Lux presents Hollywood. The Lux Radio Theatre brings you Barbara Stanwyck, Brian Ahern and Ida Lupino in Wuthering Heights. Ladies and gentlemen, your producer, Mr. Cecil B. DeMille. Greetings from Hollywood, ladies and gentlemen.  It's already stirred many millions as it will target your heartstrings tonight. And it's an actual fact that you, our listeners, are producing it, for it was your letters asking for Wuthering Heights that started me in pursuit of it. May I read you one of the many letters requesting Emily Bronte's masterpiece? This letter says, I'm wondering, would you, could you give us Wuthering Heights? If I thought my request could be granted, I should be thrilled to the skies. I think it's one of the greatest love stories that's ever been told. And leaves you with something to think about. Then our fair correspondent adds, with a twinkle in our pen, if Wuthering Heights is staged, I shall use two cakes of luxe-toilet soap each week instead of one. It's very gratifying to me that the products behind this theater, luxe-toilet soap and luxe flakes, are of such incomparably high standards that I have no hesitation in asking you to purchase them. And now just a word about how I came to cast tonight's play. The part of Kathy requires great talent in an actress, but above all, it demands sincerity. And that alone was enough to make Barbara stand with our choice. I telephoned Barbara at our ranch to ask if she'd like to do Wuthering Heights, and found she was playing nurse in one of the stables where a blessed event had just taken place. Barbara reported that both mother and son were doing nicely and gave me a confidential tip that the son looked like a sure winner in the Kentucky Derby of 1942. But of more immediate importance was Barbara's eagerness to do Wuthering Heights. Then we discovered that Brianna Hearn had cramped over the Yorkshire Moors many times, and that one of his secret ambitions was to play the part of Heathcliff. That solved a very naughty problem, for it's a role that demands rare strength and fire. For the part of Isabella, we selected Ida Lupino, one of Hollywood's rising young dramatic actresses, because she too has that emotional power, which every character in this play seems to require. From the motion picture cast of Wuthering Heights, we have Serita Woonton, Douglas Scott, and Rex Downing to play Kathy, Hindley, and Heathcliff as children. Now the players are on the stage, and our drama's about to begin. We raise the curtain on Act One of Wuthering Heights, starring Barbara Stanbick as Kathy, Brianna Hearn as Heathcliff, and Ida Lupino as Isabella. Desolate and lonely are the rolling moorlands of England, where in the winter night, the snowdrifts stand like giant specters in the teeth of screaming gales. About 100 years ago, a stranger lost his way on such a night. Freezing, blinded, and stumbling, he saw at last the fitful lights of an old manor house, aged and crumbling on the hillside. He knocked desperately at the door. It was opened by a doddering old butler who stood staring at him. Then without a word, he led the stranger into the musty living room, where in the dim candlelight, a man and two women sat without moving. The man was tall and dark-skinned like a gypsy. On his face, the stamp of years of bitter suffering. At his feet, a great dog growled ominously. At last the man turned and spoke. And what do you want? My name's Lockwood. I've lost my way on the moors. Down, Wolf, down. Strangers have no business on the moors at this time of the year. And no business here at any time. But I couldn't find the village.  I'm afraid I shall have to presume on your hospitality until morning. Do as you please. I beg your pardon. I don't keep accommodations for visitors. You'll have to sleep with one of the servants. Heathcliff, you can't. He's our guest. Then suppose you attend to his comfort. Ellen, show the gentleman to the guest room, please. The guest room, mistress? Yes, there's no other place. Yes, mistress. This way, sir. And, sir, will I unlock the door? Of course. Mind you don't stumble on the carpet. It's about falling to pieces, I'm afraid. You call this the guest chamber? It was once. It's a trifle depressing. May I light a fire?  The chimney's been clogged for years. Must have been a fine old house once. What's it called? This is Wuthering Heights, sir. And the tall chap downstairs with the piercing eyes. That'll be Master Heathcliff, sir. And the once lovely lady who looks at him with fear. That's his wife, Mistress Isabella. Well, good night, sir. If you want anything, there's the bell roll. My name is Ellen. Thank you, Ellen. Good night. Good Lord. The window, the catch broke in the window. Stand back. I'll close it. Listen. I heard a voice. A hand. A cold hand grabbed my wrist. A hand? Didn't you hear the voice? A woman shouting she's lost on the moor. What's going on in here?  Then she's come. She's come again at last. Kathy, wait for me. Wait for me. He's not going out there in that storm. I'm coming. I'm coming, Kathy. This is insane. Come back. Close the window, sir. But that man out there. Close the window. Why did he go out there? What was that voice?  It was Kathy. Kathy? Who's she? Kathy is a girl who died. I don't believe in ghosts if that's what you mean. I don't believe in phantoms sobbing in the night. You don't believe.  Tell me her story. It all began the time I came to Wuthering Heights as a servant. That was 50 years ago, in 1790. The place was different then. A sweet, well-capped estate. Mr. Ainshaw was master. A fine man who dearly loved his two children, Kathy and Hindley. One day, Mr. Ainshaw returned from Liverpool with a foundling boy at Picton. A dark, savage-looking gypsy lad with the most dreadful marks of beatings on his back. Mr. Ainshaw called for me the moment. Give him a good scrubbing, Ellen, and put some of Hindley's clothes on him. Sir, come with me, lad. Don't touch me. Let me alone. Goodness.  You're safe here. No one will ever beat you again, and you'll always have plenty to eat. Papa's home! Papa's home! Hello, father. Well, Hindley. Well, Kathy. Papa? Who's that boy? Yes. Who is he, father? Children. This little guest will pay us a visit for a time, since he wants a home here. He's dirty. Look at him. Kathy, don't make me ashamed of you. Hindley, he will sleep in your room. In my room? I won't let him.  That'll do, Hindley. You must learn to share things. Take charge of the lad, Ellen. And what's his name, then, Mr. Ainshaw? His name? I think we'll call him Heathcliff. Yes, sir. Come, Heathcliff. That dark, brooding boy, brought a subtle change to life at weathering heights. The three children seemed to play together well enough, going scamping off over the moors on their ponies and climbing the crags together. But neither Kathy nor Hindley were open and frank anymore. And one day the children fought... Oh, give me that pony. It's mine. Give me that pony, do you hear? Or I'll tell father how you boasted that you'd turn us out when he died. That's a lie. I never said it. Of course he didn't. He did. He worms his way into everything father does for us. He'll cheat us out of everything.  And you'll never get mine. There, fancy that'll teach you. Hindley, he's bleeding. Serves him right. Gypsy scum. He... he hit me with that stone. Please, Heathcliff, don't look like that.  Why should I cry? I'll pay him back someday. I don't care how long I wait. I can only pay him back. Heathcliff, don't say those things. Come, Heathcliff, now Hindley's gone and we can have our game at the castle again. I don't feel like playing at the castle. Please, you always smile and laugh when we're there. Come on, Heathcliff. We'll play our game. I'm frowned so, Heathcliff. Sit here beside me on the stone and we'll look out over the moors. This is our castle. Castle? It's Peniston Crag and you know it. It's not either.  Tell me about it, again. It's all true, crossed my heart. Your father was... was Emperor of China and your mother, a Queen of India. You were kidnapped by wicked sailors and brought to England. Don't make fun of me, Kathy. I'm not making fun of you.  And I'm your slave. You're not my slave. You're my Queen, Kathy. And you'll always be my Queen. Nobody else will make you his Queen. You hear? Yes, Heathcliff. You're Queen. And that's the way they grew up. Wild and free roaming the moors together. But Hindley grew more and more jealous. Kathy was 18 and Hindley passed 21 when Mr. Ainshaw died. Good man. He never saw the evil of life and it raised Heathcliff like his own son. But on the very day of his funeral... You're not going into that room to look at my father's body. Why? He loved me more than he did you. He's passed your weedling now. I'm Master of Wuthering Heights. If you want to stay here, we need for a stable boy. A stable boy? That's... or get out! And that's what he became. Heathcliff, who had lived under this roof as one of the family, who was a stable boy. Hand up to my saddle. Very well. Sir, you gypsy beggar. How many times must I tell you? Sir? That's better. By the time I come back in the morning, I want these stables scrubbed.  