Each one-hour session will focus on a different individual and the intersectional leadership work they're doing in their community and beyond. This month's speaker is Josh Wood, a 16-year-old racial and climate justice organizer based in Sanford, Maine. He is the co-organizing director of Maine Strikes and served as the youngest communication director in the state's Black Lives Matter movement over the summer. He has worked to remove school resource officers in his hometown and to create equitable changes to schooling. He was recognized by the Portland Press Hale for his contribution to small-town movements and now continues to work with Maine Climate Strikes to pass county-wide climate emergencies and manage statewide action teams. Welcome Josh and thank you so much for coming to speak. Thank you for having me, sir. So, for those of you who are new here, the next hour will be divided into two parts. First, Josh will speak about his experience and his work, and then we will have a Q&A session. Please feel free to put your questions for him in the chat at any point throughout the session and we will come back to them at the end. While Josh is giving his talk, we encourage you to turn off your cameras and to use speaker mode as to see him more clearly. And Josh, as soon as you're ready, we can get started. All right. Well, hello. I'm extremely happy to be giving my first Future Focus talk. In case you are not aware, as Sirui just pointed out, I'm Josh Wood and I am a 16-year-old high school senior. I've been organizing for around two, maybe three years now, and I use he-him pronouns. And throughout that time, I served as the youngest state communications director in the U.S. for Black Lives Matter, Maine. Organized to walk out with March for Our Lives in North Carolina and have done various climate justice-centric work, especially in my role as co-organizing director of Maine Strikes. We're going to talk a little bit about building resistance to oppression in small communities today. And we're going to talk about how big words may be isms, affectables of our climate and our environment. And this talk is being centered around my demands from local and national groups like Black Lives Matter, Maine, the National Movement for Black Lives, Black Power, and youth-led groups that I've seen, like the gorm anti-racism development. So there's a lot of stuff we'll be touching on, but hopefully we will learn together about anti-ism stuff. I'll get to tell a little bit of my story and why it's important that we collectively come together to save the world. Just kidding, unless you want to. So first, I want to acknowledge that I'm speaking to you from stolen Wabanaki land and doing activism work here. When organizing groups I've encountered speak about racism, there's often a disconnect. Older in both membership and experience, entire climate groups don't often acknowledge that the land that we're fighting for is stolen by settlers of what we now call the United States. This in itself is perpetuated by racism. So I'm not acknowledging that we do this work on land that was not their ancestors is an ignorance fueled failure of our systems. I think organizations can remedy this failure by listening to the guidance of indigenous communities in Maine and always keeping this paramount to activism. Awesome. Now that I've gotten into small discussion about environmental marginalization. I want to talk about it in the context of the protests that we saw this summer. When I heard the term environmental racism. I said but wait, how can nature discriminate without thinking. And instead of thinking about it in that context, I learned about the systems that keep my people oppressed, even though I had been conscious of it my entire life. In the areas we live, for example, there's an act lack to access of clean air. And we know this, but there's a reason for it, which is the fall of many factors, often being gentrification. And for those of you who don't know what that is. It's the process where a corporation in a poor urban area is changed by wealthier typically wider people moving in, improving housing and attracting new businesses and displacing people in the process. And these are most often black communities and these are most often indigenous communities and people of color. So, because corporations can profit off of pushing black people out of our neighborhoods and instead build these giant retail spaces, the quality of our neighborhoods becomes poor and poor until we are pushed out. And as a symptom we see things like poor air quality, invade what once used to be our homes. This is an example of environmental racism. It's part of the reason why we showed up in the streets this summer, and why we showed up as youth, especially, and why we'll continue showing up in different forms. Now, for a second, I definitely want to talk about my own experience with gentrification. When I was a self from middle school, I lived in what's what's called, what's called sometimes the poorest parts of Boston. Matapan. And this is what I'm talking about, although I think people make Matapan vibrant. Matapan's known for a lot of things. But what most struck me was how I saw people lack access to basic necessities like clean water, wider parts of Boston didn't have this problem. They had an overabundance of water. And before I moved, sure enough, I realized that corporations had moved in, as usual, trying to make a profit. In Maine, the white estate per capita, with 98.2% white people, and the most geographically marginalized, well, behind Alaska State, is not necessarily going to suffer from this type of stuff, or at least I thought. When I moved to a small community called Samford, Maine, I realized that I was so wrong, and I realized that I was wrong when I walked in the streets this summer. As I walked in the streets this summer with my friends, screaming stuff like black lives matter from the top of my lungs. These white guys with guns greeted us at the sidewalk. White lives matter, all lives matter, and hateful forms of speech that affected us as we carried our protests out through the summer. They didn't necessarily realize how speech hurt. And unfortunately, I think that that's a missing part of our rhetoric today. I learned throughout what seems like a lifetime that making connections is so important, and seeing things from a different point of view is so important. And this is what's most important in my activism. I've met many youth activists like Sarohi, I've met Amara Afiji, and Anna Siegel, all who've been given talks here before, and they all have such unique inspiring stories. And seeing climate justice and environmental justice through the lens of their perspective is truly what inspires me to keep going. Climate justice is more than about just our planet and its inhabitants. It's a movement of love, and it's a movement to decry things like hate, and it's a movement to decry things like white supremacy. I realize this more and more every day, and that's what's beautiful about activism. I think what's beautiful is that I can make small changes in my communities, and I can see the people who are affected by it. Whereas if I try to make change on a national scale, or if I try to make change everywhere, then I don't see the fruits of my labor all the time. So, I really want future activists to realize that making change in communities of their own is paramount, because often there's not enough people who want to help, or there's not enough people who are eager to