Add to Watchlist

Lecture 12. Protein Functions.


Citation of segment
Embed Code
Purchasing a DVD Cite video

Formal Metadata

Title Lecture 12. Protein Functions.
Alternative Title Lec 12. Introduction to Chemical Biology -- Protein Functions
Title of Series Chemistry 128: Introduction to Chemical Biology
Part Number 12
Number of Parts 18
Author Weiss, Gregory Alan
License CC Attribution - ShareAlike 3.0 Unported:
You are free to use, adapt and copy, distribute and transmit the work or content in adapted or unchanged form for any legal and non-commercial purpose as long as the work is attributed to the author in the manner specified by the author or licensor and the work or content is shared also in adapted form only under the conditions of this license.
DOI 10.5446/18871
Publisher University of California Irvine (UCI)
Release Date 2013
Language English

Content Metadata

Subject Area Chemistry
Abstract UCI Chem 128 Introduction to Chemical Biology (Winter 2013) Instructor: Gregory Weiss, Ph.D. Description: Introduction to the basic principles of chemical biology: structures and reactivity; chemical mechanisms of enzyme catalysis; chemistry of signaling, biosynthesis, and metabolic pathways. Index of Topics: 0:15:32 Protein Conformation 0:18:28 All B-Sheet Proteins 0:27:13 WD Proteins Scaffold Together Large Assemblies 0:28:12 Collagen is Formed from a 3-Stranded Coil 0:29:21 All a-Helical Proteins 0:31:40 a/B Proteins 0:33:21 Peptide Binding Domains 0:39:05 Higher Order Assemblies of Proteins 0:41:23 Equilibrium Constants to Describe the Strengths of Non-Covalent Interactions 0:46:54 Following the SPeeds of Reactions 0:49:25 Rates of Non-Covalent Interactions 0:50:16 Typical Rates of Binding 0:53:09 Measuring Biological Potency Through Dose Responsive Curve 0:56:26 Measuring Biological Reponse by ELISA 0:59:36 Steptavidin-Biotin Offers Near Covalent Binding Affinity 1:00:39 Biotinylated Reagents Used Extensively in Chemical Biology 1:03:47 Enxymatic Catalysts = Catalytic Receptors
welcome back this week we get to talk about protein struck function which is the follow-on to last week's discussion of protein structure and I would say last week was all about pretty molecules we saw beautiful structures architectures that were fascinating but I did tell you what makes them so special I didn't tell you why it is that those structures allow proteins to do unique things and proteins really are the super heroes of biology these are the molecules that make it possible to change around hold solutions and the transformations possible that otherwise would be genetically and thermodynamic lead accessible to the cell so they're doing reactions the catalyzing reactions that otherwise without these biology would not be capable of taking place OK
so specifically this week we're going to be talking about us of pharmacology were going to be looking at dose-dependent response were going to look at non-covalent binding and then by analogy to non-covalent binding were going to make the leap to catalytic finding evidence to try to understand how and science work I want to talk about how we're going to measure enzyme activity will talk about how the regulated will talk about the mechanisms and we'll talk about the genesis of engineering so these topics in here we are going to give you the foundation that you need to make predictions about how enzymes work and the overarching goal is for you at the end of this series at the end of this to lectures for you to be able to look at some reaction at the end he not design the perfect enzyme that makes some predictions about how that enzyme might work we give you an example of that and this is the enzyme that we will be talking about but it's 1 that I want you to think about what this is actually "quotation mark from the Iliad and then the quote is as the juice of the thing tree curdles milk and they considered it a moment through the Though it is liquid even so instantly to pay on cure Mars this fascinates me so in this reaction you can take the form branch off a fig tree break it open and actually this kind of milky liquid flows out and you could walk that is a big bucket of milk and will happen is exactly what's described here in Eleele now Our goal is to understand how an enzyme like this might work if I tell you that the milk is getting on and is is getting sold solidified by certain reactions that you might be able to predict what the enzyme is the enzyme mechanism is that makes that reaction possible presidents herbal will be mistreated set things up when we talk about some announcements 1st promulgated to the the meeting of the discussion so this week please read Chapter 7 work .period problems as usual ,comma term is going to be a week from Tuesday it will cover through chapter the is going to cover through Chapter 6 not 7 by their and it will it will
be comprehensive in the sense that there might be some concepts from the 1st 3 chapters but it will focus largely on the more recent material OK so when you're studying what I'd like you to focus on ideas and problems that are in the assigned homework such as these are problems every chapter I'd like you to focus on 1 of the problems that are discussed discussion and then also posed a simple mid-term which will give you an idea of the types of problems I'm expecting you know kind so hopefully Yuri starting to study for this and the that will be coming up pretty soon also coming up abstracts for the proposal the final proposal reports are due this Thursday at 11 AM and we've already talked a little bit about the format of abstract but I've also released on the website more details about the proposal assignment and I'd like to take a moment just to review those with you now so I'm very briefly
let's take a quick look at hand on the website here's the website of course here the proposal Simon and I have a very detailed description of the chemical biology proposal that you're going to be right In brief what it tells you is that you need a simple idea peso in this 1st paragraph it says you know don't come up with something that's next Manhattan Project don't tell you know Tell me if I get a billion dollars I could ask you know you do something like you know solved told off on fungus or something OK tell me something that I can do for say 100 thousand dollars or or even less 10 thousand dollars let's say those are the kind of proposals that will attract attention clever ideas things that people have a lot of that that shows brilliant such creativity at the start of the big ideas to the sequencing of thousand dunams and there are people who are doing that and they're on their creative in their own rights but that's not necessarily what this class is not so simple creative ideas that interests me OK and let me talk to you about us of ground rules you must choose a topic that will improve human health and well-being broadly if there's lots of ways to do this or that they could be on things like improving energy situation on this planet if you have a new way of generating and energy using enzymes I would love to see it and and that would improve human well-being broadly so the focus so of your proposal must be squarely qualified as chemical biology if your proposal does not fit the topic of chemical biology I will know an abstract not given back to you and tell you to changes that largely states very important that it fits the definition a chemical biology your proposal must have a hypothesis for very good reason not to be driven by testing a specific hypothesis and then after that you need to think about backup plans created variations and further inside so good proposals are a little bit like an onion at the heart you have some clever ideas you have something that if you could do this clever idea it's going to change the but equally importantly you have a lot of little backup plans and contingency ideas so if the mean idea doesn't work out you have a bunch of backup ideas that are waiting in the wings they're going to rescue the whole thing and I turned around and make phase OK so that's ultimate goal of a good proposals and arms along those lines but you really should you really should be having more than 1 idea peso proposal as 1 great idea and then there's a bunch of other little ideas kind of supporting it but do not