Heathcliff. Yes, Cathy? Heathcliff, I heard him. How much longer are you going to stand for this? I don't know what you mean.  Why aren't you a man? Why don't you run away? Run away? What, from you? You could come back rich and take me away. Why aren't you a prince like we said long ago? Why can't you rescue me, Heathcliff? Cathy, come away with me now. Now? Where? Oh, anywhere. You mean leave as we are? Live in haystack, steal our food? Oh, no, Heathcliff, that isn't what I want. Oh, you just want me to go off alone? Well, it won't do. I've stayed here since your father died, been beaten and cursed like a dog, abused and driven mad, just because I could be near you. And like a dog, I'll stay to the end. To the end. Yet Heathcliff did run away. The curses and insults were too much even for his great love. And he disappeared. Cathy found a new world in her first introduction at Linton Manor with Edgar Linton and his sister Isabella, both Cathy's age. Music and laughter were there, shining eyes and dancing, and it was escaped from bothering heights. Edgar Linton fell madly in love with Cathy. They were constantly together. Then, one evening, Edgar brought Cathy home in the pony cart as quickly as I could. My hand down from the cart, Miss Cathy. Thank you, Edgar. Oh, Ellen, we had the most marvelous time. Judge Linton had guests from Liverpool. Cathy, come here. Excuse me, Edgar. Certainly. Something wrong, Ellen? Heathcliff's back. Heathcliff? When did he come? Last night. He talks so strangely. He is now. Hello, Cathy. Heathcliff. You said you'd stay away until... Why were you gone so long? I didn't expect to find you here. Why were you gone so long? Because I've met the Lintons. Because I was at their house. Because I've learned to dance and had a wonderful, delightful, fascinating time. Are you the stable boy? Would you mind putting my horse up for an hour? Yes, and you might wash your hands and comb your hair, Heathcliff. So I needn't be ashamed of you before a guest. And look after Mr. Linton's horse, please. Let him look after his own horse. Hmm. Doesn't, fellow. How can your brother allow such a beast of a gypsy stable boy to act like this? Beast of a gypsy stable boy? Of course. A roadside beggar giving himself heirs of equality. What do you know about Heathcliff? Judging from this performance, all I need to know... He was my friend long before you. That black-eyed... Black-eyed and all, he belongs under our roof and you'll speak. All of him will get out.  Get out, I said, or stop calling those I love names. Those you love. That's stable boy? Yes. Cathy, do you realize what you're saying?    Some of that gypsy's evil soul has got into you, I think. Yes. Some of that beggar's dirt is on you. Yes, yes, now get out.  Cathy, Cathy, please, come back. Mrs. Cathy. Oh, Ellen, where is he? Where's Heathcliff? Tell me. He's across the moors, towards Peniston Crag. Heathcliff. Heathcliff. You've forgiven me. Oh, see, you've forgiven me. It wasn't your heart that spoke, Cathy. No, my darling, because my heart is yours. Cathy, the clouds are lowering over Jimmerton Head. Yes. Oh, see how the light is changing. Cathy, you're such a part of all this. Perhaps we belong to the moors, you and I. Oh, Heathcliff, make the world stop right here. Make everything stop and stand still and never move again. The moors never change. You and I never change. The moors and I will never change. Don't you, Cathy? I can. No matter what I say or do. This is I forever. Cathy. Oh, smell the heather, Heathcliff. Fill my arms with heather. All they can hold. Cathy, you're not thinking of that other world now. Oh, don't talk, darling, don't talk. All this might disappear. Hurry, please. My hair's not nearly done yet. What's the matter, supposing you're not ready when young Mr. Linton gets here? Any young man will come snivelling back out of the way you treated him. But I sent my apologies to him, didn't I? Of course he'll come. Cathy, I can't believe this change in you.  That's my other nature, Ellen. I still have it. It used to fly around wild, but now I can coax it into a cage whenever I want to. Heathcliff. Since when are you in the habit of entering my room? I want to talk to you. Get out, Ellen. I will not. I take orders to...  Now that we're so happily alone, may I know to what I owe this great honor? He's coming here again. Who? You know who I mean.  You're unbearable. Utterly unbearable. Why are you dressed in silk? Because gentle folk dress for dinner. And why are you trying to win his peeling flat? I'm not a child anymore. You can't talk to me that way.  I'm talking to Cathy. My Cathy. Oh. I'm your Cathy. Yes.  Cathy, where's your heart? You had your chance to be something else. You left here once. Oh, why didn't you stay away? Now, let me alone. That's right. That's right. The dirty, stable boy can't come near you unless he soil your dress. But who soils your heart? Who turns you into a cheap, vain, ambitious fool? Linton does.  Stop it! See for beggars all you were born to be, kneeling beside the road, begging for favors, not earning them, but whimpering for them with dirty hands. I see. All I am to you now is a pair of dirty hands. Well, have them then. Oh? Have them where they belong. Thank you. It doesn't help to strike you. Just stay out of my kitchen. Is...is Cathy still with him? Yes, she is. What's the matter with you? What are you staring at? I...I want to crawl to her feet, wimp her to be forgiven for loving her, for needing her more than my own life, for belonging to her more than my own soul. I want to beg for a smile. I...I don't care if she loves Linton or whom she loves. If only she'll look at me and say my name. Oh, Heathcliff, you... Ellen! She's coming out. Get out, Heathcliff. I'll wait outside the door. No, you can't. I want to be where I can see her and hear her. Heathcliff! Ellen, where are you? Oh, here you are. Oh, yes, Miss Cathy. Has...has Mr. Lynch and gone? He just left. Oh, Ellen, I've some wonderful news for you. Well, the kitchen's no place for that dress. Come inside. No, no, listen. Edgar has asked me to marry him. What did you say? Well, I'm to give him my answer tomorrow. Cathy, do you love him? Of course. Why? Now, isn't that a silly question? Because he's handsome and pleasant to be with. Not too enough. Well, and because he'll be rich someday. And I'll be the finest lady in the county. Oh, Ellen. It would be heaven to escape from here. What about Heathcliff? Heathcliff.  It would degrade me to marry him. I wish he'd never come back. What was that? I think, oh, nothing in the wind, perhaps. Well, my darling, if Master Edgar and his beautiful home mean heaven to you, you'd better enter that heaven and take your place among the Linton Angels. The only thing is, I wonder if I belong in heaven. I dreamed once I was there, and I broke my heart with weeping to come back to earth, to the bleak moors. And I awoke, sobbing with joy, on top of weathering heights. And so, you see, Ellen, I suppose I've really no more business marrying Ed Galinton than I'd have in heaven. But, oh, Ellen, what can I do? You're thinking of Heathcliff? Who else? He's sunk so low, he seems to take pleasure in being mean and brutal, and yet, yet he's more myself than I am. Whatever our souls are made of, his and mine are the same. Everything he's suffered, I've suffered too. The little happiness he's known, I've known. If everything else in the world died and only Heathcliff remained, life would still be fuller for me. Who was that? Ellen, I heard... Miss Kathy, Miss Kathy! Yes, Joseph? Heathcliff's taken Master Hindley's best horse, and he's gone. Gone? Ellen, did he hear what I said? Yes, Miss Kathy. How much did he hear? I'm not sure, but I think to where you said it would degrade you to know. No, no, Heathcliff! I'm the first act of weathering heights. Stormy conflict and adventure lie ahead for Kathy and Heathcliff in the second act of our play. But this is intermission time, and Mr. Roick is going to tell you a little story that he told me at rehearsal yesterday. Ladies and gentlemen, in this brief intermission before our stars Barbara Stanwyck, Brian Ahern and Ida Lupino return in act two of Wuthering Heights, I should