing their configuration. The iron atom without oxygen bound is pulled out of the plane and as soon as you bind oxygen in one of those four subunits, it gets pulled out or gets pushed to the center of the plane and the other ones also feel that effect. So now iron atoms are moved in the unoccupied, the sites that don't have oxygen, they're moved to the center of the plane and when they're in the center of that plane, they can have a stronger binding affinity to oxygen. So it helps the second, third and fourth binding of oxygen to have that first oxygen bound because there's a conformational change making the rest of the subunits in a more optimal configuration to bind to iron. So these are the interfaces here which these conformational changes are transmitted. And so as we already said, if that oxygen is in the middle of the plane, it has a higher binding affinity to oxygen if the iron atom is in the middle of that plane. So in the R state, that's the state that's relaxed in relation to the porcelain ring where the iron's in the middle. And so if we put one of these subunits in the R state by having it bind oxygen, the others will tend to move towards the same relaxed state. And then when oxygen comes along, they'll have a higher binding affinity to oxygen. So this is a fundamental concept that we'll see throughout the semester of allosteric regulation. The binding of a molecule to the protein at a site other than the active site causes a change of a shape in a protein. And here it's a little confusing because we do have oxygen binding at what you could say as an active site, that the binding, the ligand binding site, but it's also affecting the other sites that aren't binding ligands. This is a positive allosteric regulator. So allosteric means something that changes the shape of a protein. It's a shape-shifting influence. So let's look at the binding curve of hemoglobin. This figure is confusing. I updated this slide a little bit. So if you don't have these words written on there, if you printed it out early, you should add them. So the actual binding curve of hemoglobin for oxygen is in this S shape. But if all of the hemoglobin subunits were locked somehow in the R state, this is what the binding curve would look like. If, hypothetically, all the subunits were locked in the T state, this is what the binding curve would look like. The function of hemoglobin is not just a bind to oxygen, it's to bind it and then release it. And so it binds it somewhere. It binds it in the lungs. So where the partial pressure of oxygen is pretty high. And then it releases it in the tissues. So if we're all locked in this R high affinity state, then you would bind it in the lungs, it would get to the tissues and it would just say, uh-uh-uh, I'm not gonna give it to you, it's mine. It's selfish, selfish little protein. But from the T state, it would get to the lungs like, hey man, there's oxygen and I'm not just hanging out, I'm not gonna party with that oxygen. So it was only just still maybe, you know, I don't know, what is that? 0.5% of the hemoglobin molecules. And then they say, okay, I'm going to the tissue now. And only half of the active sites are full, what good is that? They get to the tissue and say, yeah, man, I'll give you, maybe I'll give you some.  But look at the difference here. The actual binding curve of the hemoglobin that has this shape shift goes between these two curves. So in the lungs, it has extraordinarily high affinity for oxygen, but then it gets to the lungs and that affinity is greatly reduced, right? So that helps it to, in its function, it's not just storing oxygen, it's delivering oxygen to the tissues. Does this figure make sense? I'm going excessively slow today because I have a limited number of slides, so. This is not the normal state of things in biochemistry. Any questions? Okay. Yes, online question. Just one clarification about the R and T states. The R confirmation triggered by oxygen binding to hem, which? The R configuration becomes more favored in the unoccupied subunits when one subunit binds to an oxygen. So when the first, you know, normally, we'll get to sort of ways to think about the ways in which the subunit shifts state in a moment. There's different models for this, concerted or sequential. But for the first binding event, there would be a larger proportion of the subunits would be in the relaxed state. But as soon as you bind one oxygen molecule in one of the subunits, that would pull that iron out and the other irons in the other subunits would also shift slightly more into the plane of the ring so they can make tighter affinity bonds. And that's cooperative. So that's the binding of the first oxygen helps in the binding of subsequent actions and that's why the shape of this curve is sigmoidal. Yes, that's a good question. So you mean on the release? Yes, you would imagine so. Especially, well, it depends. The thing is the problem here is we don't have data. We have an X-ray crystal picture of the R state and an X-ray crystal the picture of the T state.  