propose experiments that require human subjects were samples obtained from humans this is important I know many of you want to go to I don't know dermatology and the Argentina you know medical school become dermatologists later and I've got proposals about taking steps and things like that that is still not interested and that is not what this class is about you know that's not this about so I will not accept any proposals that require you to collect samples from humans your proposal must include control experiments we've discussed these positive and negative controls we've discussed that and and then the next part is coming up with ideas how you the 1st thing you need to do is come up with a clever idea here is 1 very simple formulaic way to be brilliantly creative for the rest your career if you learn this formula you can be incredibly creative and I'll be honest has always worked for me and all I do is taking a new a new technique and then I simply apply this new technique to problem that's already exist and you do digits we've been talking all quarter about new techniques you have found new ways of screening libraries we talked about activist for example we talked about display and we're going to talk today about measuring enzyme activities there are all kinds of neat new techniques that you could use you simply apply those new techniques to an existing problem and build new creative that's all thinks that's all you have to do to be creative is you take what you stand on a list of all the new techniques we presented this class you stand on list of problems you take Holiday Inn you take all of the 1 from anyone from being put him together and again move your creative that's all it will take for you to come up with a creative new idea but it is essential for this idea to be creative for it to be novel and if it is not I will return at your downgraded kind on fiercely defensive about creativity this must be created OK now but the other thing is actually come of this idea you need to verify that affect its original and this idea can be so outlandish has to be a possible but only if and only if that's already been done it doesn't count created even if it's brilliant but what I usually do next effort ,comma the creative ideas simply type it into pub that I simply typed into Google and see what else has been done in that area if it turns out that someone else has done this idea that I thought was brilliant not Nazi I think that's fantastic that tells me that my idea was brilliant enough that someone's willing to invest their own money and it doesn't bother me at all if I'm coming out of ideas that other people are willing to invest in and do in fact if anything that tells me that I'm on the right track and that should also tell you that your other retract so don't panic if it turns out that other people have come up with the idea before that's perfectly acceptable and I it's actually kind of normal and it's a good sign it stronger right you're going in the right direction OK so yeah OK so we talked a little bit about different ideas and Mommy scroll down a little I am OK so here after you finish screening Eureka about the idea that the real work begins you have to dig into literature were in the field just a little bit and I know something about the area that you propose a peso and that's really the real work of this proposal if you spend all your time trying to think of the original idea but you're wasting time that 95 per cent of effort comes after you have idea so only give yourself a limited amount of time to think in new idea and then after that start doing research and get that idea to share don't spend a lot of time to cycling for ideas and and you know taking yourself also not the world's greatest fine you don't need the world's greatest idea for assignment which indeed is an ability to argue successfully for that idea that's what I'm greedy 1 of so along those lines you know OK so yeah so anyway focus on that sort of thing and 95 per cent of the work is comes after you have idea but I'm OK we talked a little bit about the format abstract that's listed here and here's some stuff about the format of this assignment this time is going to be around 5 pages of not more than 10 I'm wondering if it's longer than 10 and no 1 wants Street office on somewhere in there there should be lots of figures and this is important the level of detail should be sufficient for someone in this class to understand what you're proposing you should be able to hand it to a random stranger in the fastest turn to the person on your right hand and hand him or her the proposal and then that person should get some idea of what's going on and should be able to judges and that's a level of detail you don't need 1 to tell me about every experiment you don't have to tell me where you must materials and stuff like that and most of all you don't have to do the actual experiment good this year of Visa card because all tell you those experiments are expensive and then finally if you'd like to have your agreed proposal returned to you with comments aplenty then you have to give me in advance attached to the assignment a copy of the and self-addressed stamped on below if there is no self-addressed stamped on below then I'm going to assume that you don't want any written comments back on your proposal and that's quite all right and I just don't wanna spend time reading comments it turns out there are Connecticut up but if you comments that I will take the time to comments on the proposal which he is will take time integrated and comment on it as well and so you'll get back something that has some feedback to you but I know not everyone set so it's totally up to you and last thoughts but it's important as usual that you turn it in through the tournament . com website this'll be scanning for plagiarism that is Ivory picked up plagiarism on the last assignment which I think is disappointing to me and every year low drive me that we talked about and not to spend more time delivering OK now now is the time I usually ask for questions if you have questions don't hesitate to shoot me an e-mail acid tears and Miriam critical no huge amount about this assignment they can help you OK that's move on so next I want to
talk to you about the office hours I have opposite hours this week you know I'm not here on Tuesday but I will have office hours at the usual time is unusual places Thursday immediately after lecture and then Wednesday 1852 315 in my office Miriam has office hours Friday and critical has office on Tuesday usual times you play OK that's it for the money announcements let's get on with it regularly scheduled program I want to talk about enzymes in enzymes actually remarkable
they are attractive to celebrities like these to but and there are also attractive to chemical biologist and I would say the attractive for the same reasons there are attractive because they do transformations that would otherwise be inaccessible enzymes make possible the things that otherwise would be impossible that would otherwise just take too long or required to much energy and will talk a little bit about how they do that and understand you know so entertained when I find them you know celebrity endorsement of my favorite topic here in New York Times last year actually on the scene there was delivering a lecture last year February 27 to those of enzymes try to grab the spotlight and there are tons of enzymes that are found in the pile call Pat and on the notable for digesting proteins that notable for digesting skin for example there actually uses as treatments for people who were undergoing therapy after that birds after bad you know high-temperature Burns and it's a way of digesting away a chronic tissue and these guys have clearly some good facial structure here so maybe they're onto something OK let's let's talk