like to say a word about the product which brings you tonight's play, Lux Toilets Ope. I just want to tell you what a lovely and intelligent lady whom I know here in Hollywood said to me the other day. She knows that I talk to the Lux Radio Theatre audience each Monday night, and we chatted about the program for a while. Then she said something I'd like to pass on to you. She told me that she thought I ought to tell our women listeners of her own experience with Lux Toilets Ope. You'll be interested to know that the lady in question was Joan Bennett. She said, I think you could be even more emphatic than you are about your soap. Here's what I mean. For example, some years ago, I used to buy an expensive soap. Then I found Lux Toilets Ope in the neighboring stars dressing room and tried it. I liked it better than the soap I'd been using. It seemed milder, gentler, and it agreed with my skin. Well, since then I've been one of your most enthusiastic users. And here's what seems to me so marvelous. For a few pennies, literally, I get a beautifully made, subtly perfumed complexion soap that I like better than the very expensive one. Why don't you tell women what a bargain Lux Soap is? It is interesting to realize that for a few pennies, any woman can buy the very same soap that the lovely screen stars use, 9 out of 10 of them, to help keep their complexions lovely. I'm sure I don't need to remind the women in our audience that taking care of your complexion, making the most of your looks, pays rich dividends in the admiration and love of those around you, and in bringing you romance and keeping it. No woman ever quite outgrows the longing for romance, the need for it. No woman ever grows tired of hearing the lovely things that loveliness makes people say. There's a song you may remember that says the kind of things I mean, exceptionally well. If there is someone lovelier than you Then I... If there is someone lovelier than you But no, I am not blind My eyes have traveled everywhere In hopes that I might find A creature half so fair If there is someone lovelier than you I am as beautiful Such beauty comes with... Our producer, Mr. DeMille. Act two of Wuthering Heights starring Barbara Stanwick as cafe Brianna Hearn as Heathcliff and Ida Lupino as Isabella. And that stormy winter night, a century ago, the traveler Lockwood sat in the musty guest room at Wuthering Heights, and the story told him by the old servant, Ellen. Outside, the winds swept over the moors, shaking the ancient house to its foundations. The lamp on the man-for-peace flicker, casting eerie shadows. That night, Heathcliff went away. Kathy ran far across the moors after him in the bitter cold, calling his name into the wind. Hours later in the morning, Edgar Linton found her, half-frozen in her silken party dress, unconscious on the crag the children used to call the castle. For weeks after she was ill, the Lintons took her to their manor house. And I was glad for day by day the strange influence that Heathcliff had had upon Kathy wore off. And she was happy with it. Why can't you remain here forever, Kathy?  I'm too happy by just being kind. That should be enough for me. After all, what else can I give you? What else? You've given me a great deal, Elf, Edgar. You've given me your own self, your strength. My...my strength? You'd understand if you really knew what my life was before. It was like the moors, endless and desolate. And I was lost in them, calling for someone in the darkness to save me. Nobody answered. I couldn't even see the trace of a path. I was so frightened, so terribly alone. And suddenly you were there, Edgar. You held out your hand and led me back to a way of living I thought I'd lost forever. What you said long ago is true. There was a curse on me. It kept me from being myself or at least what I wanted to be. And that kept me from living in heaven. There. Do you understand now? Kathy, my darling, let me take care of you forever. Let me guard you and love you always. Would you? Would you love me always? Always, Kathy. Then let's be married, Edgar. Quickly. Quickly! Linton home had seemed heaven to me before. I don't know what to call it after she became its mistress. The only one who did not seem completely happy was Edgar's sister, Isabella. A year or two after the marriage, the three of them sat in the drawing room, Edgar reading, Isabella playing the spinet. Kathy quietly doing petty point when the door knocker sounded. When I saw who it was, I went back into the drawing room. What was the matter, Mrs. Kathy? Heathcliff. Let's go back. Tell him I'm not at home, Ellen. Not at home, darling? To whom? It's Heathcliff. Heathcliff? Well, does he seem the same, Ellen? No, sir. I hardly recognize him. Fine clothes. He seems quite the gem. Don't prattle, Ellen. I said I didn't wish to see him. Oh, nonsense, Kathy. It's been a long time. Bygones must be bygones. Oh, yes. Let us see some kind of column. Show him in, Ellen. Yes, sir. Edgar. This is a mistake. Why, Kathy, your hands are trembling. Are they? The past is dead, dear.  Thank you, Edgar. You always understand. Mr. Heathcliff. Come in. Hello, Kathy. How are you, Heathcliff? Have you met my sister, Isabella? I'm sorry. How do you do, Miss Linton? We are glad to see a guest, sir. Thank you. Well, Heathcliff, I must say I've never seen such a complete change in a man. You seem to have prospered.  I did. We wondered where you went. You must have found at least a gold mine. No. I merely remembered that my father was an emperor of China and my mother a queen of India. I beg your pardon. So I claimed my inheritance. Kathy will understand. It's an old joke between us. I see. Are you staying long in the neighborhood?  Really? I've just bought the horses, the cattle and the moors belonging to the estate known as Wuthering Heights. No! You mean Kathy's brother Hindley sold out? Yes. But he doesn't know it yet. I imagine it'll be a shock when Hindley discovers that his gambling and drinking debts were paid up for him by his former stable boy. Heathcliff, you can't have done that. That's as underhanded a piece of work as I've ever heard of. If I'd known that his holdings were being stolen by a stranger... Can I remind you, Mr. Edgar Linton, that I am not a stranger? I'm merely a neighbor. So now I'll say good night. Wait, Heathcliff. Well? I... I want you to know that we sometimes have friends who come here as guests. Edgar and I. You're welcome to come, too. But not with the old scowl on your face or the old bitterness in your heart. Thank you, my old friend Kathy, for the warning. Or just remember I forgot to congratulate you on your marriage. I've often thought about it and I can assure you. May I now express my delight? Good night. Edgar, I think you behaved abominably. What? And you, too, Kathy. What in thunder do you mean? You could at least have been civil. You'd dismissed him as if he'd been a servant. Well, do you consider him anything else? Yes, I find he's grown fascinating and distinguished. Really, Isabella, I hope I misunderstand. Well, you don't. We see all too few people. And I, for one, shan't be rude if he ever calls again. Edgar, I greatly dread what the future will bring. Oh, nonsense, darling.  That's all, Joseph. You'll stay on, of course. Thank you, sir. Shall I pack Master Hindley's things? Oh, just move him out of the master's bedroom. He'll remain under this roof. Master Hindley, sir? Oh, yes, he gave me a roof once when I needed it. I, uh, I take it he's drinking a great deal. Uh, yes, sir. Though Dr. Kenneth has ordered him not to. Well, give him all a drink he wants. Yes, sir. Uh, a lady is waiting to see us, sir. A lady? From Linton Manor, sir. Why didn't you tell me? In the future, announce visitors at once. Oh, Miss Linton. Are you disappointed, sir? Oh, not at all. Well, I, I was passing by and my horse went in. I see. Mr. Heathcliff. I want to tell you I'm furious with my brother and with Kathy. They received you most shamefully last night. Your brother didn't send you with this apology, did he? Oh, no. No, in fact, he's forbidden me to. To speak to me, huh? Well, yes. And, uh, and Kathy also forbade you? Yes. Then in all the Moorland, you're my only friend. Well, I, I would like to be. Thank you. Miss Linton, I enjoy frankness. You didn't come here to apologize and your horse didn't go lame. Why? You came because you're lonely. Because the house you live in is too happily witted for an outsider. And because it's no joy to ride the Moors alone. Is that right? Yes. Then you needn't be lonely any more, my dear. Oh.  