save the world, like I said in the beginning. Alright. Now, using my power as youth, I've come up with ways to organize around these issues, but supporting marginalized communities in our organizing is very important in creating an equitable society for everyone. Like I was saying, Black Lives Matter is as much of an environmental movement as it is to decry police brutality, it just says the movement for climate justices. And it's my hope that organizing and youth activists will eventually make environmental movements more accessible, anti-racist, and intersectional. And I think that the notionality means recognizing that factors like racism, ableism, sexism, wealth and more, and gentrification like I was talking about make the current environmental issues even worse for marginalized communities. I remember standing on the steps of my city, Holland, North Carolina greeted by friendly onlookers as I gave a speech about remedying environmental issues. I was in contrast this summer when I was marching in Portland, Maine, and snipers pointed at me and my friends as we scream the words Black Lives Matter from the top of our lungs again, because officials were not hearing us. The system was not hearing us. So we kept screaming. And that is such a good metaphor for continuing how we do activism, and how we really advocate for our communities. We keep screaming. We keep bringing things to the table and we keep putting our foot down. We make sure that we get stuff done. I want to point out some of the ways that youth have organized in Maine with the intersection to environmental and racial justice. So, thanks. What we, what we were doing was supporting marginalized communities in their work by meeting them at their intersection, and by meeting them at their intersection. What I mean is supporting projects by tribal communities supporting projects by Black Lives Matter and supporting the work of marginalized organizers, particularly marginalized youth organizers. And I don't know. I don't want everyone to be like main strikes. I just want everyone to be able to support the work of marginalized organizers. And I think it's important that when we talk about organizing in rural communities, we recognize that we have to support people where they are, we have to support marginalized folks where they are. We recognize that if an organization came into my area in Sanford, Maine, and started talking about demilitarizing our police department, or getting SROs out of schools, which are school resource officers. I wouldn't like that because these organizations don't know our communities. And that's really why change starts with youth organizing in their own communities, because we know our communities best. And we can reach places that frankly adults can't. We can reach people on social media. We can reach people where they are. But I have to give a little credit where credit is due. Adults have definitely supported us in ways that are unspeakable. They've supported me in ways that are unspeakable. Whether that's giving rides, whether that is providing funds for our work, or whether that is speaking on behalf of youth when we're not able to speak in spaces, or really just delivering what we want to say to people. And that is a way in which I've seen adults step up and be really good allies throughout this whole process. And I'm definitely thankful. So, being thankful is a privilege, I think, because there are so many people here, and there are so many people across the United States that are still suffering, and they can't be thankful they can't see the fruits of this work. And that's another reason why I keep organizing. When we discuss social movements, less wealthy people of color have been traumatized by things like discrimination and generational issues. So, making space for people to breathe in these small role movements is such an important part of making my movements accessible. Yes, we take into account things like able body privilege, meaning someone is not physically disabled. And making our own movements accessible is a way for us to hold each other accountable to that work. I've personally seen it. My grandparents went to an environmental protest way back in like the 1900s that was such a long time ago, but they went to an environmental protest back then. And they saw an able-bodied speaker platformed before a disabled speaker. And that story has resonated with me throughout the time that I've been doing activism work. I've used it as a call to make sure that I am holding myself accountable while I'm doing this work. So you have a disability in the United States, you're twice as much likely to be a poor as someone without a disability. As such, disabled people can be put at risk of living in underprivileged areas left with lower quality environments. This is stuff like ecoabilism that we have to be cautious about ecoabilism is such a big word. But truly learning what it means means learning about the experiences of disabled people and environmental movements and the impact of climate justice on disabled folks. We also need to be vigilant of the effect that this has on queer and trans and black indigenous people of color. The rippling effect of environmental crises, homophobia and transphobia. Fixing all of these at once is why we hold these accountable, these officials accountable. Because people who hold these identities die sooner. And this is something important. It's something so important. It's not something you just put on a resume. It's making sure that we are being accountable. It's making sure that the climate crisis is fixed and treated with the same urgency as say, World War Two, because we can pay for the climate crisis. The effects will be everlasting. But if we don't fix it right now, we'll die. And realizing this and seeing this and reflecting on it, it makes me sad. But I have hope. I have hope from people who I meet every day facing these crises and seeing them overcome, seeing them overcome oppression and seeing them overcome accentuating circumstances to ensure that other people do not have to ever again. And that is why I keep organizing. And that is why I encourage you to organize in your own community as well. Think of a change right now in your area that you want to see. I'll give you like five seconds. Think of people who will be affected by that change. Now, how will you do it? Will you support the work of marginalized organizers? Will you build a new campaign in your town? Or will you rally people up? Or will you inspire them? Inspiring people is another way of organizing. Lifting up people with your words is another way of organizing. And that's why I'm preaching tolerance here. I'm preaching seeing stuff from different perspectives. Because that's what's so important in recognizing the effect of the climate crisis on our communities. And that's really what I wanted to emphasize in this talk. Thank you. Thank you, Josh, for those incredible, incredible words. I have a few questions for you right now, and I encourage everyone who's listening to put their questions for Josh in the chat so he can answer those as well. Firstly, you mentioned at the very beginning of your talk that you served as the communication instructor for Black Lives Matter Maine over the summer. In my experience, a common misconception about the Black Lives Matter movement is that it's about hating people and hating institutions, which you and I know is false. But for those who don't know, what does the Black Lives Matter movement really stand