next about them uh about how this topic ties in with what I told you about on the bus Thursday last Thursday I was showing you how simple rules dictate protein structure and in a moment we're going to then be applying the same rules to understand enzymes protein structure leads to protein function so the shapes that the proteins were assuming on Thursday are what allow the proteins these enzymes in this case to actually acquire their unique functions and what I found so beguiling about this idea of conformational analysis is that the rules are so simple were talking about something as simple as just the clips versus staggered at bay or gauche persist Yantai this fascinates me this will keep the right to work for a really long time because that such a simple idea these you know such foundational concepts in a modern chemical biology that I can explain to my grandma these are things that only makes sense right it makes sense to you that you would want to avoid electrons which are all year of electrons banging into each other right electrons head banging into each other it makes sense that things to try to spread furthest apart and if we think about things in that way then such complex structures as the proteins that were about to look at their structures start to make sense as well and we talked about how as a consequence of the simple rules some amino acids can be found in specific types of secondary structure and at their tables of this and you can even make predictions about the secondary structure of proteins based not on nothing more than the amino acid sequence and surprisingly these predictions turn out to be pretty good about 80 per cent accurate herself and from there people of that attempting to bridge protein structure for a long time and quite a bit of progress has been made in recent years toward that goal and we also discussed by sulfides which provides spot welds that hold together independent regions of protein structure that otherwise would sort of flop apart and we also talked about how readily exchangeable East bites were to allow confirmation of me I sulfides and exchange of 1 vessel for another and the next topic we discussed was this hierarchy of protein structures from primary structure of secondary structure to domains to tertiary structure to assemblies etc. and so on this helps us to organize our thinking as on these assemble into complex architecture so we're going to be making reference to this as we start talking about and enzymes and then we ended with the concept that a relatively small number of protein domains are found very commonly human proteome now the truth is I can finish the discussion of this so I
need to pick up just very briefly with just a little bit more about protein structure and then again want enzymes so enzymes are aware of the last thoughts and protein structure and this is an example of all beta-sheet protein called an immune globulin domain these oftentimes assemble into long strings and these are used in proteins like Titans tight it is found in muscles have wonder why muscles which is so strong so you know you've probably seen for example on Olympic 16 alleged that a lot weightlifters you've seen flexing the steel box right there lifting the stuff and the Steelers flexing and that tied in the muscle is hanging on what would happen says there are long strings of these immunoglobulin domains that are lined up like beads on a string so have domain that's another word for for immunoglobulin so you have to buy by gene by gene by GE and as you pull 1 ends up 1 of these can unfold without stopping the whole muscle fiber so the Titan actually is holding things together so that individual domain can unfold without breaking the arms the entire protein shakes and that gives muscle in that specifically gives Titan found in Mosul as some remarkable properties as a material OK so on the left this is the protein that's found muscles and each only 1 immunoglobulin domain noticed that the ends are 180 degrees apart this then sets up these long strings of tight that can extend you know up to the roof up here in down through the floor down here you can have many many numbers large numbers of these like that I'm on the right here is an example of an enzyme along the lines of the kind of thing on the talked about today this is a really remarkable inside this is 1 of those enzymes that makes it possible for you to live on this planet and this is an enzyme called superoxide mutates and its active site is actually not where you expect it to be I think when you look at these immunoglobulin debates these are happy to see which your expectation is that the insiders have decayed and that's not the case instead this insight is chock-full of cheese this is full of stuff it's only actually on the outside where the really action is taking place outside over here somewhere is where the active site of the superoxide dispute cases found and incidentally it's mutations in this enzyme that are responsible for diseases like Lou Gehrig's disease which is which is you know you truly terrible disease and so mutations these these kinds of proteins and enzymes have very very serious of constant medical consequences OK last fall on a new immunoglobulin demands and the year also named after the into bodies of 4 men for which the founded and I that this is generally is truly ubiquitous domains each 1 of these lobes over here is immunoglobulin domain and notice how these are just all strung together into the into body it should come as no surprise to those of you who were attending last week's lecture that the business end of the 2 bodies which are professional mining proteins is our Founding Act belt loops OK so these antibodies are designed for binding things that's their role like the professional binding proteins and in order to get that kind of binding the loops out here are exactly where he you pick up that kind of binding those loops are flexible member we talked about the low number of hydrogen bonds the backbone we talked about how the loops can accommodate many different shapes and sizes that's what equipped enter bodies with the ability to recognize foreign attackers raped he cannot set up in advance other body against everything on the planet these have to be just ready to pick up random things that they might get you might encounter when you visit and I don't know the taco acted out here something like that so you have to be ready for that kind of thing not knowing advanced with the shape is going to be so having these flexible and molecular recognition versatile domain with these loops over here to accommodate diverse partners turns out to be key to understanding their activities erect and another beer she protein and good friend of mine when I published many papers either using studying is a protein called struck and good news for me talking about this again in about 15 sites for now so it is a great pleasure to introduce you to the wonderful struck out and struck out and is charged with binding to Pieter it again this is an albeit a she protein so there's a little tiny alpha-helix but it's largely albeit a sheet and it forms these wonderful little bit barrels at 1 end of the bearded bear all this small molecule called by its assets and will look at the structure by items greater detail the moment this is an example of a small molecule the molecular weight of Pieters 254 GM from all but a tiny tiny and by and is tracked by the Quaternary structure of stripped out the Singapore
concept if we look here that say but the secondary structure Our Ahtisaari the tertiary structure Pieter to outstripped Abbott on your expectation is that the fighting is not to be very firmly held in this debate there right I mean look at all the space you can imagine the Pieter just floating away and and coming out of the the stripped out and without very much trouble case tertiary structure doesn't begin to hint at the extraordinary abilities of this molecule to grab on to buy it and 2 bites at end here's what I mean OK so now this is the Quaternary structure of struck and stripped out and consists of 4 it consists of the homo touch Rumor of War stripped out and a art non covalently joined together to trap itself and there is therefore quieted down here now what's happening is the alpha-helix from a neighboring struck evidence is sticking down over the top of my head I've highlighted that for you and black over here and you could see it's actually forming at door To text slammed down over the top of the buyer and prevent the Pieter from floating away that turns out to be key to its activity so strapped Avedon defaulted .period piety with astoundingly high affinity and will talk about the exact number of moment and but it is really