I don't know. You hated it? No. I see you like frankness too. Oh, come in, Kathy. Wasn't it a lovely dance tonight? Though I think you could have been more gracious to my guest. Isabella, may I speak to you for a moment? Well? You behaved disgracefully. How may I ask? It was bad enough asking Heathcliff here without consulting us or preparing us. You'd have forbidden me. But to make a spectacle of yourself, to throw yourself at him the way you did. Well, nobody else would pay attention to him. You refused to dance with him. I had to dance every dance as a result.   Really, Kathy? I'm going to open your eyes, Isabella. He's using you. Using you to be near me. To smile at me behind your back. To stare at me until I guess fear his murderous gypsy look. To try to rouse something in my heart that's dead. And I'll not let you help him any longer. So that's what you mean. It's you who are vain and foolish, Kathy. Heathcliff's in love with me. It's a lie. It's not a lie. He told me so. He's kissed me. He's... Yes, kissed me. Held me in his arms. Told me he loves me. Oh. I'm going to your brother. Yes, go to Edgar. Tell him Heathcliff asked me to marry him. And that I've said yes. You're here, yes. You can't. He's not a man. He's something horrible and dark to live with. I know why you say these things. Because you love him. How dare you say that. Yes, you do love him. You're mad with pain and jealousy. The thought of my marrying him. Because you want him to pine for you. Dream of you. Die for you. While you're safe as the lovely Mrs. Edgar Linton. You won't have him happy. You want to hurt him. Destroy him. But I want to make him happy. And I will. You hear? I will. I won't say. I'm not surprised to see you. Heathcliff is a true. It's what true. Did you ask Isabella to marry you? Oh, Heathcliff. You mustn't do this villainous thing. She's never harmed you. No. But you... Then punish me. And that's what I intend to do. I don't understand. Every moment I hold her in my arms. When I kiss her. When I promise her life and happiness. You'll be punished. You'd marry her to do that? Yes. To teach you the ways of pain.  If there's anything human left in you. Don't make me a partner to this crime.  Your heart were only stronger than your dull care for the world and its conventions. I'd live silent and content in your shadow. Begging for an occasional word or thought as I used to do. But now.  You have to keep me tormented with that cruelty. You think so pious. Oh, have I been cruel? You wish to have been known as the finest lady in the county. You wanted your luxury and your light. And at the same time, you wanted to keep me your despairing lover. Well, now that I am returned, had you given me the smile of love, I might have been content.  You can think of me as Isabella's husband. And be glad for my happiness. As I am for yours. So ends the second act of Wuthering Heights. In a moment, we hear act three. And the dramatic way in which the love of Kathy and Heathcliff seeks its own solution. But before we continue, let's listen to a man we all like here in the Lux Radio Theatre. In our brief intermission before act three of Wuthering Heights, starring Barbara Stanwyck, Brian LaHerne, and Ida Lupino, I want to remind the women in our audience that keeping as attractive as you possibly can is not only a wise thing to do because it helps you to be more successful, but also because it gives so much pleasure to those who know and love you. However, to make the most of your lux, you must give regular attention to your complexion. Isn't it true, Mr. Roy, that to really care for your complexion you must thoroughly cleanse your pores of dust, dirt, and stale cosmetics for there's danger in imperfect cleansing? Yes, that's very true. Poor choking causes cosmetic skin, the dullness, little blemishes, and enlarged pores. But with regular lux toilet soap care, you can use cosmetics all you wish because lux toilet soaps active lather. Give your skin the very necessary protection of gentle but thorough cleansing. I'm always impressed, Mr. Roy, that 9 out of 10 screen stars use lux toilet soap. They can afford to spend any price on soap, yet almost all of them use lux toilet soap. It's the highest praise a toilet soap can receive to have the world's most beautiful women so overwhelmingly prefer lux toilet soap. And clever women everywhere by the thousands use lux toilet soap too for the same reasons the screen stars do, to help keep their complexions as lovely as possible. So, I hope you'll buy at least three cakes of lux toilet soap and begin tomorrow your regular lux toilet soap care. I promise you that before the three cakes have been used, you'll thank me. We pause now for station identification. This is the Columbia Broadcasting System. Once again, Barbara Stanwick, Brian Ahern, and Ida Lupino in Wuthering Heights, in the crumbling house on Wuthering Heights a hundred years ago. The candle burns low in the dreary guest room. There's no sound but the wind outside and the hushed voice of the old maid servant as she tells her story. Isabella Linton came into this house as a bride. Yes, Heathcliff married her out of revenge. The same revenge that made him keep Hindley here.  Isabella learned the reason for her marriage to Heathcliff, but she was powerless to do anything. Then one day, Dr. Kenneth, an old friend came to see her. He had come that morning from Linton... Isabella, go back where you belong to Edgar's house. Edgar disown me, Dr. Kenneth. I know, but he needs you now. Kath is gravely ill. Really? Didn't you know? It's a matter of days now. Perhaps hours. She can't be dying. Fever, inflammation of the lungs, this intense cold and something else. Something else? I'd call it the will to die. Kathie dies. I might begin to live. Isabella. Ah, begin to live. In this house with Heathcliff, nothing can live. No, Hindley, nothing but hate. Goodbye. So you think you'll begin to live when Kathie dies? You won't? Oh, Hindley, what is it?  Of course you can. And you. He hates you even more than he does me. Stop it! He loves you. Every time you kiss him, his heart breaks with rage that it's not Kathie. Isabella, why don't you do what I've been to weak to you? Kill him. Don't talk to me. Get away! While there's still time to save your immortal soul. Well, Hindley, remarkable. Heathcliff. Hindley, the first coherent speech I've heard from you in weeks. Heathcliff, please don't. I tried to stop him. Thank you, my dear wife. Your loyalty is touchy. Your curses will come home to feed on your own heart. Every agony you've given will return. Laugh now, Heathcliff! There's no laughter in hell! Heathcliff, why do you have him here? I can't breathe with him in this house. Oh, existence would be so much less without my boyhood friend under my roof. Oh, Heathcliff.  Send him away. And love will come to this house. Kiss me, Heathcliff. Tell me you love me. Tell me, darling.  Heathcliff, let me come near you. Please.   All twisted inside. I can make you happy, my darling. Let me please. You'll never regret letting me try. I'll be your slave. I'll bring life to you. Life and sunshine and freshness. Put your arms around me. Look into my eyes.  Like Clintons. They aren't empty. If you'll only look deeper, Heathcliff. Look at me. I'm pretty. I'm a woman. Let your heart see me just once. It's no use. Why did God give me life? What is it but hunger and pain? A naked runner in a storm of spears. Mr. Isabella. Ellen. What do you want here, Ellen? I've come from Linton Manage to speak to Mr. Isabella. You'll do so in front of me. Her brother's asking that she come home for a visit. So he's lost some of his pride, has he? Well, there's none gone in this house. Please, Mr. Isabella. He needs you. He needs her. What is this, Ellen? Why does he need her? Let go of me. Kathy's ill. Yes. She's dying. Tell me the truth. Yes. She's dying. Die? Jose! You're not going, Heathcliff. Yes, Mr. Heathcliff. Settle my horse at once. No, she belongs to Edgar. She belongs to me. If she's dying, let her die in his arms where she belongs.   Now who speaks of hate? Get out of my way. Heathcliff. From the height. A faraway and wild heartbroken moan. The wings of Lucifer beat on the night. The soul of Lucifer wept all alone. Shall I read some more, darling? No, Edgar. Will you open the window? Won't it be too cold? Please. Of course, darling. Oh, now I can smell the heather.  