for? What is it about? Black Lives Matter movement is a movement of love. It's a movement to undo 400 years of oppression. And it's a movement to combat hate. And the words Black Lives Matter in themselves really show what it's about. And misinterpreting that to say all lives matter or white lives matter is a prospect of ignorance. And it's ignorance to the fact that we are not seeing other people's perspectives like I mentioned. So to remedy that, we open people up. And we have platforms like these, which allow folks to really see the impact of movements, to really see the meaning behind movements, and to understand. I definitely agree. And I would also add that I feel like people who purposefully misinterpret the intent of Black Lives Matter, the Black Lives Matter movement are people who are afraid of seeing how their own actions have been attributed to hate. They're afraid of seeing their own internalized biases. So it's easier to lash out at an outside organization. But you're completely right that Black Lives Matter is about love and about fighting hatred. Exactly. Fear definitely breeds ignorance. And I think that quote holds true as we think about Black Lives Matter and its impact this summer. Definitely. So some of the people in the audience here are young people who are interested in hearing the voices of other youth activists across Maine. For those young people, what advice would you give to youth who are interested in getting involved with social justice but don't know how. Well, definitely don't stand in front of a line of police and shout Black Lives Matter. No, I'm just kidding. Stand in line of police and shout Black Lives Matter. Stand on the steps of your city hall and scream from the top of your lungs. What you want your officials to hear. Make sure that you are being loud. Because if we're not loud enough, then they won't hear us. Just make sure that you are being heard constantly because being heard and sharing the perspectives of others, sharing the perspectives of yourselves. Most importantly, sharing the perspectives of those who can't is so, so important in this work. So one start in your own community to be heard. Definitely. I really like the point you made about starting in your community. I remember at some point a while ago and I remember this all the time that starting locally is incredibly important. Like you said, in order to see the fruits of your labor but also because you can make interpersonal connections in a way that statewide or nationwide campaigns just don't allow. I think that holds true. For the other half of our audience who are older people who might be interested in helping youth activists. What advice would you give them on how to be a good adult ally. What I think is really helpful by my grandmother all the time is that I either forget to shut the door to the pantry, or shut the door to my bedroom, or switch the laundry or something. That's because I'm not using my sensory awareness. I think for older folks using your sensory awareness and meeting youth where they are is super important. If someone needs a ride to a protest, give it to them, or if someone needs a donation to continue seeding a project, do that. Use your awareness to see what people need and adhere to what people need. Sometimes we'll say it blatantly and sometimes we won't. Being a good ally is definitely meeting people where they are. And I think that's so important to emphasize. That's a great answer. And I would also just add for my own for my own experience and incredibly important part of supporting youth is to pay them for their work to stipulate them and support them financially because a lot of us are doing our work in addition to being full time students are holding down jobs. Right and if you pay us we can continue doing the work. And that's super important. Absolutely. So, we talked a little bit in your introduction I mentioned that you're 16 which I think a lot of people find is really stunning. I certainly find it inspiring because you've done so much at such a young age. But as such a young activist. What is it like being an organizer at this age.  I've learned from other activists as I go. And I've learned from other organizers as I go. I really think that keeping your perspective open is super important to learning. And just making sure, again, that you're holding yourself accountable when you're opening your perspective is super important. I guess I'm just continuing learning. I'm continuing learning from activists like you. Or even other folks sometimes who are willing to lend a hand. Definitely. That's a very good point. I'm always learning from everyone around me. I would add back. I'm also learning from you. So, at this point, I encourage everyone to add to put their questions for Josh in the chat. One of the questions earlier from Michael done to paraphrase, are there laws in Maine you see as inherently racist, or how does racism manifest itself in law or in the legislation in legislation. This is a very good question and it ties into systemic racism. It ties into a lot of what I was talking about experiencing gentrification growing up in Boston, making sure that we root out systemic racism and all of our policies is definitely important. I know that in Sanford that they have at least two armored transport vehicles sitting in the police parking lot on full display for all citizens to see. And for as long as I've been living here, that's intimidated a lot of the black folks here that's intimidated the whole black community here, because police essentially have high weaponry, and we saw this at the protest. We saw police brandishing these huge guns and huge ammunition. Unlike stuff that we've even seen in big cities. So, I think recognizing the ways that people have too much power or recognizing the ways that systems have too much power is a way to make equitable legislation. And it's a way to take into consideration the stories of other people and how they feel. Definitely. You make a very good point about systemic racism. I think it's important also to remember that the majority of people in power are not people who are part of marginalized groups the majority of people in power are older people white people generally male people generally are not people there's a whole, there's a whole lot of privilege there, and that privilege often leads them to create laws that are blind to the sufferings of marginalized communities just because they haven't experienced it so they're not aware of how their laws might impact those people so as Josh said earlier it's important to broaden your view and support the voices of those who might not have a voice in those decision making spaces. I have a question from the chat. How would you describe the difference between being an activist and being an organizer. Great question. Being an organizer is definitely jumping on projects organizing stuff. Being an activist is being outspoken speaking places, making sure that you are being involved in projects at the very least. In activism, there is often a very blurred line between the two, especially in youth organizing. Just because we as youth activists don't really tokenize or commodify terms that we call people because we recognize that whatever folks can contribute is super important either way. That's an excellent answer. I have another question from the chat. Can you talk a bit more about what accountability looks like in organizing. How do we hold ourselves and other organizers accountable without cutting