extraordinarily high and it evolved to bind this Co after call by itself as a way of killing any bacteria that happened to be present so for example on a related protocols Avedon is found In egg whites saw a white had a high concentration of Abadan and that means that if any bacteria trying to colonize the egg you don't talk about headaches here if any of those of bacteria try to get in there and do the town and all the juicy richness of of a wonderfully but they're going to die because they abiding will get sucked up by the struck out at and then wants almost permanently and that turns out to have been consequences of the bacteria it turns out that it also could have fairly fatal consequences for humans if you live on a diet of nothing but a lights from Europe by itself will also get pulled out of your body and this is 1 of a kind of an astonishing fact because it turns out it doesn't take a lot of Pieter 4 by using humor to survive by essential Coke factor in the synthesis of lipids and I but however there center people out there who counseling do experiments like this and I when they show up in hospitals after eating a diet of nothing but a writes that physicians tend to be totally baffled because I'm not used to seeing such bizarre symptoms and so and there was a case of send around I think this the New England Journal of Medicine of this English guy who showed up it was living on a diet of nothing but tea and egg whites and arms he the analysts like bleeding out of his guns and porous and the way he was no falling apart basically and astonishing things would take just a few micrograms a day of Pieter for him to be totally all but there's such a high level of of avatars and its hold on what against struck out at present an egg whites it was actually will be all of the blighted out of his body OK pretty extraordinary of biochemistry pretty extraordinary letter recognition it actually fascinates me to understand how this works better and it's something I've spent a lot of time thinking about OK so I'd get another example of a very common protein poll that found in the human proteome the IWG proteins consists of these beautiful propeller light assemblies where there's actually 7 1 of these little triangles they look kind of like slices of pizza and that are that come together to form these large assemblies scaffolds to organize big machines found inside yourselves so this on each 1 of these bases over here might binds to a different protein and bring it together like it's an assembly line for putting together really complex things inside yourself actually fascinating stuff but you know something else is truly bizarre what's up with the 7 symmetry you know and I think I'm ready for a fourfold symmetry and but we humans don't want to think and 7 fold symmetry so obviously that is something we puzzle over but also another very common and I'm switching gears were going down the chart the most common protein structures found in in humans and collagen is a very common protein structure and it's actually an unusual 3 stranded coil it differs from the Albertville oil so we saw the twist is different and it and it's a little hard to see but there's actually 3 different colors there's a green color purple and blue collar so there's 3 different colors here
these 3 strands that are winding around each other and this makes a very strong framework for college collagen and is another ways ubiquitous structural proteins found in the body of notably this protein requires a post-translational modification of introducing a hydroxide into probably residues and that has the effect of setting up 1 particular preferred structure of particular twist and in the collagen triple helix without this hike hydroxide the protein is unable to assume that confirmation by switch gears I want to talk about the GPC this is the class of proteins that makes it possible for you to see me and that what makes it possible for you to smell the taste and pretty much any of your senses are totally dependent upon this classic proteins so I think we should take a moment to be grateful for their existence Hi how these things work this is a really you know how is it that you get a sense of the longtime have happened you actually um you don't respond to satellites which is you know you can only light freight so on how these things work area so In short the sensing proteins again the called g protein coupled receptors at TPC odds are all out the killer cold and and noticed that you there's these and they each have 7 property was seized the 7 LP helices transit of plasma membranes in the self-serve going through here it here's the plasma membrane so there's an outside an exterior and there's an interior space in the cytoplasm exterior suspects facing extracellular milieu and on these change confirmation upon finding things in the case of photons the GPC are responds by having a baby summarization of the core of the carbon-carbon double bonds so the photon hits that I summarizes carbon-carbon double bonds flips it from 1 configuration to another configuration from Trans desist and back and in doing so that rearranges the confirmation of the these from these residues down here that are found on the inside of the cell OK last thought that this that this is a quilt quail and if you look carefully at this it is a left-handed quilt Quayle almost all the quilt oils found in nature a left-handed Minnesota and can trace the out in a left-handed manner doesn't want trace out of right OK so these are very common because was in general are hydrophobic secondary structure they fit nicely into membranes and this property makes some very useful for sensing what's happening outside the cell and communicating it to what's happened to the inside of itself the next 1 here's another example of an out-there sorry amid talk very briefly about alpha-beta proteins that this is an enzyme that will talk more about the very shortly this is a a barrel like protein here's the barrel in the center of this is the active site and so you can mix and match articulacy than I'm showing you as you said you wanted him oppression that secondary structures never mix in fact they're very commonly mixed together I repeat proteins so repeat proteins are another very common assembly of proteins on the surface is an example of an anchoring repeat protein notice that it has 2 sides the concave side has the leaps and wouldn't you know it the loops turned out to be the key to this mining activity this is approaching its used a very versatile it's used in many different contexts and what happens is the loops served mutated to give them a particular buying property that's and useful for the sale of its corollary it is Hussein richer peak which now has loops on the convex side in place of the Ankara patents on the concave side this 1 has loose on the convex side and these lives then conclude ground on to the binding partners pay analogous to what we saw with into bodies and so on this is a very the Stewart very versatile it's structures that can be a ball through organisms pretty readily and that gives us that equips organisms with new binding activities which in turn can be used to respond to environmental changes at 7 right last structure that I want to