And isn't the snow almost gone? Yes, quite gone. Edgar, will you get me something? Anything you wish, my darling. Some heather. There's a beautiful patch near the castle. Will you get it from there? What castle, Kathy? The castle on the moors, of course. Bring me some from there. You're in a fever, dear. There's no castle on the moors. There is. There is. On the little hill beyond Wuthering Heights. You mean Peniston Crag? Yes, yes. Please go. Why do you call it a castle? Because I was a queen there once. Will you bring me the heather, darling? If you rest while I'm gone and sleep. You're so kind. So good. My darling. You made me the finest lady in the county. Oh, go now, please. Get me the heather so I can have it on my pillow. Sleep, my dear Kathy. I'll be back with the flower you want. Where is she? Where's Kathy? She has not to be disturbed, sir. Master Redger's gone for the dock. Get out of my way. Kathy. Kathy. My life. How can I bear this? I dreamed you'd come before I died. And when you came in my dream. You scowled at me once more. Does it hurt so much to see me dying? Please, Kathy. Don't hurt me. Oh, how strong you look, Heathcliff. How many years do you think you'll live on? After me? Kathy.  My soul. Oh, my darling. Don't let me go. Oh, and finally I could hold you too. We were both dead. Ladies, please don't speak of death. Will you forget me and be happy? When I'm in the earth. Ever. Ever. Will you say this is the grave of Catherine and Jor? I loved her long ago and wept to lose her. But all has passed.  If you die, Kathy, if you die, tell them no peace for me ever. Poor Heathcliff.  Oh, why did you betray your heart, Kathy?  My darling, hold me. Just hold me. No, no. I'll not comfort you. You deserve this. Heathcliff, don't break my heart. I never broke your heart, Kathy. You broke it. My tears don't love you, Kathy.  You love me. What right had you to throw my love away for the poor, fancy thing you've felt for him? I found out, Heathcliff. Misery and death and all evil could never have parted us. You did that alone.  And mine. I know, Heathcliff. Oh, forgive me. Oh, Kathy. Your poor, wasted hands. Oh, why didn't someone tell me? I forgive you. I forgive you for what you've done. I love you. I love my murderer. Carry me to the window. Let me look at the moors with you once more. All my darling ones move. Yes, yes, yes. And over there is our castle. I'll never forget. The castle, Heathcliff. I'll be waiting there until you come. I'll be waiting there. Kathy? No! They're coming. Master Edgar, offer heaven's sake, go away and... You're dead, Kathy. May your soul hear this before I go. May you not rest so long as I live on. I killed you. Then hold me. Be with me always. Take any form you wish. Only don't leave me when I can't find you.  I can't live without my soul. Though that was twenty years ago, I can still hear and see that wild hour with poor Heathcliff holding the dead body of his beloved, crying out to Kathy's soul to haunt and torment him till he died. Then the strange hand that gripped my wrist last night, the strange voice that called whom he was Kathy, and Heathcliff went to follow her into the storm. It wasn't Kathy herself, but her love. Kathy's love, stronger than time, sobbing for its unlived days. Who's there? Dr. Kenned, open the door. Dr. Kenned, what's the matter? I was just crossing the moor on my way home. Did you see Heathcliff? Yes, I saw him from my carriage. He was wandering through the snow with a woman. A woman, you say? A young woman, she seemed, as mad as himself. They were walking through the storm with their arms about each other. I shouted at them, but they didn't hear. I was nearly up to them when, of a sudden, my horse reared and plunged. And then, then I found him alone, lying in the snow. Heathcliff was dead. Dead? Was it by the castle? By the tennis-tuned crack? Yes. However did you know?  Into the dimness of a hundred years ago. We're back in the present with Barbara Stanwyck, Brianna Hearn, and Ida Lupino at our microphone. But it seems to me that they all are just a little bit stirred by the drama they just brought us. It's a moving play, Phoebe. But more than that, I think it has a lesson today for every woman. It shows the necessity of holding on to one's ideals. But Kathy gave up what she really wanted for position and security. That's a common failing among women, isn't it, Mr. DeMille? On the contrary, Ida. I think women are less likely than men to sidetrack their ideals. Whatever kept his faults. She was one of the most real characters I've ever come across. Yes. If you know the story of Wuthering Heights, I imagine it would be pretty easy to hear the voices of Kathy and Heathcliff out on those moorlands when the winds begin to hover. What about it, Brian? You've been there? Well, that wind is one of the weirdest sounds I've ever heard. It's no place to be at night. No place for my Irish imagination. No for anybody's without a good, big flashlight. You ask me, the safest way to cross those moors is in an airplane. Yes. What have you been doing with your airplane, Brian? Oh, very useful, C.B., very useful. Last time I used it, I went up to Northern California and got myself a marriage license. Yeah, that's right. You've taken on a co-pilot. Congratulations and happy landings. Thanks. Perhaps my co-pilot and I can take you for a ride sometime. Or how about you taking us?  But before we fly away from here, I want to tell you how much I've enjoyed working with you again in the Luxe Radio Theater, and that I'm still absolutely loyal to Luxe soap, too. It was a number of years ago that I started using it, and I've been a faithful Luxe fan ever since. It does a swell job for my skin. Well, I agree with Barbara. Luxe soap is a wonderful beauty care, and I know we're all looking forward to the Luxe Radio Theater next week, Mr. DeMille. I see by the rehearsal schedule backstage that you're going to have Ginger Rogers, George Brent, and Edith Fellows next Monday night, C.B. If you'd read a little further, Barbara, you'd have seen the name of the play, too. She married her boss. A story about a girl who thought the boss's home should be run as efficiently as his office. Ginger Rogers, George Brent, and Edith Fellows, and she married her boss. Well, I'll be waiting for that. And good night. Good night. Good night. Good night. I wish I had you three in a picture. Our sponsors to make it the Luxe Toilet soap joined me in inviting you to be with us again next Monday night when the Luxe Radio Theater stars Ginger Rogers and George Brent with Edith Fellows and she married her boss. This is Cecil B. DeMille saying good night to you from Hollywood. This is Fowler W. Asking you to be sure to listen to the Luxe Daytime Radio Program, The Life and Love of Dr. Susan. This human and gripping story of a young attractive woman doctor is brought to you every afternoon, Monday through Friday. For the time in station, see your newspaper. The Life and Love of Dr. Susan comes to you in addition to the Luxe Radio Theater. Also heard in tonight's play where Thomas B. Brent Smith as Edgar Linton, Kemble Cooper as Hindley, Marga Ann Dyson as Ellen. Barbara Stanmick's current film is the Columbia Picture Golden Boy. Brianna Hearn will be seen next on the screen in the RKO production, Vigil in the Night. Ida Lupino's latest picture is Paramount Studios' The Light That Failed. Lewis Silvers is from 20th Century Fox where he directed music for The Reigns Game. Here is important news for those of you who live in communities not operating on Daylight Saving Time. If you live in a community which does not operate on Daylight Saving Time, watch this program beginning next Monday night one hour later than here to four. And now, ladies and gentlemen, may I take this opportunity to announce that there's a very great treat-and-store for you. Tomorrow evening over the Columbia Network, a full hour of top-notch entertainment. First, Edward G. Robinson, dynamic star of stage, screen and radio, returns to the air, starring again in a new series of the dramatic expose Big Town with lovely owner Munson. Immediately thereafter, the Tuesday Night Party, starring Walter O'Keefe, with special guest star Sophie Tucker and Bobby Dolan's Orchestra. A half hour of thrills, excitement and dramatic action, followed by a half hour of laughs, song and music. See your local paper for the exact time and station. Melville Royce speaking, this is the Columbia Broadcasting System.


President Emanuel Macron has warned that France would withdraw troops from Mali if political instability there leads to greater Islamist radicalization. It follows a second coup in Nainmont in the West African nation. Macron warned of the risk of Mali moving toward greater Islamist influence. Now France has 1,000 or 5,100 troops in the Sahel region which has been a front line in the war against Islamist militancy. France troops have been supporting forces in Mali, Mauritania, Niger, Burkina Faso and Chateau-Barton militants in the Sahel region since 2013.