Are they gonna shift independently or all together? Now definitely, when you get to the last one, you would think that it would help but we don't have data. That's a wonderful question. We don't have data because it's flexing and it's not crystallizing, that's one of the reasons. You know that question? And so one more. How does the first oxygen bind? It binds chemically, I don't know what else. Yeah, so it, well, I guess it's in the lungs and the oxygen becomes dissolved in solution. It gets into the red blood cell and it binds to the hemoglobin, one of those iron atoms in the heme group. So diffusion, I guess, is important. That's a good question. Okay, that's why we have discussion. Okay, any other questions? Yes, all right. So we have the shape shifting. We're gonna see examples of positive allosterism where you're helping by the regulator binding in subsequent binding events. We have negative allosteric regulation. We're looking at all types of regulation. Today we'll see different examples beyond just oxygen binding. So but now we had this nasty stuff that we did for myoglobin. We can do a similar mathematical treatment of describing the equilibrium between hemoglobin and its ligand oxygen. So here we could have more than one ligand. So we have to take that into consideration. So we can define a association constant, products divided by reactants. Here we have to raise to the ends of power because we have the possibility of more than one ligand. We can define theta. Theta is defined similarly, right? So remember theta is the percent of active sites that have bound to a ligand. And now we're gonna do something that makes no sense whatsoever. I'm gonna take theta and I'm gonna divide by one minus theta. You're like, oh yeah, that helps, that's good. So we took this nice, tidy little equation and we divided by one minus theta. But then we started to rearrange and I'm not gonna go through it because I'll get you all confused. But if you go through it on your own, you'll see that this nice, tidy equation got even more tidy when I did this manipulation. But this has no real physical meaning. What is fractional occupancy divided by one minus fractional occupancy? All it does is it makes the equation look nice and neat. So, okay, this is a hill equation. And so, but this is in perfect. We want it to be really simple. This is not linear. So back a while ago, they didn't have computers. They had slide rolls. So it was often helpful to make things the slope of the line. Was it me? Oh, it's okay, okay. Can't come to the phone. All right, so all you do is you take the log of both sides. Remember, that's one of the things you're allowed to do. And if you take the log of this side, you get rid of the power. So it becomes a linear relationship between this odd combination of fractional occupancies and ligand concentration with slope of N. So I can determine what N is because I can measure fractional occupancy at a variety of ligand concentrations. You're like, right, yes, you can do that. But what does it tell you? You can think of N as the measure of cooperativity of the binding of a protein for its ligand. So as you increase the concentration of ligand, here it's a gas, so it's expressed as a partial pressure, as you increase the partial pressure of oxygen, you get to a certain point where one of those four subunits has bound an oxygen atom. And when you get to that point, the binding of subsequent oxygen atoms to the remaining three subunits is assisted. It's cooperative. So until you bind that first oxygen atom, it's not cooperative. All of the iron atoms are in this tense state. They have a lower affinity for binding oxygen, but as soon as you bind one, it becomes cloppy. And so the slope goes up. So perfect cooperativity would be a slope before, but what does that mean? I don't know. It's just sort of a metric of how much you're assisting. The degree of the allosteric regulation. Okay, and so for hemoglobin, it's pretty cooperative. If you look at, you can do this type of analysis for lots of different proteins that are regulated allosterically, and you can calculate these hill coefficients for different proteins. And as it turns out, this is very cooperative. So for myoglobin, it's not cooperative. This just is not allosteric regulation. It just binds oxygen and then the story's over. So the slope is one, irregardless of the concentration of the ligand. But here, we're measuring the degree of cooperativity. And so this is just a useful thing that one could do in the lab to measure how allosteric, how much allosteric regulation is changing, the ability of a protein to do its job. Okay, theta divided by one minus theta, right? That's cool. Okay. You have a question? Actually, I should probably just dwell. Yes. Yes. Yeah, exactly. So what this curve, this is not a theoretical of the actual curve, suggests that when the first one binds, it's pretty much already at its maximal level of cooperativity. So this is really useful to compare different proteins, like how much allosteric regulation impacts it, but the actual like three versus two, the physical meaning of that is hard to understand. Because you would imagine that, we have not just one hemoglobin molecule in our experiment, we have lots of them. And not all of them are gonna have the exact same number of ligands. There's gonna be some distribution. The other question, this is called the Hill Equation and the Hill Coefficient. And he just got it by playing around with the numbers. Yes, Art. Yep. So what do you think would move and depend on, or depend at all on the type of ligand? I would imagine so. So if we