people out of decision making processes. Yeah, so I said earlier that the protests in 2020 were more than uprisings combating the wounds inflicted by police brutality. They were a celebration of the boundary is love that form that the boundary list form that love can take on. And I think recognizing that these protests recognizing that all of our work that we do is rooted in love is a great way to start holding yourself accountable but not stopping there is super important. Continuing learning from folks, continuing learning and broadening your perspective outside of spaces that you do activism and super important, super important. And just really making sure that you are bringing yourself, you got to bring yourself in order to hold yourself accountable, because if you're being inauthentic in spaces. People will realize. I know that when I first joined these activist spaces, I was super shy. I honestly, I did not not know what to say to people. I didn't know what to do. And recognizing that that's okay too. But you just got to keep learning. And that's really what I want to emphasize again throughout this talk and just be the theme. Recently, I did I resonate very much with the being very shy about speaking up in a space at first but once you get more comfortable, it can be a great it can be an amazing experience. A question from the chat. How do you think environmental organizations in Maine can be better at being intersectional in their work. this one a lot. Meeting people where they are is paramount to being intersectional in your work. Meeting people at the projects that they are doing and supporting them at the projects they are doing is so paramount to being intersectional in all of activism work. Because if we try to trample on the work that communities are doing to rectify hurt or the work that communities are doing to rectify the blindness to systemic oppression, then we won't get anywhere. We need to make sure that we are supporting young activists, we are supporting young organizers, we're supporting marginalized led organizations, and most of all we're supporting Black-owned businesses. I don't see all doing that anymore. You all got to support Black-owned businesses. That is a great way to continue being intersectional in your work, being intentional in your work, especially as an organization, just supporting people. Definitely. And I would also add if you're in a position of power in an organization and you find yourself being called out on behavior that you find that someone is found to be problematic, especially if that someone is a member of a marginalized community, don't be offended and don't lash out. I heard someone say once that being called out is a blessing because you're being made aware of how you can be better. So know that it's not a personal attack against you, but it's someone's way of showing that they care enough to try to correct your behavior. That's a really important one. Another question. What advice would you give to someone who needs to confront behavior that they consider to be harmful? If they try to bring up a way of being more intersectional, but people don't listen or they're having trouble making people hear them? So he just brought up a very good point about this. Sitting with your uncomfortableness in these situations is a way that you can hold yourself accountable. You can hold other people accountable because whoever hurt is more uncomfortable than you right now and recognizing that the situation is not about you. Just seeing again, seeing it from a different perspective other than yours is such a way to connect with people. And I think this holds true in discussions about accountability as we try to rectify again systemic racism, as we try to rectify the wounds of colonization, misogyny, stuff like homophobia, and especially in organizations, this applies to everywhere. Holding ourselves accountable ensures that we are doing the work that needs to be done. Definitely. Another question from the chat. Have you been able to connect with other young people and allies in your school community? And what kind of projects and campaigns have you been able to lead during the pandemic as opposed to in a time when we could be interacting with each other in person? Well, I often find that when I first come into spaces that seem tokenizing, they're scared of me because I leave from the heart and not the head. And I think that just being unique, being authentic, again, is super important. I can't speak enough about maintaining a sense of self-identity and activism. And being confronted on that question, I'm definitely still learning about it, especially as a 16-year-old organizer and activist. But I think that would be my best answer, being authentic. That's a very good answer. Another question from the chat. How do you strike a balance in staying physically and mentally healthy? I know personally being an activist is an incredible emotional strain. And what are some of your tools you use to keep yourself mentally and physically sound? Grounding myself in this work is super draining sometimes, because I'm essentially telling systems and I'm telling government that I matter through my work. And them seeing that I don't matter, even when I do this work, it gets super draining. So I appreciate this question. I can wholeheartedly say, after nearly three years of doing this, nearly spending my childhood on this, that I am exhausted, especially as a Black youth. I am exhausted of trying to explain to people why I matter. I am exhausted of trying to work with systems that want to work against me. But in the face of it all, I work with some really inspiring people that keep me motivated to do this work. It's the little things that remind me that I am important. I am enough to continue doing this work. Finding the right space for you is a way that you will not get burnt out. I can wholeheartedly say, again, taking breaks super important, but getting burnt out permanently in a space might not be the correct space for you. So find people who also lead with the heart. Just a quick follow up for those of the people who might not know here. Can you define what burnout is? How I would define burnout is not having motivation to do projects anymore because you've been constantly doing them. That's a really astute definition. Sort of a related question. Someone in the chat said, I deeply appreciate leading with love. How do you deal with folks who are steeped in misinformation about climate change and racism? How do you approach having a conversation with those people? Yeah. And thank you for this question because this happens in my family so much. Having conversations is important, but doing the work is also very important. And I need to focus on the work that I do, especially as a marginalized youth. But with people who hold more privilege, I think that having these conversations and being intentional is super important. I know that I express when I am uncomfortable, but I should not again have to explain to individuals or systems why I matter. So confronting people where they are is super important.  Broadening someone's perspective can brighten someone's day someday. And you will see that. That's an excellent answer. Another question from the chat. How do you feel about the intersection of poverty and race in rural places like Sanford? In rural places like Sanford.  And they, I would say that at least 75% of the neighborhoods that I've been in that are super nice, super ritzy, have all these big houses are white.  