Thank you very much for inviting me to give the talk. Right, so, the gallery at the bottom really shows an evolution in our structural biology efforts. And the title is really referring to what we had to do to be able to get these initial crystal structures. And at the end of the talk, I will take you all the way through to our latest structures where actually there's not much whipping involved. So we'll see how this evolves. So I thought I'd start off with a little bit of an introduction to GPCRs, because okay, you understand that they're really important and for drug discovery, 34% for FDA-approved drugs are target GPCRs. But I think the important thing to recognize is that how dynamic they are. And this has been really instrumental in trying to understand how you can get structures. If you understand your protein, the dynamics that undergoes, then you're in a far better position to be able to engineer your protein and to get structures. Oh yeah, let's get my interruption. So in the inactive state, which is here, there's no ligand bound. Obviously the GPCR just sits in the membrane quite happily. Binding and vaginas though induces a conformational change which allows G protein coupling. And the alpha subunit then exchanges the bound GDP for GDP, you get dissociation. This is the active form goes off and does all its intercellular signaling. Beta Gamma can also do signaling. And then this signaling is turned off through phosphorylation through receptor kinases. Normally phosphorylation of C-terminus or psychopathic loop three. This then allows coupling of arresting and shutting off of G protein signaling, but arresting can signal in its own right. And I'm not gonna go too much into the pharmacology here, but what's really interesting from the drug discovery perspective is that we know that some of the therapeutic effects in many GPCRs may be driven, for example, through the G protein coupling, but adverse effects may be driven through arresting coupling. So there's a lot of interest in comparing different structures with arresting bound versus G protein and whether we can develop really tailor made drugs which only signal through a specific pathway. So there's still a lot of interest in GPCR structures. And obviously we want to get as many as possible to be able to understand this pharmacology. So I'm gonna be talking about ligands, ligand efficacy. So in terms of biological response, a full agonist is maybe 100% biological response. So think adrenaline or adrenaline, but then a lot of drugs may give a partial response. So even though they've got full occupancy, they may only turn the receptor on partially. And then antagonists can be divided into a number of subgroups depending on their activity, weak partial agonist, neutral antagonist. So inverse agonist. And it's only the inverse agonist that really inhibits basal activity and inhibits the receptor fully. Now, the thing to bear in mind is that the receptors undergo a number of conformational changes. And so the inactive state R can transition spontaneously into R star. So this isn't even in the absence of ligand, the small proportion can conform this active state. And then these states are stabilized by the binding of an agonist. But you can have agonists bound to an inactive state, it's low affinity. You have a high affinity state here. And of course, when a G protein binds, you get even higher affinity. So the key part here is that this equilibrium is going on the whole time, even in detergent.  And this has been part and parcel of learning how to deal with GPCRs. So let's just, sorry, I can't stop it, hold on. So this is basically a movie of a GPCR going from the inactive state to the active state. And what we have is just three crystal structures of the A2A receptor going from the inactive, active, intermediate to active. And what you see is this cleft is open that allows the coupling of the C-terminal alpha helix of the G protein actually in here. And mainly through hydrophobic interactions. And so we understand a lot about how the GPCRs are activated now from all the structures that we've done. But that gives you an idea, it's a fairly small change. It's not a dramatic change that we actually see compared with more transporters. Okay, so how have we gone about getting structures? This is basically a rather steep curve that has been happening over the last few years in terms of numbers of unique GPCR structures. So we're up to about 70 of GPCRs out of 350 or so, ligand binding GPCRs in the human genome. And what you see is that virtually all of them have been done by HVAC crystallography. And it's only in the last few years we've been getting unique structures bound by cryoEM. The first structure was done of rhodopsin. And this was a structure that was a purified protein from native sources, so bovine eyes. And the engineering required for all of these subsequent structures, virtually all of the structures have ligands bound as well. So what have been considered over the years? So if we think that most of them have been done by crystallography, I'll mention probably about crystallography initially, it still has a lot of advantages and go on to cryoEM at the end. So what we had to do was think about removing flexible regions, obviously post-translational modifications, as Aaron mentioned, these fusions to the soluble proteins to improve crystal contacts and also using binding partners. And then the two key parts here, reducing conformational heterogeneity and increasing thermostability. You see actually you're using the same techniques to improve both of these. However, you have to be very careful because you can add a ligand, which may increase thermostability, but may not reduce conformational heterogeneity enough to be able to get crystals. So these things have to be considered independently and you've got to find the right combination to allow both of these two to occur. So I just want to say a few things about expression before we go on.  And I just want to remind you, this is about GBCRs. So hopefully I won't upset too many people by what I say again, but of course, as I mentioned yesterday, you know, you've got a huge variety of different expression systems that you can use. And basically you can get anything to express in any expression system, but it's the amount of time and effort required to be able to do that, and also the quality of material that you actually get at the end of it. So if you think of the complexity of the expression system in terms of expressing the mammalian GBCR, so I'm thinking in terms of how well does the system cope with folding, eukaryotic membrane proteins, post-translation modifications, et cetera, then clearly the best systems are on the mammalian cells. If you then look in the literature and see what has been happening, and look at the ratio of the functional to non-functional receptor. So to me, this is really important. If I'm going to start a new project, I really want to make sure that the protein I've expressed has the optimal possibility of being folded correctly with not too much misfolded material. I'll just mention that in a subsequent slide. And in our experience, it's really the mammalian cell systems which have been the best. But even transient transfection in mammalian systems can cause a lot of problems. And then the, I forgot to write this. Sorry, let me get that out of the way. I can't see one of the slides. Then you've got the potential functional expression levels of the receptor. And what is surprising is that actually a lot of the mammalian systems can express them very, very well. And same with baculope. So it's a trade-off very often between these two, these two things that we think about. That's interesting. It seems that my slides have stopped moving forwards. Maybe you can re-share once again. Oh, there we go. That's it. So the other thing to remember is that expression is receptor dependent, but also user dependent. And I speak from my own personal experience of when I was sort of around about 2000 to 2003 developing and setting up mammalian expression systems for use in the serotonin transporter. It took me a while to actually get mammalian cells growing effectively for the different systems and to get good expression. And the key thing for all of these particularly for baculoma mammalian, if your cells aren't growing healthily, your expression will be bad. So I learned that the hard way. So I just wanna say a couple of things about the transient expression systems. An inducible expression. So this is an inducible expression in the T-Rex H2O3 cells. So these are tetracycline-induce balls. We used this in 2002, 2003 for expression of the serotonin transporter. We've always found this to give very, very nice expression. And in baculoma, what you find is, you often find is a lot of stuff is intracellular and not on the cell surface. Actually cell surface expression is always a good surrogate for deciding whether your protein is folded correctly or not. What we came up with with this, so this is actually expression of a GPCR and a Tinsim receptor. And it was basically a quick way to be able to ask the question, have you got misfolded material present? And this is basically a differential Western blob. So basically you just take some cells, same number of cells, solubilize them in different detergents.  So there's no activity there. And then we've got these detergents, very mild detergents, which will maintain your protein probably in an active state. And we should get full extraction of functional material. So what you see, if you look at SF9 cells and you just see what you extract from the membrane, you get a huge amount of material extracted with SDS and FC12. And apparently on the initial, there's virtually nothing here. And if you expose it for longer, then what you see, yes, there is some actual protein extracted. But when you do the binding assays using a specific ligand for the receptor, then what you see, yeah, SDS as you'd expect, nothing there, FC12 a little bit, but then you're getting extraction of large amounts in DDM and Digitome. And just to say, so this is your 100% level that you'd get from membranes. And you're wondering why do I get double? Well, you get double because this ligand won't cross a membrane. So when you make and freeze membranes, you get inside out and right side out vesicles. When you solubilize, of course, the side of the scopes. So you only measure half actually here in the membrane. Digitonin is not quite so good at extracting material as DDM, but you still got a lot of functional material.  