had an example like for, I bet there would be a different, there could potentially be, well, I would guess for the example of hemoglobin, like if you look at carbon monoxide binding versus oxygen binding, I bet you would get a similar Hill curve. Because I bet the binding of carbon monoxide is enhanced by the binding of the first carbon monoxide molecule. But that would have to be experimentally determined. So the Hill coefficient's a bit of a pain for all bioconstrict. Okay, so we can think of, remember, we only have data in each of these two states. We have X-ray structures where all subunits are in the T state, where all subunits are in the R state. So we don't necessarily know what's happening in between these. One possibility is the so-called concerted model, where there's only two possible states, either all subunits are in T or all subunits are in R, and there's nothing in between. In other words, each subunit cannot act independently. Or the other model that fits the existing data, because we only have data on the ends, is that each subunit can individually oscillate between states, right? And so there could be mixtures of subunits, where one subunit has iron atoms in the center of the ring, and the other subunits have iron atoms, not in the center of ring. This is to raise a hypothetical possibility to you. We don't have experimental data yet to differentiate, which is the case. We didn't catch a naughty subunit doing something different than the others in a crystal structure. So this is one way to think about things. Okay, that's also very often confusing. Circle is one state, square is the other. Let's look at some of the other amazing ways in which hemoglobin is regulated. We've seen one positive asteric regulator, oxygen, affecting the binding to the other subunits. Let's think about what hemoglobin is doing. It's taking your oxygen from the lungs and bringing it to the tissues. And so, do you guys remember, if you take CO2 in solution and you shake it up, and you put a pH probe in there, what would you measure? It's acidic, so it's about pH of three. If you take CO2, like the Coca-Cola you drunk today, if you put a nail in Coke, after three days it'll be dissolved. I don't know if it's an old wife tale. That's acidic. It also has phosphoric acid, it adds a flavor, but the carbonic acid is also acidic, so just seltzer water has a pH around three. And so, one of the byproducts you'll see throughout the semester of metabolism is acid. So when cells are respiring, one of the waste products is acid. The other waste products is fully oxidized form of carbon, carbon dioxide, okay?  And so, and obviously there's more CO2 in your tissues than in your lungs. So, hydrogen, as it turns out, binds, makes a covalent bond to a particular amino acid that by, I'm sure, not just random chance, exists right at the interface between subunits and affects the interaction of those subunits. We'll see that in a second. So the binding of either protons or carbon dioxide, which also makes a covalent bond to the protein, either of those binding events decreases the affinity of hemoglobin for oxygen. You're like, isn't that bad? Well, so you can look at the binding curve here. So as you decrease the pH, this curve shifts to the right and that means that you're decreasing the affinity. Why? Because KD is increased. KD is one over KA, KA is the reflection of affinity. I just said that really fast. You might wanna process. So as this curve shifts to the right, right? It takes, because it has the hemoglobin, it has a lower affinity for oxygen, it takes a higher concentration of oxygen to fill half the binding sites. Remember, theta of 0.5, that partial pressure of oxygen is the P50. That P50 is shifting up and that's equal to KD. KD is one over KA, KA is the measurement of affinity. Are you with me? So the pH is affecting the binding affinity of hemoglobin for its substrate or for its ligand. So here we have the two states. Remember this amino acid that we tugged on the alfie helix and this one amino acid was going whoop, it's going from the center of the donut hole here all the way out to here. Well as it turns out, this is where the proton is ending up. So in your tissues, hemoglobin comes to the tissue and the tissue says, let go of cargo now please. And there's lots of acid being produced in the tissue, protons, and so this is a histidine. What's the PKA of histidine? Six. And so it's gonna pick, this particular histidine picks up that proton. What can you say about the charge of a histidine when it becomes protonated? It goes from what to what? Okay, remember the structure I might ask you this. So histidine, unpronated is, is uncharged or positively charged? Uncharged. So when you add the proton to histidine it becomes positively charged. Now if you take an amino acid from an uncharged state to a positively charged state, can you imagine that, oh, now it has a new positive charge and that positive charge could potentially or possibly have a charged charge interaction with a negatively charged amino acid. So look what's hanging up here, lurking, right? And a spartic acid.  