And this shows up very prominently. So in Sanford particularly, when we speak about the intersection between race and poverty, we have organizers like us trying to bring awareness to that. We have people like Anne Hanselman who is a city counselor trying to make more equitable legislation. We have people working with us in schools to redefine the curriculum to be more anti-racist. And we have more people ready to inspire others and just have those hard talks with their family. So I think broadening that in Sanford has been really important for us as we continue doing anti-racist work. That's an excellent answer. You mentioned earlier that you had moved to Sanford that you didn't grow up, you weren't born there. How have you connected with and learned about the community that you're in and the people in that community as someone who's coming to this as a new, coming to a new place? Love this question. So I've lived in a lot of different communities and finding people through social media is so easy. But for those of us who it is not easy for, there are definitely community centers. There are youth centers and in maybe a bigger city, there are opportunities to connect with youth. I know that I started originally March for Our Lives in our school. In middle school in North Carolina, I saw a poster for it and I got so interested immediately because I knew that helping people was the path that I wanted to go on. So finding your own path, super important. But then using the resources available to you, also very important. If you are not mobile or if you cannot go places right now, I definitely suggest talking with other kids in your area who might have access to these opportunities and I'm sure that they will be eager to help out. Definitely. I think that's one of the many beauties of community organizing. When you come to a new place, you don't have to where can I solve the climate crisis or where can I solve racial justice? If you're able to immerse yourself in the community, be it the school community or your work community, you'll see the problems manifest themselves and you can work with the people around you to create local solutions. And again, that's one of the big differences between local organizing, grassroots organizing, and other types of statewide or national organizing. 1000%. It's all grassroots. You know your community the best. Absolutely. Everyone, please feel free to put any more questions in the chat, but I think we are almost done. Well, thank you all for your time. Before we let you go, we just want to give a quick shout out to Amara Efeji, who just won a very prestigious award from the National Geographic Organization. Let's do a virtual round of applause for Amara. Go, Amara. Amara, if you guys don't know, was one of our future focus speakers and she's, I think everyone who's ever met her would tell you that she's an amazing person who's an inspiration. She really is. And she's in the chat right now. So thank you all for coming. Thank you for your time. A recording of this presentation will be made available on the Future Focus website, the link to which it should be in the chat. Please join us next month for our next speaker, who will be Jess Cooper. Jess is a climate activist based in Norway, Maine, who will be talking about the importance of community building to the climate movement. Thank you again to Josh for speaking and thank you all for your time. Thank you, everyone.


So, we have been discussing about the protein folding phenomena to just to have a quick recap. We said protein folding is described by this sort of a diagram, this is called as a folding funnel. So, we can look at the folding transition, this is the native state here and then the top you have the unfolded state which consists of trillions of millions of million different conformations and each one of the molecule in your ensemble may have a different conformation and it will eventually start folding down the funnel causing through various energy barriers and eventually lead to a single conformation or maybe a related small number of conformations here. This is called as the native state ensemble here. So, it may be few conformations which are very similar to each other by and large. So, to monitor these folding transitions, equilibrium folding transitions, they will also belong to the entire set of conformations that are possible for any protein. We can start with the denatured state here, we created denatured state by using urea or gonadine hydrochloride or whatever some of the denaturing agents or DMSO and then you slowly start changing the conditions to induce the protein to fold that means induce the protein to go down the funnel here and then you will start seeing changes in the HSQC spectra. These are proton N15 HSQC spectra, amide proton N15 HSQC spectra and you will see that there will be small shifts in the peaks and the peaks will start becoming narrower and things like that. See this spectral dispersion is will increase as you go down and this is at zero molot. This is the folded state. In the native state the spectral dispersion is very good, it goes all the way from 7 ppm to 10 ppm and you see here in the unfolded state is only 0.3 ppm dispersion there and therefore the peaks are looking broader in this situation. And you start going down, there will be changes in the chemical shifts, there will also be changes in other parameters like the relaxation properties which we will use because as the protein starts to fold some new contacts will be made when the contacts are made between different residues, they will put restrictions on the free moments of the individual residues. Therefore the dynamics in the protein gets reduced. So this will reflect in the relaxation times, the relaxation rates, particularly the R2s the transverse relaxation rates they will get reduced. Therefore changes in the relaxation rates will be good monitors for seeing the folding of the particular protein. The motions will start slowing down and that will be reflected in the individual residue wise changes. So that is how we can identify which residues are folding and what is the pathway, how the protein is trying to fold. So this is what we showed here in this particular slide. You start from an unfolded state which is completely random and very high frequency motions are present and as you start going down the motions will change. So this is indicated by the change in the color here and the motions will start slowing down that will reflect in the change in the R2 values, R2 values will start going up. And then as the structures are getting formed you will start seeing secondary structural preferences. In these areas there are no structural preferences but here we will get some structural preferences. They are not permanent structures still, they are structural preferences. So you get helix made here and the motions will further increase I mean get restricted and as you go further down 5 molar you will see more helices are formed transiently. So they make and break, make and break that sort of a situation happens. Go to 4 molar some of these, one of these helix actually disappears and these two helices are there. When you go to 3 molar so all the helices have disappeared and then you will see the structures has no structural propensities. But you see here the R2 values have increased at various places and this is the final structure of the protein and what is colored in red here these are the places where the R2 values have gone up. That means these