talk to you about peptide binding domains this is an example of the S to demand this tiny little domain shown here and yellow lines Foster tyrosine proteins of proteins that have been post-translational modified using class of enzymes called kind users which will talk about a moment of a blind to this sh to and fit into the theory deep pockets as the deep-pocket down here and so these evolve basically to have specificity that bind to a particular sequence not blaming Gary Foster tyrosine picking out specific binding partners another peptide binding domain of note they are H 3 domains the spine polypropylene helices this is the right plus 3 hillocks that 310 hillocks that we saw on Thursday during Thursday's lecture and on this tiny pocket is a very shallow once it almost looks like the peptide isn't resting on the top of this SH freedom and it's it's like a butterfly kind of alighting on the top of the 3 domains and notice how delicately folded it is into this three-star threefold symmetry elects right so you can actually looked down on 1 axis of this he you can see how it's forming this polypropylene plus 3 Felix OK I know it's hard for me to talk about this without having a few favorites and I know as a chemical biology section have favorable chills but I and this is 1 that I spent years of my life thinking about in my wasted years and I was interested in these MHC receptors for reasons that are too bizarre to explain right now but suffice it to say these are the receptors that your immune system relies on 2 let us know when the records are coming this is the use of the receptors that raise alarm when foreign invaders are trying to take over and you're Europe physiology and the way this works is a small percentage of peptides that a synthesized by the cell are digested and then displayed what on the surface of the protein just like a little flat and idea is that if the virus has taken over the cell the but little flags of virus will appear outside on the services of the cell and immune system and knows no no that sellers been infected with viruses I better kill itself I better mount a strong response this is a very effective way of alerting immune system OK here's some old friends as well notice on down here to recognize that the main descent look familiar yes that is the same domain we saw few slides again this is the biggest sand wedge immunoglobulin domain and here it is making a cameo in a slightly different but equally important role here it is actually a lofted the peptide Al off the surface of the cell down here is the surface of the cell fears that the scaffold at this immunoglobulin them a kind of holding it above the surface of the cell making a little easier for the plant to be seen by passing T-cells especially cytotoxic T-cells that take an interest in these things and they can go into action until the seller necessary right 1 last example of this would be last example is a slightly different variants of these MHC receptors in this case this is 1 call class to the 1 in the previous items called Class 1 the details not so important but this guy actually displays peptides not that are being the spider sell necessarily but peptides that are being in bowl by itself so with the cells kind of randomly taking of extracellular material and so again this gives the immune system a different look so the MHC class 1 tells about what's reports on what's happening inside the cell the Class 2 reports on what's happening outside the cell and notice that the this structure of this peptide when view down the same access that we looked at earlier from Street it again and starts to bear some hallmarks of seeing notice that it also has that sort of threefold a kind of looks like a triangle type of geometry and yes this is also assuming a polypropylene type Felix analogous to buy plus 3 helices that we saw earlier in this class OK so hopefully some of the concepts that we saw earlier finally come into play and what I want you to do so would you 1st have an esthetic appreciation in things these are beautiful because I like to think of this 1 as like a hot dog in a hot dog bun and look at this stage it's so juicy acting unit but but equally importantly has immunoglobulin remains again that have a wonderful function of lifting it off the cell surface but at the same time is presenting what's a surface area out here so cell can recognize whether or not this flag once itself non self even interrogate of 1 receptor and determine whether or not the regional sales are from South versus non self and this is really 1 of the challenges for organ transplant patient is deal this class of proteins where everyone there word different humans have different MHC receptors and this is 1 way that means that he's in on Morgan said it had been transplanted and knows to kill them OK now obviously I can talk about this particular topic for hours but we don't have ourselves and we will have to move on right I I wanted to finish our discussion of protein structure and
by talking about higher order assemblies and I guess the best example of this is a structure that I introduced you earlier collagen collagen again places key role structurally strengthening bones and joints and doing all kinds of important things this is not yet again 1 of those proteins that makes it possible for the week lifted whole East you don't use corporate debt left over his head and have the ball flax I've always wanted to do that but I don't think it's going to happen but just to see the bar flexing his you know a thing of beauty right and will how was that possible OK so obviously these things are really strong collagen is remarkably strong and it also is assembled into an ordered structure where that ordered structure supports each of its constituent fibers and I guess the best example of this that you'd be familiar with the something like a rope break the individual strands of the rope of a fiery road not so strong but we knew going in together and they're all supporting each other something get something really strong backing anchoring aircraft carrier to a doctor something like that right so here's collagen works and you have to control its assembly in a way so that it doesn't get it doesn't know kind of prematurely whined itself up the legal tangle OK so what happens is that the Triple Helix is formed with some caps on the end and on these caps on and remain for quite a while so big the intact pieces are then brought together and then the whole assembly with the capstone place is secreted outside the cell and where these red heroes are these caps are then snipped off using produces the scissors of the cell and that gives you the formation of Bibles without that this does not happen because without that you get this kind of sticky tangled up mass but what ends up happening instead is a very detail oriented assembly where each step in the process is carefully controlled and that's essential to making structures that are really strong OK now I want to move on I want to next about insights we've seen pretty instruction and beverage instructed let's talk about what they're good for obviously good for strength and structure I wanna talk about catalysis that's OK so enormous talk about tells us I have to introduce you to some measurements of street the binding of catalytic efficiency and so the 1st thing I'm going to have to do is to find a few equilibrium constants for the 1st of these is used to describe the strain of the non-covalent receptor and interactions with the receptors indicated are and the white in full sphere indicated L now view of a bunch of receptors on the surface the cell that want to bind to like and the like and it's going to hop on it's going hop off it's going to hop back on his behalf back off so we need some way of describing the occupancy How many the receptors are actually grabbed bounce along an avenue wagons are free in solution so underweight chemists do this is using equilibrium constants of these equilibrium constants are kind of special sleeker special name but there were less the case the EQ that backing can 1 so here's the way this works we can describe some receptor alike and interaction that's form as having a dissociation constants in which the receptor and the like and dissociate from each other and that dissociation constant the take the is equal to 1 you know constitution receptor times constitutional again divided by the Constitution of the complete receptor like interaction on the inverse of the dissociation constant is the the association constant abbreviated KA yeah but again this is just fancy equilibrium constants however they tell us quite a bit about the strength of an interaction and I'm going to be referring to the 1 thing you need to know is that a lower Acadia lower dissociation constant means a stronger interaction and we're going to stick with taking take everyone out and the pharmaceutical world than the biochemical world discusses things in terms largely of Katie's is a you basically every time he hears on I'm mentally take the inverse that number and then think about in terms of case is just a convention OK but what matters is is that a lower Katie needs a stronger equal a stronger interaction that needs more of a Weigang's found your rights you have more complex form a bigger number down here lower let's take a look at some of these OK so I be talking to you a little bit earlier about organ transplants and rejection so after people received a transplanted livers they're given a class of drugs called immunosuppressants that suppress the immune system and we know quite a bit about receptor like interactions through classic studies done by Stuart Schreiber and others that looked at how these immunosuppressants work on fears of the euro are 2 examples of