But if you have a positively charged histidine, it's like, yeah, and it locks onto that thing, favoring the T-state, right? Do I have this right? Yep, yep, T-state, it's up here. And so that's gonna decrease the binding affinity of hemoglobin for oxygen. What about this CO2? You're like, that's inert, man. It just gets dissolved and fizzles in my drink. No, it can convert, be converted into a reactive form through the action of an enzyme. You can carbamolate an amino group. And the amino group that gets carbamolated in hemoglobin is at the interminous, the actual interminous itself. So we can take a uncharged or a, I guess you would say positively charged a physiological pH amino group and put a negative charge where a positive charge used to be. And that, as you can imagine, you're changing the charge there. And that could affect the interaction of amino acids at this interface. So between subunits, you see the new carbamolation, that negative charge makes a salt bridge with this basic amino acid lysine. Again, affecting which state is preferred. Okay, you with me so far? So subtle little covalent modifications. Even though it's covalent, it's still an allosteric regulator. It's a small molecule that binds to the protein, changing its shape, affecting its affinity for its ligand. In this case, it's a negative allosteric regulator because the binding of a proton or carbon dioxide at this interface decreases the binding affinity of the unoccupied oxygen binding sites to oxygen. That makes sense? Great. Okay. Any more questions on that? Yes. Yes. Yes, but what happens during respiration? Why is this also beneficial? In terms, think about what's happening here. Do we want that CO2 to just accumulate and get all puffy in your tissue? So it's hitching a ride on the protein. A lot of the carbon dioxide has been turned into carbonate, bicarbonate, right? So it's an ion, it's in the blood itself. So that's one way that CO2 can be transported out. But erythrocytes are full of tremendous amounts of hemoglobin, so they can also hitch a covalent ride out of your body, right? And then when you get to the lungs, you can release the carbon dioxide and you can remove this proton, too. So only the, I believe it's the beta subunit becomes covalently modified. And there's two of them. Yes. Yeah. So is that gonna help you in the lungs? No. Aha, exactly. It's not just an oxygen storage molecule. It aids not in the pickup of the oxygen, but in the delivery phase. It allows the tissue to communicate to the protein saying, please give me some of what you have, right? Because the tissue is respiring, it's making these byproducts. And those byproducts cause hemoglobin to increasingly release their oxygen, decrease their affinity for oxygen. Does it make sense? Okay. Any mountain climbers in the room? Anybody climbed? Cool. So when you climbed, did you go to different base stations or you just did a one day climb? Yeah, so if you wanna climb Mount Everest, right, you have to go up a little bit and up a little bit. And the reason has to do with oxygen. So you might think, well, hemoglobin probably has some role to play. So not only can your tissues communicate to hemoglobin, they can, hemoglobin protein can respond to the environment. So when you're climbing up a, yes, hi. A different amino acid, the picture here is confusing. Cause the histidine that gets protonated is actually on the C-terminus, but they're actually really close together, the C and the N-terminus. The N-terminus gets carbamylated and the histidine gets protonated. And they're actually, if you look at the primary, so you can see it here, it's confusing, they're far apart. But in the tertiary structure, they're right on top of each other. Okay. You have a question? Yeah, okay. So why do you have to go to these different base stations? Why can't you just go marching all the way up? Well, it's because your body needs time to adjust. Well, what is it doing? Well, as you climb the mountain, there's less oxygen, right? So to get more oxygen, you have to breathe quicker. You have to hyperventilate. If you hyperventilate, what's gonna happen to carbon dioxide levels? It's gonna go down. What happens if it goes down in terms of the acidity of your blood? Think about it. Yes, acidity, Lord, the pH goes, yep, you're right. Yep, sorry. So what does that mean? Well, there's actually a small molecule called 2,3-bisphosphoglycerate. In this semester, we'll learn about glycolysis. One of the metabolites in glycolysis is 1,3-bisphosphoglycerate. This is a byproduct of glycolysis. It's made by an enzyme that is regulated by pH. So as the pH goes down, the enzyme makes less of this 2,3-bisphosphoglycerate. You say, okay, yeah, right. So what does that mean? So if the acidity, as you climb the mountain of your blood, or if pH is going up, right, so you're gonna make more of this 2,3-bisphosphoglycerate. And this, do you see how much charge is on this molecule? This is just a little ball, intense ball of negative charge. This thing very precisely nestles down into this open donut hole in the T state of the enzyme, the low affinity state. And it binds, it makes non-covalent interactions with all these histidines at the center of the donut hole. And it tends to reduce the ability of the hemoglobin molecule to go to the R state. Like, yeah, and what does that mean? Well, let's think about this. So as you climb the mountain, the acidity of your blood goes down, the pH goes up, right? The enzyme gets turned on to a higher degree that makes this bisphosphoglycerate. The