This is the band of interest, these are contaminant bands. So virtually the same amount of material is extracted with both systems. And again, no functional material here in SDS, FC12, nice functional material in DDM and Digitone.  And this is really important. You wanna be purifying good stuff for structural studies. So we found this actually very, very useful in our work. And this is something is also worth remembering. At the end of the day, membrane protein folding is complex and poorly understood. So I'm always a great advocate of expressing it in a system where folding is going to be most likely to occur in the most optimal way. And I'll be happy to answer questions more about the, of why I think the mammalian systems are better than say bacula virus or membrane proteins. And certainly from our perspective, we found it's more simpler just using bacula and mammalian systems as much faster. Okay, so where did we start on this story? Of course, we didn't start on the river punting, although that's always quite nice. We started with the beta receptor. This is where we started in Gephardt-Schertz's lab. And when he started in Cambridge, what was about 1990. And this was expressed in the bacula viruses. But in terms of X-ray crystallography, now with hindsight, you just look at it and you think, well, this is not going to be a good one. You've got potentially flexible regions, N-terminus and the C-terminus, perhaps other loops as well. And you can see from a disorder prediction that these regions are actually quite distorted. We've got multiple phosphorylation sites and end glycosylation. So lots of potential problems. This is Tony and he actually spent seven years trying to get this into shape. And what he did was for the beta one receptor constructs, he made up to 36 constructs, deleted the N-terminus, C-terminus, ICL-3, he could purify a fair amount from insect cells, from each of insect cells. I think you'll agree that he can purify it quite nicely in a truncated form, nicely non-dispersed and non-gel, it's nice and clean, fantastic. However, what is clear from this slide, no crystals. So why? And what is apparent now is that the receptor is basically too unstable in short-chain detergents. So I'll come on to why that's a problem in a minute, but if you just put this, take it from DDM and do the same prep in some, like, decommaltes on it, then it'll just crash out overnight in the fridge at four degrees, so it's that unstable. So this was a real problem. So subsequently, we've done some work in looking at the, of why these GPCRs run stable in short-chain detergents. And so these are ND simulations, which have been done either with dodecal multicide or with octal glucoside.  If you compare that now to the OG, you know, the OG is on speed. I mean, it is just going around all over the place. And if you look at some of these individual molecules of OG, they are really rotating in the plane of the hydrophobic, where the membrane would be, they're jiggling about. And now at the end of the simulations, they're starting to intercalate between the helices. So this is like the first steps of denaturation. So it's because you've got these enhanced movements of the receptor in short-chain detergents is probably why they're so denaturing. Okay. So how did we get round this? So we got a team of postal. So this is Tony. We've got Yoko, Maria and Francesca. And they worked on developing this conformational thermostabilization. So the idea was to make the receptors more stable in short-chain detergents. And this was a very straightforward, simple process. So we did alanine scanning throughout the receptor. So basically you change every amino acid into alanine. If it's already an alanine, you change it into a lucid. You then express each of those receptors so you can either express them in manian cells or actually originally we decided to use E. coli. We thought it might be easier, but it actually isn't. It's just as fast doing it in the manian cells. And then after expression, you then do the assay. So this is the heart of the whole process, the thermostability assay. And so what you're doing for each of your mutants is then saying, asking how thermostable is it in relation to your wild-type receptor? And so when we say functional receptor, this can be measured in a number of different ways. So initially we did radioligin binding.  And you heat the sample to different temperatures and quench and then just measure how much is bound, draw a curve and that to your parent. So very, very simple, albeit somewhat tedious. So a heptare is now they have a whole suite of different methods for doing this. So like aggregation assays, fluorescent assays because in many cases we don't have a radiolabeled ligand or the properties of the ligand are good for these assays. But what you end up with is basically round a banal, I don't know, 6 to 9% of the single amino acid point mutations being thermostabilizer. And then what you have to do is to be able to put them together. We've worked out a number of ways of efficiently being able to put them together. And then you end up, in our case, found about four to six mutations being put together and gradually increases thermostability. So all of this, we've summarized all of our experience in this publication here if people are interested in the details. So this is the first example that we did, the beta one receptor. This is when we got into the stage of having single point mutations and we're putting them together in a whole number of different combinations. So these are just six mutations put together in different ways. And then what you see, when you look at the TM, so this is the wild type, all of these are all much more thermostable but there's one which is absolutely amazing compared to that, so it's a 21 degree improvement in thermostability. And what does this mean? Well, the original wild type receptor, which is shown here, we'd only measure thermostability curves in dodecoma altoside and in decoma altoside. Whereas the thermostable receptor in 23, not only could you measure it in DDM and DM, which these curves, so you can see that they've shifted significantly to the right, but now you can actually measure thermostability in octal glucoside and non-al glucoside. And of course, these are short-chain detergents and ideal for crystallography. But of course you have to think about what you've done to the receptor. And what we found was very interesting. So what we've done is biased the confirmation of the receptor towards the inactive state. And so these are just pharmacological, pharmacological gases binding by agonist or antagonist. So antagonist binding is unchanged. Agonist binding is shifted significantly to the right. So it's a key thing. Your structure tells you something about the antagonist bound state, but it won't about the agonist bound state. So yeah, be wary of that. So in the end, it crystallizes in octal-fired glucoside. And so in summary, these are all the things that we had to do to be able to get these crystals. So we have the deletions in orange boxes. We've got the thermostabilizing mutations in red. We've got another mutation, PSS116L to improve expression. We've deleted the palmatulation site at 358. And yeah, so lots of engineering actually required to be able to get that. But what's been nice about this is that once that engineering has been done and we understand it, what we've been able to do is to be able to get a whole multitude of different structures with ligands of different efficacy. So this allows us to understand why it is that a full agonist is a full agonist and why a partial agonist is a partial agonist. We can understand why it is that when you go from the inactive state to a state coupled to a G protein, why you see this increase in affinity of the agonist and also why that increase in affinity is weaker when you actually have a resting bound as opposed to a G protein. So the structures have been incredibly informative. So, as Adrian has mentioned, you can also do these gene fusions, T4 lysozyme. It's another way of doing it. Actually, this methodology was developed by Gil Privey and for trying to get crystals of lack permeate, which never worked, but it worked unbelievably well when put in GPCRs. But the position of these fusion point is absolutely critical. You can change these by one amino acid and it can be the difference between getting a crystal and not getting a crystal. So typically people may be screening a hundred or so different constructs to get the best crystals here. And all the crystals have to be grown lipidicubicite. You don't get good crystals in detergents. You also need high affinity ligands to be able to stabilize the center. And now what people are doing is basically combining the methodology here, the T4 lysozyme and the thermostabilizing mutants and having good ligands. And all of that has come together in a very robust system for structured termination of GPCRs by X-ray crystallography. And these are just some examples. So this is the same receptor. It's been crystallized in a variety of different ways, either with a fat fragment bound, either with one of these gene fusions. This is T4 lysozyme or by thermostabilized. But here you need very high affinity ligands. The advantage of thermostabilization is that you can now actually crystallize the presence of very small ligands with very low affinity. Okay, so I'm running out of time. There we go. So we can do exactly the same thing for looking at GPCR G-protein complexes. So in 2011, I'm sure you realized the, we have this amazing structure came from Brian Kabilka's lab in collaboration with Roger Tsunahara. And yeah, it won a Nobel Prize. No question, it was a real milestone in the field at the time. But nobody has been able to repeat this for any other receptor. And obviously people wanted to get active state structures. And what we noticed was that when you looked at this structure, virtually all of the interactions between the G-protein and the receptor are driven by GES. So I'm sorry, by the alpha subunit, not by the beta and gamma subunits. And virtually all of that is driven actually by the GTPase domain, right? And not the alpha helical domain. So we thought, well, we can simplify this. And so we started engineering. So we don't need T4 lysis, and that was required for contacts. We don't need beta gamma or MB35. We don't need the alpha helical domain. And that's what you end up with. This is the minimal unit of the G-protein that you actually need. We call this mini-GS. And you think, well, let's make this. So to simplify the crystallography. Easy to say, difficult to do. So first of all, we devised an