residues are coming closer and going away, coming in contact and going away things like forming and breaking, coming close and going away and things like that. So there is exchange here. This is the kind of a folding unfolding transitions happening here. These residues are coming close and then going away. So therefore these residues are experiencing an exchange process. The exchange process results in increase in the R2 values. And therefore you see that is an interesting thing that at this point the protein is getting ready to form the native kind of a structures. These structures were non-native therefore those structures were removed and then once it is formed here then this kind of a structural transitions are happening inside the ensemble and then slowly further reduce the urea concentration you start seeing the beta sheet formed and then the two helices are the native helices and then it forms a very stable structure at zero molar urea concentration. That means in the absence of the urea. So this is how you can monitor the residue wise changes that are happening in the particular in any given protein. Now here I show you an example of the folding done in the similar manner it was in gonadine hydrochloride but this is with HIV protease. HIV protease it has intrinsic preferences here. Initial preferences are these are called the fold-ons. So these are the areas which the folding can get initiated. This is a dimer here. So this is a dimer this is called the flap here. So individual two monomores are held together kind of a beta sheet at the terminal here and then the seat terminals come close. And so therefore this is the initial folding preferences in the protein. So and then you start folding initiate the folding here then you start getting more folded preferences folding preferences that is again indicated by the color code. So you get the blue ones and then the green ones and then the red ones you start getting these. On what basis? This is on the basis of the slowing down of the relaxation rates the R2 values. Now you see here the ones which all these are adjacent to each other or the changes that are happening they are adjacent to each other which means when a particular portion of the protein folds it induces the neighboring fellows to fold and this is called as the cooperativity. So there is a cooperativity in the folding this is clearly demonstrated in this example of HIV protease and this is the final structure of the protein. So it is a beta sheet these are all beta sheets here what is shown here is of course I have shown the same structure not that these ones are in the initial phases these structures are present. But these are the preferences which are present and where the protein gets kind of propensity is to fold in that manner. So those that is why these ones are indicated on the same structure here. So it shows that there is a cooperativity of the structure formation. The initially it will be preferences and then eventually they will form the structures. And this is the flap area here this is the so called active site of the protein at the active site of the protein is very at the interface of the two monomers and this flap is also very dynamic and this dynamicity of the flap is responsible for the protein function. The substrate will have to come in here and then go out the flap opens for the slippage to come in the flap closes and then the cleavage happens this is the protease right. So it is supposed to cleave the proteins. So the substrate comes here and once the reaction is over the flaps open it goes away. So therefore dynamics of the protein also plays a very crucial role in the biological function. I will also illustrate this to you later with another example and this is how the protein folding happens. So you have initial preferences and then you provide the folding conditions there will be cooperativity in the folding transitions and they will eventually find way to the final folded structure. And the final folded structure here is it has one helix and it is a whole series of beta sheets. The beta sheets they are two beta sheets here over left of the two monomers and it forms a flap. So now so far we are looking at small proteins. Now we see how NMR can be used to study large protein assemblies when I say large not some 100 amino acid residues or 150 amino acid things like that but these are of several megadaltons, hundreds of kilodaltons of molecular size. How do we study such large protein assemblies? So what is the problem in NMR to study such large protein assemblies? So here is the slide which shows the line widths in the protein signals according to the molecular size. What is plotted here is line width on this axis, this is for the amide protons and this is for the N15 and on this axis there is spectrometer frequency in megahertz. And this various numbers indicated on the curves these actually indicate the correlation times. The correlation time reflects on the size of the molecule. 20 nanosecond correlation times it is a relatively small molecule, 60 nanosecond is larger and then this is 320 nanosecond this is an extremely large molecule. They can be assemblies these are multiple copies of the same protein can assemble together in an ordered form to form large assemblies. In such a situation we can see how the line widths change in both the proton axis as well as the N15 axis. The line widths are so large you see the 50 hertz and it is 30 hertz and things like that. So these are very large line widths. So when that is the case it is very difficult to see the signals in the HSQC spectrum. And of course this is also field dependence it is also indicated here. So if you see the line widths are smaller at this particular frequency this is almost 1 gigahertz actually this makes the case for going to larger and larger spectrometer frequencies especially if you are going to larger molecules. Of course when you are dealing with small molecules it does not matter. So small molecules are 20 nanosecond you can anyway from here to here if you go it does not matter the correlation time is 29 seconds and the line widths have not changed much. So from here to here it is about the same line width so therefore it does not matter. So it goes to larger molecules it becomes very crucial to go to higher magnetic fields where you have a smaller line width. So you see it is 10 hertz here at 1 gigahertz and it is 50 hertz here at if you take at 500 megahertz it is about 30 hertz and you go further down of course it goes to 50 and so on and so forth. So therefore as you go higher in molecular size you also need to go to higher in spectrometer frequency to get sharper lines. It will have important implications as we will see. This is true for both proton line widths as well as N15 line widths. What is the consequence of this we will see here. So therefore to take make use of this make use of this different line widths depending upon the spectrometer frequency to generate a better spectrum with a better line widths as a technique was developed which is called as the TROSI. This is transverse relaxation optimized spectroscopy. This was developed by Mr. Bhushan in the laboratory of Kutvitrik and this actually became extremely useful. This is called as attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anesthetropy indicates an avenue to NMR structures of very large biological macromolecules in solution. We will not go into the theoretical details of this. Basically it is make use of a kind of a interference between