higher levels of bisphosphoglycerate interact with their hemoglobin, increasing and decreasing the affinity of hemoglobin for oxygen. So as you climb the mountain, your hemoglobin will have less affinity for oxygen. How could that be good? Doesn't that seem paradoxical? Yeah, you got it. It's a two-picture story. You pick it up in the lungs. So if you have lower affinity in the lungs, that's not necessarily good, but the partial pressure of oxygen in the lungs is pretty high. So having a little bit lower affinity for oxygen in the lungs would be not so bad. But in the tissue, they would be more efficient at delivering it. This figure is very confusing. So let's consider the concentration of bisphosphoglycerate that occurs in your cells, in your erythrocytes, at sea level. So at sea level, you pick up oxygen pretty well, and then you deliver about 38% of the oxygen to your tissues, right? So this is the partial pressure of oxygen in your tissues, and so the delta here is 38%. Now, if you climb the mountain and you have the same amount of bisphosphoglycerate, well, you would pick up less and deliver the same amount. So only 30, so each hemoglobin, trip of the hemoglobin protein from your lungs to your tissue would only deliver 30%. So your tissues are starving for oxygen. But look what happens when we add bisphosphoglycerate. Yes, we pick up less, but we deliver more. And the degree of decrease here is not as big as the degree of decrease here. So overall, you're back to the efficiency of delivery of oxygen to your tissues that you had at sea level. So as you climb up, you have these physiological responses because you need more oxygen. There's less oxygen, you're gonna breathe more, you gotta get it in there. But there's a negative consequence of that, right? You decrease the acidity of your blood, that's particularly bad, right? What does acidity do? So if we have decreased the acidity of the blood, what state will that favor? R, the high affinity state. So if our blood pH is changing, that's gonna make it less efficient to transport the oxygen, right? So this thing is compensating both for the change of acidity, but it's also aiding in addition to that. So we're balancing things out. Your tissues are your tissues. Your tissues are the same no matter whether you're on top of Mount Everest or at sea level, but it takes a while. This bisphosphoglyphus rate is produced metabolically, so it takes days for you to hyperventilate, pants a lot, and then eventually build up the concentration. Smokers have a higher level of bisphosphoglyphus rate. Any medical condition causing you to hyperventilate could potentially cause this alkalosis, this increase in pH in your blood, which would need to be compensated metabolically, okay? Is that cool? I think that's just amazing. Does this make sense? What an amazing thing. There's all this sensing of the environment. Okay, there's also problems that can arise with hemoglobin. You can have hemoglobin proteins that have a mutation. So you guys have heard of sickle cell anemia, right? I had a good friend with this, and it's a horrible disease. And it's caused by a mutation of a glutamic acid in the beta subunit of hemoglobin to a valine. And this glutamic acid, you might imagine, is on the surface of the hemoglobin protein. Where a lot of the charged amino acids tend to hang out. But if you put a valine on the surface of an amino acid and on the surface of a protein, and you have two of these subunits, think about what you've done. Sticky patch, sticky patch. Two sticky patches, polymerization, right? So we have a valine, a glutamic acid cannot make any kind of productive interaction with another glutamic acid on a different hemoglobin protein molecule. But two valines can have a hydrophobic interaction. And there's two subunits. So this occurs just on the beta subunit. And so you have sticky patch. So you begin to form a polymer of hemoglobin.  And they get all stuck to each other, and it affects the circulation of blood in your body. And so you might wonder, this is awful. Why does it exist? Why does evolution allow this to exist? It's a horrific disease. Well, there's a really neat discovery that they made. They look at the incidents of sickle cell anemia in the world, and then they looked at the incidents of malaria. And they say, ha, it was nearly overlapping. So although having, being homozygous for this mutation infers sickle cell disease, sickle cell trait where you're heterozygous for this mutation, you have a reduced concentration of the mutant protein. And this actually is good. And the reason it's good is because of the way in which a plasmodium protozoa interacts with your cells. It affects cellular pH. And for whatever reason, the shift in pH, having a one copy of the mutant hemoglobin, the shift in pH causes aggregation of the hemoglobin and change of shape of the infected erythrocyte. So it basically has a malaria detectorometer. And so malaria comes in to cell changes shape because it has this valine, and for whatever reason the change in pH causes this aggregation. So it's a positive effect is that people don't get malaria when they have sickle cell trait, when they have sickle cell disease. It's not a good ending. Any questions? So there's all kinds of medical implications. Perhaps there's some pre-medical students here. I'm not