assay. And this was basically an assay which allowed us to measure how good a mini-G-protein is. We know that an agonist shift happens when you couple to a G-protein. So this is the inactive state. So anything that we engineer has to do exactly the same. And this is what Byron did, Byron Carpenter. And I'm not going to go through all the gory details because I don't have time. Suffice to say, at the beginning, this is the best he could purify it. Because if you purified it anymore, the band disappeared because it's so unstable. You could only do the assays at four degrees. You could see an agonist shift, but at 20 degrees you couldn't. So this was very, very unstable. But you could do engineering. And you could do engineering based on the structure of a small GDP age. You could compare it and engineer the protein. So about 300 or so mutants later, you ended up with this mini-G-protein, 28 kilodolton protein. It's got eight mutations, three deletions. It recapitulates all of the pharmacology that you would want for the receptor. So you see an increase in affinity when you go from the inactive state to when you actually bind the mini-G-protein. And even better, you actually increase the thermostability of the protein compared with when you do the G-protein itself. It's absolutely ideal for crystallography. And we ended up being able to solve a structure here. So, you know, G-protein engineering worked just as well with the soluble proteins as with membrane proteins, and they can really help you be able to get structures of membrane proteins in different states. So in the last few minutes, I just wanted to say a little bit about the, about cryo-erea. Because, you know, what we've worked on initially of the simplest proteins, this is the way biology works. You work on a simple system, but there are some real beasts out here. So this is a sort of proteins we're sort of interested in now. The ligand itself is 28 kilodolts. The ligand is, it's got 11 disulfide bonds, 3-ended-guide-cosylation sites. This is just the ligand. When you look at this, you've got a large extracellular domain. This has got 6-ended-guide-cosylation sites, 5-disulfide bonds. There's a cleavage site in here. If you're a mediate cleavage, which requires for activity to remove the C-peptide, this is a nightmare. So how would you actually do this by extra crystallography? I don't know. You need a lot of work. I'm sure you could do it, but it'd be a lot of work. But the key thing with cryo-EM is that, as I'm sure you're aware, you can use any detergent. So that means instead of worrying about trying to crystallize things in O-G-N-G, or using lapidic cubic phase and optimizing the packing in there, you can just go straight for very mild detergents, very, very large. So you've heard all about FSEC before yesterday. So I'm not going to dwell on this. Suffice to say, it is a real, really important tool in our lab. It allows you to assess the receptor quality, the receptor quantity. It allows you to assay a whole variety of different detergents and stability, and you can get, in an afternoon, a huge amount of information, which really helps you be able to take your structural biology project forward. So for example, in this case, this is a typical of what I mentioned about the problems with transient transfection. You transient transfect, you get nice green cells, what would you do? But you see you've got a lot of misfolded material here, and the peak, this peak doesn't look too good either. You use lentiviral transduction, you can make stable cell line here, and now you look at the peak there. That's what I want to start with, is to look at the purification and not this, much more dispersed, much less aggregate there as well. And then you can go through and test a number of things. So for example, we know from high-resolution structures that we've done, sodium is now a steric antagonist. You can look at different effects of different concentrations of sodium, and you can pick the best concentration for purification. You can look at the best used FSEC also for looking at many G proteins. Okay, we've done structures, they all look amazing, and the resolution is getting better and better in terms of just the sheer number of particles you can now actually get. I think this is worth thinking about, and really the importance and use of cryoEM, because when you think about it, you've spent so much time engineering the G protein required for X-ray of removing flexible regions, post-translation modifications, optimizing crystal contacts, thermostability, all of these things we don't have to worry about now with cryoEM. Protein dynamics, yes. Here we want to lock it down. CryoEM maybe, maybe not. In some cases we've been able to separate out different conformational states, but you don't want too many of them and you don't want to continue them. So this is something you may need to think about, but of course the real advantage, you can use virtually any detergent. Size may be a little bit of an issue. I think at the moment, we're around about 70 to 80 kilodolton in size, which of course for 35 kilodolton, GBCR is a bit challenging. And yeah, there's no worries about shape or the complex dissociation perhaps isn't come up in the discussion again, but yes, this is a real problem. So this is just one example of a one problem we have, preferred orientation. This is a nightmare and you can spend a lot of time. This is equivalent to your crystal problem in X-ray crystallography. Your crystal is not diffracting properly. You've got a lovely sample. You only get one view and that's it. You can't get a structure from that. And there's a whole variety of things you can try. And in one example that I'm going to show you, you've got to collect tilted data sets, which is a bit of a nightmare, but it was absolutely essential to be able to get this particular structure. And this really shows the power of cryo-EM. Could you ever get a structure like this with a nanodisc, liquid nanodisc, which is essential for arrest in binding? You're not going to be able to do that by X-ray crystallography. I don't have time to go through all of the purification stuff, but I just want to finish off with, you know, the revolution which is occurring in cryo-EM and make no mistake, it is a revolution. It's going to get faster and faster. There are loads of more improvements which are coming online and small membrane proteins, people are now working on these 50 kilo-dolton membrane proteins or smaller, just putting on a fab or a nanobody, you can increase the size and do the structures. This really shows the way forward if you like the GPCRs. So this was done by a PhD student. He started without having ever worked on a membrane protein and within 14 months he had gone from absolutely nothing. We'd never worked on this protein before to the density for this structure. And what is really nice is that this is a wild-type receptor. So there's absolutely no deletions, no mutations, and all the post-transitional modifications are present. We've got 125 of amino acids hanging off here, which is unstructured. And actually, if we tried to engineer it, we probably would have got everything wrong because this was thought to be a really unstructured and terminus and actually is absolutely essential in a domain-swapped method of forming the dimer interface between this GPCR dimer. So there you go. It's my favorite cartoon. You have to think, you have to work hard and be meticulous and never ever give up. And well, as the frog found out, there's always a way forward in the end. So thank you very much for listening. I'm sorry I ran over a little bit. And I'd like to thank everybody who was involved in the work and of course all the funding over the years. Thank you very much and I'll be happy to take a few questions. Thank you, Chris, for this interesting talk and showing us so many strategies to purify and mix table GPCRs. So we can take a few questions. And the first question is like, what do you think about like, what is the reason for better yield of functional protein with stable expression rather than with the transients expression in mammalian cells? Yes, a good question. So with transient transfection, you cannot regulate how much plasmid goes into each individual cell. And there's probably a range of over 10 to the 5 difference between 0 to 1 molecules and 100,000 plus molecules of plasma. And what that means is that you can have a vast overproduction of messenger RNA. And that just totally overwhelms the secretory pathway in the folding systems for the membrane protein. And so all that happens is that if that happens, you end up with misfolded protein. Whereas if you do a lentiviral transduction, you can determine you have one, two, three on average. Lentivirus is going into the cell. So that means that you're not overwhelming the cell with mRNA. You get proper folk. So we have another question. At what stage in purification can you test the receptors for thermal stability? Oh, yes. That's a good question. So actually we test them for thermal stability all the way through. So you can actually just take cells, solubilize, because they've got GFP on, an F-set, you don't need to purify them. So you can do a thermal stability asset. Oh, sorry. Yeah. Sorry, I was thinking about something else, not F-set. So you can use F-set, heat F-set, which David mentioned yesterday, but you can do it immediately on solubilization. You can actually do it in membranes if you want. And you can do it after purification. And very often what you see is that your most stable protein, maybe in the membrane, when you solubilize it becomes less stable.  So yeah, you can do it whenever you like. Okay. Then one last question. What is the physiological relevance of thermo-stability? I think it's quite similar. The physiological relevance of thermostability. Well, obviously the mutations we put in have no physiological relevance whatsoever. They're just basically a tool to be able to get the structure. But the corollary is, is that some receptors are more thermostable than others. So for example, we worked on the Turkey beta-1 receptor initially because that was far more thermostable than the human beta-1 receptor. Why that is, I have no idea. And I don't think there's any particular correlation between an organism and thermostability. So it's just what evolution has come up with. You know, people have looked in thermophilic bacteria for, for transporters that are much more thermostable. Some are, some aren't. So yeah, I'm afraid there isn't anything. Thank you.


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