immunosuppressants on the left is a small molecule called at the time of 6 on the right is a small molecule called recognizes they both work by targeting of a binding protein that the Schreiber laboratory called named F K B P 4 in the 6 binding protein and here it is neatly fit into this receptor which is F K B P so the light and is the immunosuppressant drugs the receptor is that KDP and on notice that this is finding a really deep binding pocket 2 . 2 but Simone effect but take a closer look so imagine now that we can zoom in and just looking at the green light beyond what we would see it's something like this where in blue this is the region of the small molecule that's bound by F K B P OK so again on the left deceptive father 6 on the right is if it is wrapping license notice some similarities to obtain noticed that in blue these largely have the same structures because that's not a coincidence that same structure and helps story and the molecules and make it up so that this half of the molecule can very readily blind to know this is the part that's not shaded from these 2 are wildly divergent a molecule left the park accentuated looks completely different than the thing on the right OK and on that's not too much of a surprise either because it turns out that these 2 molecules affect different pathways in T-cells to suppress the immune system and our the the widely and act as sort of like the meat in a molecular Sam Wyche and they were crude to different on top layers of bread this 1 over here recruited different protein than this 1 over here however both of these molecules bind to F K B P III with very high affinity and the way we know this is -dash any is we refer to the KGB the dissociation constant as having 87 animal where Katie range that's really get that's really really tight binding and it turns out that most of the and pharmaceuticals that are approved tend to have affinities for their targets in this kind of very low key the range why that is will be apparent to you in a few slight OK so this is 1 way of describing non-covalent interactions and next have to tell you how we're going to be describing speeds of reactions the kinetics that's make reactions possible 2 kinds of reactions that were going to be sitting in this class kind number 1 you know molecular reactions these reactions we have some reactants and on it goes through transformation and that's OK there's no other the other of some species that's implicated in the reaction mechanism that's it assesses 1 Tagesspiegel intermediate falling apart collapses such regional immediate the rate of the euro molecular reactions equals summary constant little tiny K times the the concentration of the starting material this came over here I'm emphasizing tiny gave reason locate indicates rate constants it's truly different than the equilibrium constants I was showing you on a previous life never the twain shall meet that do double a different banks addressing crazy there but the both symbolized by and there's nothing I can do about that kind were stuck with that's old-timey nomenclature are next 1 were also going to see by molecular reactions these reactions that have 2 reactants better colliding with each other and in enact collision and resulting in the formation of a new product the rates of these by molecular reactions and are going to be equal to some little tiny K the rate constant times the concentration of reacted 1 in this case hydroxide times the concentration reacted to which recalling wireless makes sense OK I expect that this has been a review this is something I know you've seen before In biology that these rates vary enormously OK check this out this rate constant K 1 ranges from 10 to 13 per 2nd to 10 to the minus 7 per 2nd that's 20 orders of magnitude difference in 1 speech OK this this is at the wild you know fast end of the scale and these are things that are a total blur and the Super Slow end of the scale these are things with geological times that are so slow they simply don't even matter and biology without some sort of catalyst speeded up OK so these the parameters were going to use so let's now think about kinetics 1st of non-covalent interactions and then we'll talk about inside
OK so non-covalent interactions you can imagine the life against hopping off of the receptor when that happens and there will be some some speed of this that will have a rate constants of little chaos similarly like instant hopped onto the receptor and again and there's a rate constants that OK on naturally if this is an equilibrium then you can actually you can work out that the KGB equals the ratio but the payoff to the chaos 10 there are also again this is an equilibrium where 1 there also was no OK good here's the thing but the typical
rates of binding are again wildly different and this is a cable from the book a table 6 . 1 and would you take a moment to do just that days at the the truly awe-inspiring nature of this differences in the year focus was just take a moment to appreciate that but what I'm showing you is a series of different receptors and over here a series of different like him at the top of the stairs small icons uses small molecules might buy it and then we saw earlier and the bottom and these are large like this opinion check this out This is really cool on notice that the on rates for all the small roughly the same very very little difference there is no there all right and that tend to the strange and hate guess what tend to be it's kind near the speed limit the speed limit for resuming through the cell is going to be somewhere around 10 to the nite promoter per 2nd said that tend to the tonight is a physical constant Yukon downstream through water and faster than that and so these small molecules are there zooming along about as fast as they possibly can to fit into the ear of receptors but check this out off rates these off rates vary enormously they range from saying 9 over here up 200 thousand and so on down here and the big things huge changes in work and in off rates as well OK so what this tells us is if you're trying to design the perfect pharmaceutical the perfect therapeutic to treat 1 a muscular dystrophy or something and you want to spend a lot of time thinking about operates off rates are where the big money is when it comes to therapeutics .period comes the pharmaceuticals they all of roughly the same on rates which differs is off rates and those off rates how you can determine whether or not a pharmaceutical that can be given a low-dose forces the high but I'm getting ahead of myself we don't talk about this again OK let's talk about the large like large light hands have enormously variant on on rates you know there's too many zeroes here count and and then over here enormously variable operates the new that should make sense to us just intuitively because large molecules are going to be able to zip through the cell as quickly as small molecules right they're going to get you know sidetracked gonna try to bind other things they're going to do but you know they're diffusion rates are going to be slower for example OK now let's put it all into effect OK that's that's that's put everything we've seen so far but it's going to 1 that you know summary that tells us on what it is that we care about in terms of treating patients and so on the way it works is what we want to do is we want to have some biological response OK if our goal is too cure patients say on toenail fungus then are biological response is going to be you know what percentage of their toenails are clear from the fungus here's the latest works on the Y axis this is represent biological effects that biological effect results from 1 against buying into some receptors Hey we've seen for example in about better binding but to the riders up in that case survivor zone would be your biological receptor and the biological with with that