being sarcastic. Okay, I love you guys. Okay, so I do. I'm actually gonna teach medical biochemistry next year, so I have to learn this stuff. All right, so I'll learn with you. Maybe some of you will be with me. Okay, so another protein whose function is to bind other things is immunoglobulin. And this is a structure of immunoglobulin. It's a hetero-tetrameric protein that has, it's very similar to alpha-2-beta-2 hemoglobins in the fact that it has two so-called heavy chain polypeptides and two light chain polypeptides. And here's the structure of an antibody. At the ends of these structured regions that's hyper-variable regions that flap around, okay? And so here is another picture of this. And you could see here, these hyper-variable regions. It's all random coil here. And there's a huge amount of genetic variation of the types of amino acid sequences that occur in these variable regions. And so what's gonna happen here is these antibodies are gonna bind to their substrate, the ligand to the antibody, the epitope, and these variable regions are very flexible. And they're gonna actually wrap around the ligands. So this is not a static Y shape, and it's not peace for us all. This is some kind of, look at the motions that occur here. So here's an antibody without binding to its ligand. And they've highlighted some of the charged residues here on the surface. And then this is the structure of the antibody bound to the ligand but hiding the ligand. So here's this here. And look at the movements here. So these things, this protein is wrapped around there and it makes the tightest interaction if it's the right type of substrate. It has complementarity in terms of the non-covalent bonds that it makes. And so we'll look at lots of practical examples of immunoglobulins throughout the semester. Many of you do biomedical research where you exploit the flexibility of this protein to recognize the presence or absence of certain proteins in a solution. And so one of the ways you do this is using something called immunoblot. And the majority have done immunoblots? Yep, yep, okay. But some have not. So immunoblots, if you're from the 60s, you call it immunoblot. These days we call it a western blot. I find the terminology a bit arbitrary. It's not a southwestern blot. I mean, okay. So, but to do this, we separate the proteins based on their size using SKS page gel electrophoresis. We talked about this previously. Then we use the flexibility of these immunoglobulins to bind so we transfer the proteins from a gel onto a nitrocellulose paper which is also somewhat arbitrary but it's sticky of proteins. So then you have proteins on a piece of paper.  So these proteins are separated by molecular weight and you'll have a signal here where this antibody is bound selectively to its substrate. We then have, there's many ways to do western blots. One way is to have a so-called secondary antibody binding to the constant region of the heavy chain of the antibody. And this antibody could be covalently conjugated to an enzyme. So you guys might have done ECL. Have you guys done ECL using X-ray developer? Okay, I'm not that old. So these days we use fluorescence blots, something called Lycor. But so this enzymatic reaction converts a invisible substrate to a detectable product. And so you get these bands that show up. And the reason you need that is because it amplifies the signal. If you don't, an enzymatic reaction converts more than one invisible substrate to a visible product. And so we'll learn more about that later but this is just a side. So any questions from today's lecture? So when you think about your blood you have new thoughts about what's happening in your body. So just one more. One more question, guys. Why the valines on the surface of hemoglobin lead to the subunit sticking? I didn't hear the last part. Why the valines on the surface of hemoglobin lead to the subunit sticking? Yeah, so valine can interact with other valines with hydrophobic interactions. Whereas the glutamic acid, the wild-type hemoglobin glutamic acid would actually repel another glutamic acid. It would be a negative charge on a negative charge. So it's a hydrophobic interaction. OK, goodbye. No, excuse me. OK, I have to make an announcement here. So last lecture, there were five people that turned in their clicker votes at the end of class after I announced the answer. And so we're not creating a curve. We're not competing against each other. But that's not allowed. Just to be clear, it might be a little stressful because it doesn't work and you've got to run down. But you have to make it to Alex before we tell you the answer. OK, turn in your votes. Also, maybe while we're polling, maybe we could talk to Alex after polling because some people need to come in and deliver their vote on a piece of paper. Has anybody not voted? Anybody not voted? Like, here comes a paper. Alex, you've got to let people in with their papers. We can't tell the answer until everybody's voted.  You've got it awfully, OK. OK, everybody, if you vote, if it receives your vote, you should have seen a green light show. Alex's laptop was pretty far closed. So I want to make sure everybody got their vote in. How many total? 246. All right. This is Go Find a Slide Scavenger Hunt clicker question. So what's the answer? Thank you. You guys are geniuses.