chemical shift anesthetropy mechanism of relaxation and the dipole-dipole coupling mechanism of relaxation. So the two things go in opposite directions. At some stage they will sort of cancel to produce a very small line width and that is indicated here. So suppose you are taking in an HSQC spectrum. In the HSQC spectrum if it will have on the proton axis you will have proton amide proton is a doublet with proton N15 coupling and N15 is also doublet with the same coupling provided we did not decouple this amide proton nitrogen coupling. If we did not decouple it will have four components here the particular correlation peak proton N15 correlation peak will have four peaks. But all these four peaks are different characteristics with regard to the relaxation and that is indicated here. So if you see here so typically in the normal HSQC spectrum this is what we will see because we decouple this we remove this coupling we remove this coupling therefore it will appear a peak at the center here this is one peak is what we will get in the normal HSQC spectrum which is what I showed you earlier. Now if we did not decouple then the couplings are present in both cases there are four components here. Now all these four components of the peaks have very different line weights and therefore you can see their intensities are also very different. So among these this is the sharpest this is the sharpest and these ones are relatively less sharp and then of course is the broadest. So therefore different ones are different components and this is the point one makes use of in the Trozy to say let me pick up this only forget about these ones I will do a technique whereby I can pick up only this component of the cross peak of the correlation peak. Then you see that is what we will get in this spectrum here this is the so called Trozy spectrum and you will have only one peak here this is much sharper in intensity and much larger we will see that example immediately in the next slide see here. So this is a normal HSQC spectrum it is also called as COSY because it is a N15 proton correlation spectrum is also it is called COSY but you see this is the normal HSQC spectrum which is looking like this. Now in the Trozy the same spectrum looks like this because you have picked up the sharpest component of the multiplied notice therefore that you cannot afford to decouple the proton nitrogen 15 either in the F1 or omega 1 dimension or in the omega 2 dimension and then you devise the pulse sequence in such a way that you pick up only the one component of that one 4 and that will be the sharpest signal and that is what is represented here it will reflect the denominator should do anyway. So therefore you see how sharp these ones are now you take this cross section there are 3 cross sections are taken this peak this peak and this peak these 3 cross sections are shown here this is in the COSY so you take a cross section like this horizontal cross section in all these positions so it shows the line width along the F2 dimension. So here it is case of the proton so COSY all these 3 look at the Trozy peak intensity this is so much more larger the noise level is the same this is so sharp similarly in the N15 axis also so this is the COSY and this is the Trozy such a sharp line here for each one of these and therefore naturally so if you do this your resolution in the spectrum is going to be higher and you will be able to see larger number of peaks in the spectrum and this is the very very exquisite illustration of this gain in the resolution this is a large protein of 130 kilo Dalton or something like that so this is the COSY spectrum or the HSQC spectrum and you see here is the Trozy and the peaks are so well resolved and sharp you can count these peaks measure their intensities and all kinds of things you can do therefore you can actually work with large proteins in this situation it may be a single protein or it may be an assembly of smaller molecules but eventually lead into a larger molecular size so then either case one can use this notice however that if you use very large spectrometer frequency for small molecules you actually stand to lose why you stand to lose in this case because I will show you that here so you saw here that we are picking out one component of the four in the normal case we are getting contributions from all the peaks here if you are working at a small molecule then all these components do not have very different intensities therefore when you decouple this will be very sharp peak with high intensity and at 1 Gigahertz or something but if you did a Trozy experiment on a small molecule of 10 20 kilo Dalton then you are throwing away the intensities of all of these and picking out only one then of course you actually tend to lose the intensity so therefore we should not do that if you are working on you should use Trozy only when you are working with a larger systems and then go to higher spectrometer frequencies okay so this is what and then of course 1 21 further to develop two more pulse sequences this is done by the same group once more this is called the crypt and the crinept okay this depends upon polarization transferred by cross correlated relaxation in solution NMR with very large molecules okay so we will not go into the theoretical details of this but I will simply show you the result that was obtained in this okay now this was an application to a 900 kilo Dalton Groel-Groes complex and all of you know that this is actually a chaperon this is the Groel-Groes complex is a chaperon and this is the structure of this chaperon this is determined by x-ray crystallography this structure is known okay so it is not that you are determining the structure what is the intention here is only to demonstrate the application of the technique to observe signals even in such large complexes okay now you see here this is the structure and the same thing is played out in this particular form these are three different ones here you have a SR1 and then you have this domain here and then the domain here so all of this sit on top of each other there are multiple copies of a particular small protein here in this case there are seven copies of a small protein which is 10 kilo Dalton in size so therefore there are seven copies of this one is trying to look at the signals from this particular protein okay what you do is we can actually reconstitute this protein with particular some portions labeled and some portions unlabeled and then you can see what you are going to get so in the free this is the spectra in the free this one assembly without the without these ones there and then of course you can see reasonably good signal here now the moment this one is bound to SR1 that is one this all these are presented together then you will see the signals are completely disappeared vanished in the Trozy in the Trozy these ones are vanishing so this is what is trying to demonstrate here that where the Trozy also becomes insufficient then you can use the crypt Trozy and you see this is this spectrum which is recovered when it is bound to SR1 and this is free when it is free of course you are seeing this in the crypt Trozy this actually has much larger number of peaks compared to this and this already has shown a certain enhancement but if even when it is bound to SR1 it is bound here then you will still have a large section of the peaks which are present here so this is how this technique can be used and this was published as you can see in nature in 2002 and this was