would be the death of the cell that would be killing the bacteria OK and so in general and we see it soon will a biological responses when we look at the receptor like binding OK when it scrapped as long as the concentration drug along the x-axis and Percent biological response at the top at the very top at 100 % biological response from this rule will take a very high concentration of drug take notice that the numbers are bigger on this side and then smaller over here this is 10 to the minus 9 over here 10 to the minus 1 over here and concentration of bowler I realize this access a little confusing there with me there's a reason we get the way of OK so appear at a really high concentration of 100 per cent biological effect of cable maybe at that concentration and you end up with a drug that has to be given with pills that are like you know the size of and you know races or something no 1 likes to swallow things that really bad and so on we compromise instead what we want is we want to have a 90 percent receptor occupancy in indeed 0 to on CD some sort of effect OK that's our goal so we're going to be measuring the biological potency treaties dose-response effect and the major goal of pharmacology is to get up here into this 90 % receptor occupancy we get 90 % biological effect greater than 90 per cent biological now typically b and the number's up here you know obviously we're approaching it as something that things aren't things our you know go over a really long time up here I'm so instead we described biological potency in terms of an effective concentration for a 50 percent effect that 50 per cent of that takes place right here at the point of inflection for this same dose-response curve and so we compare 2 different drugs just by comparing the EC 50 where the more potent drugs will be the 1 that gets the same 50 % response but at a lower concentration right it means then that the patient can be treated with a lower dosage to get the the same effect in the same benefit OK so you know I think it's worth taking a moment to talk about this because I like 90 per cent of students in this class are going be spending the rest of their lives battling with this city will curve and trying to get up here and sometimes being down here OK let's talk about how you measure biological response oftentimes they can buy lavatories this is
measured using a Aliza that enzyme-linked immunosorbent passing and other before I can tell you a little bit about how that assay works I need to tell you about on some reactions that catalyzed by enzymes and their 2 enzymes are kind of the were courses for Camby laboratories on 1 of these called on peroxidase other once called positives as these are reliable enzymes that will catalyze reactions that lead to turn over a dime molecules so here are 2 molecules up here this is this is these 2 aromatic molecules these guys up here are clear OK so he made a solution these guys you would look more or less clear when we're going beyond the more less clear however after the enzymes these 2 enzymes catalyze these reactions what ends up happening is you get a has a deep color very strong color this 1 forms a dark black actually more brownish color very dark color and this 1 over here forms of bright yellow color so both of these give us reliable indicators that we can use to follow how much activity is taking place at a certain dosage so let me show you how this works and what we do is we use we plates and I think I talked about this before their eyes on the inside of their lies at the core of the wife of plates and colloquial in the lab and these plates have 96 wells on the process that's over here and each 1 of these wells can be coded so that the surface is coated with the receptor pasted here's the receptor down here but the problem is that the surface likes the buying receptors it also bind Elijah and we don't like that kind of thing so what we do instead is at a blocking agent typically something like dried milk and nonfat milk and not drives itself and so we take nonfat milk including any other place on the surface of the swell that otherwise might start to buying on specifically to like him we don't have lot idea it's stuff blinds we wash away the non-binders and then entered into body against the lagging so wearable wagon is bound were going to get into body stuck to its this antibody bodies is especially 2 bodies unlike at the bottom section earlier this 1 happens to be covalently tethered to the enzyme that I showed on the previous slide that enzyme is peroxidase and so this means when you add the guy in this proxies goes to town and turns over and the die in in this in this well and then you can look at a large number of wells to get a dose-response curve this works really well the Dudley robust you can use this in your proposals it works great OK let's talk next about about the receptor like interactions because I mean they're showing you up and Aliza as as an example of catalytic receptor like interaction I wanted him back to struck out OK so strict Avedon has this remarkable half-life of 200 recall that this is the protein that is found in analog is astounded a whites and if you live on a diet of egg whites eventually you're reaching all of the bite and upon this half-life over here of 200 days starts to make sense of of it starts to explain why it is that you can actually reach all of the bite an idea that everybody it's key happens to be an astonishing 10 to the minus minus 15 work people on some Peter Moeller really comfortable or it's would be tantalized 50 says some people Muller Katie that's extraordinary because earlier I showed you wrap mice and after about sex 55 a 6 and I said that the animal or affinity was really high that's a really great finding partner and in this case is 1 that you know a million times better and that is really strong OK now chemical biologists have learned uses for all kinds of assays is the 1 thing we do pretty often is attached by a 10 covalently 2 and small molecules and use is kind of like a worse for fishing so you'll know about fishing rights to the way this works you have allying neutral 1 water at the end of the line is the Fisher smart attended the shock and eat some random so instead will put some sort of lower on the that and bring the fish about obtaining classically I guess this is 1 final official worms official flights but I also like official small molecules and when I do I always used by at the repressive years the way this works so here is the and it's now covalently tethered to some small molecule this becomes the lower so this side over here is going to track proteins from the cell and then this side over here is going to attract the attached is going to act as a candle so that's the part that you grab onto an old and where this is really useful is if you have some new small-molecule and you don't know what it binds to and the self this happens to us all time will have some molecule that we pulled out of I don't know fruit flies were looking for some molecules that make fruit flies less drunk or something like that and we wanna know how does that work OK so the way you do this is you have some winter and then by accident and then you basically go fishing and hope for the best a quick word about the link the linker matters a great deal because you member earlier I told you about the trap door and how the neighboring stepped out and subunit slips over there vote 1 beta barrel and flaps that without this winter and it's very hard for these molecules to be strongly held by pirates and that the linker allows the trap door to close all the way and get wedged tightly shut without this then some molecule like this it has rings system nearby would would basically get into the trap door and block it and make it harder for to close all the way so the Lakers actually matter quite bit but in practice here's what it looks like in practice we