an important development so the point to remember here is that you must use this techniques only when you need to work with very large very large molecules large protein assemblies it is possible to study the association process okay and so that is the therefore the you can stretch your application to very very large systems from small molecules to large systems now going further from this we would like to see how one can see self-association in the previous case what we saw was that the associated state is is particularly known the structure is known how the association is happening is already known in these cases okay so this is we know that structure therefore we know what the pattern of association of the individual molecule the structure is not changing from individual molecules to in the associated state because the spectral peaks have the same pattern there and therefore that is this is the one application this is the demonstrate that you can actually see the signals even in large ensembles okay now when you are working with a small molecule which actually associates this is very common phenomena in protein NMR that or whenever you express proteins many proteins tend to self-associate now how do we understand the self-association process does it lead to a association which is constructive or destructive is it functionally relevant or not relevant so therefore self-association is an important phenomena to be studied by as important phenomenon in biology therefore we try to see how one can study this I will illustrate this to you with particular example here now this is a molecule this is a process called as endocytosis this is the process called endocytosis what does that mean so if a small molecule has to be internalized the small molecule comes here and then this will be engulfed by a membrane and then it actually forms a kind of a encapsulated system and then there is a protein which actually binds at the neck of this formation here and this protein's name is dynamic okay and this is the budding clathrin coated vesicle the dynamic actually forms a big rope kind of a thing and then when you have the GDP hydrolysis this actually gets separated out and then you have this molecule internalized so this is the process known as endocytosis so the interesting point here is this dynamic molecule the dynamic molecule how does it work what is dynamic what is its role okay so this is therefore a functionally relevant process okay now what is dynamic let us look at what is he present here the structure of dynamic so this so the dynamic is a protein of 864 amino acid residues it has these many domains the gt paste domain the middle domain the ph domain gd domain and the prd domain these are five domains which are there this is 864 amino acid residues and this protein actually self associates self associates to form huge assemblies several megadalton size assemblies that is how it is able to form a big rope when it forms a big rope then it can actually bind at the neck wrap around the neck of the budding vesicle and then of course it squeezes with GDP hydrolysis it forces puts pressure and then the whole thing gets separated out so this among these domains it is the gd domain which is responsible for the self association of the of the of the dynamic so therefore can we study this this domain so okay so let us see this you can I individually express these proteins produce these proteins in solution and this is what you get so you take a gd when you take the gd molecule express it and you see it has a nice structure this is the circular dichroism spectrum and you see that it has a very beautiful structure this is the helical signature this protein has a nice helical signature okay of course this will not tell you that what is the size of this molecule whatever is the size so this is expressed protein has this sort of a thing now and this is the electron microscopy image the transmission electron microscopy you can see that this is forming particles of this size so what that means this means these are extremely large extremely large assemblies associated state so typically so 20 to 30 nanometers of size you will get here so which means the molecular weight if you translate that into it will be several megadaltons 10 10 15 megadaltons in size with such a large molecular weight it will be impossible to get any signals now we look at the HHSQC spectrum of that one here so you see how many peaks you see only about 25 peaks here roughly okay typically the GED protein itself has about 618 to 752 okay so this is about 134 residues so 134 residues discount some prulines and things like that so you should expect about 125 to 130 peaks so if you see look at that in contrast to that you are seeing only 25 peaks so what is happening of course why where are these peaks coming from obviously these peaks are coming from a certain section of the protein which is flexible and therefore it has a different correlation time the flexible domain has a certain correlation time and you will see that okay so I will be able to see the signals for these ones and where are these if you look at these numbers these ones one can analyze so these are coming from a particular domain of this particular protein and these are in the n-terminal so you look at these residues number here 3 14 21 11 25 15 so these are mostly in the n-terminal of the protein so therefore n-terminal is free whereas the rest of the protein gets in a kind of an associated state to form a large assembly so when it forms a large assembly it will you will get electron microscopy pictures like this and all those portions which are in the interior of this assembly you will not see the peaks from there because that forms a huge molecular mass and that will be tumbling very sluggishly and you will have very large line weight and you will not be able to see those signals here okay in these ones you can see particular types of residues here these are mostly you can see few of the glycine's you can see the serine's here and the threonine's serine threonine's and and then you are largely charged residues okay either hydroxyl groups here or the charged residues glutamates okay aspartates arginine's okay and tyrosine here and one or two here valine and things like that fine but by and large these are more least charged residues and it is reasonable to expect these charged residues are on the surface of the assembly and the all the from the different components because their flexibility they do not show as different peaks suppose there are some 10,000 molecules in the assembly all these 10,000 molecules n-terminal segments are looking similar they are producing one peak only they are not producing different different peaks they are all producing one peak so therefore this ensemble average is what you are getting and these are the residues which are on the surface these are charged residues therefore they are expected to be on the surface and the ensemble average of all of those ones gives you one peak each for each of these residues and you are able to see those peaks and you can analyze this you can assign this individual residues by using the standard procedures which you have discussed earlier for small peptides or small proteins okay now how do we understand this process our objective is to understand this process here okay this assembly process I think okay all right so we have to use a use a particular strategy to identify this okay so I think I will take up in the this in the next class we will stop here