have these columns that we flow In the Still Life over and we were looking for His were looking for molecules that will stick to the column better buying into but not abided but to the product that the target that's then tethered to the buyer to pay so this is the way we go fishing on the handle is over here that's bite and it's grabbed onto by strict Avedon and then the Luiz hanging out and you send through jumped from the cell to cell sorter counterpart new hope that stuff sticks other molecules that don't bind to a target flow through and don't stick and again this works great press so everything you know about the cities are now being applied OK so now what I've shown you is I showed you the example of a really strong receptor like interaction I've shown you had measure biological fact let's now zoom in and start talking about catalytic receptors This is a great example of a non-Catholic receptor on enzymes are basically catalytic receptors Everything that you that we've been discussing in terms of dissociation constants in terms of mining in terms of what to architecture all that comes into play we discussed so What We Talk About When We talk about enzymes we like to talk about these in terms of a few simple parameters and again these are parameters that are familiar to us from earlier discussion so earlier I talked about receptor like interactions having answer chaos similarly enzymes substrate interactions are going to have K on the chaos making either form the complex and substrate complex sometimes called the Michaelis-Menten complex was baby decide not to end up on the key the the 1 little twist here is the fact that the enzyme is going to catalyze transformation of the substrate to some product of substrate is a fancy word for starting material and what is happening here is we're going to form this intermediate complex and this intermediate complex is going to very quickly collapsed if classes to the left if a classes to the right then and it is going to then form a product and this product will hope you hope quickly disassociate if a classes to the wall left then on the substrate diffuses away and this goes through an offering take a quick word about this but in this case from showing that enzyme product the product then has to disassociate from inside if that doesn't happen then enzyme is tracked and inhibited in this state and guess what that actually happens in effect is 1 of the reasons why oftentimes this last step can be a rate determining step for enzymes that you would not expect this to be a problem for director of MIT's zoom in and take a look at the reaction quarter diagrams for on enzyme catalyzed reaction I will tell you an answer that this is grossly oversimplified the real on the the real reaction Gordon background is complicated and messy and I'm going to show you the theoretical idealized version 1st OK so any lines work by stabilizing and that's lowering the energy of the transition states doing that accelerates the reaction OK so here's a typical reaction but on the Y axis this is that the change in energy Delta GE up Hieronymus access means less stable lower and access means more stable and as you know the higher means takes more energy which means an Internet slower on the X factors this is oftentimes call the reaction coordinate it's nothing more then on the the conversion of going far are the other the pathway between the enzyme plus substrate going all the way to enzymes plus product over here OK in here is going through a couple of different and intermediates enzyme substrate intermediate enzyme product and then also a transition state OK so in order survive so let's talk about the UN catalyzed reaction 1st on catalyzed reaction in the EU the substrates starts off over here December discovered products that takes a lot of energy OK the enzyme in this case is involved that's why it has a plus sign it's kind to hanging around spectator was just a matter that would happen so if that happens but there is this activation energy here the difference in energy between the transition state and the starting material is very high and we can actually do right this the reaction from knowing that energy right and we know of bigger energy over here higher activation energy means a slower reaction and oftentimes these reactions are too slow to be biologically useful if we relied upon spontaneous reactions taking place you wouldn't be human if OK you just know it would be possible for biology take place instead what happens is enzyme stabilize vying to the substrate lower the energy of the substrate complex and turn most importantly lower the energy of this transition state the Red here is exactly how much lower the energy at them bigger than areas the better right because that's how much how greatly improved this similar layout had been signed and binds to the product and then eventually disassociate from the product the product Associates OK let's take a look at an example of the island example is 1st you know molecular reactions and I realize this is kind of a hearing examples bear with me it's worth it and so in this case what's nice about this example is this reaction is a conservative reaction meaning goes smoothly in 1 fell swoop it's 1 you know step in this reaction mechanism this is a Paris cyclic reaction and it had it involves the transformation of Korea's may on the left to prevent a on the right and again enzymes called charisma duties and by the way this is a key step in the she can make acid pathway which although plants on this planet rely on to produce phenylalanine and the aromatic amino acids this is 1 of those things that we humans could not exist on the planet without we cannot do this reaction plants and microorganisms are the only on of organisms that have the necessary and lying to catalyze this reaction and for that matter the only other organisms that have this much the Makassar pathway which is why is indirectly musty plant material to survive OK go on check out this reaction mechanism this is cute but this is awe-inspiring a in this reaction mechanism electrons are going to bounce down your downspouts and also some or all the way up to here producing a new carbon-carbon bond between this Parliament over here and this carbon these guys are going to join together to produce a new carbon-carbon bond that has to be a wedge coming out toward us OK so in order for this to happen this part of the reaction mechanism has to be sticking out towards us and this whole thing is just a swing over the top and all 6 electrons in flying once this is fury style to give you a needs swoop this new carbon-carbon bond while breaking apart this old carbon-oxygen bond and breaking a carbon-carbon double bonds to form a new carbon-carbon double 1 this is really spectacular stuff and this is the stuff of just you know graceful electron choreography in motion OK with take a closer look and see exactly how this works
0 0 and by the way I think if you try to do this reaction when you can get it to work but it's in the slow it's going to be a dog so check this out this is the way the enzymes do and this is the way they make this happen what they do is they grab on to the and to the the handle appear and forced into some semblance of the necessary transition state and in doing so they're going to lower the activation energy of this reaction OK so here's what it looks like owned or actually I'll tell you moment what this is this is kind of like the transition state bounds of enzyme and up in practice the transition state looks like this here in -dash lines are the bonds that are being made and broken and in order to get this reaction to take place then to grab on to this thing over here and basically forces over the top so that this common in this carbon next to each other to form a new bond if that doesn't happen that no reaction takes place and so when the enzyme is doing
is is landing on enforcing this thing into the right configuration to allow the reaction to take place punitive duties after the injury of looking at here is the way we know that that's how that happens this is on classic work done by and my adviser from my undergraduate days at UC Berkeley Paul Bartlett and by the bottle laboratory synthesized and inhibitor of this reaction that looks like there's a kind of mimics this transition state and guess what this mimics the transition state so well that it sticks in the air and just plugs up it bombings really well for this enzyme because the enzyme of fall to buying into this transition state that's how it works and so since enzymes buying transition states with the highest affinity this strategy could potentially yield the best inhibitors binding to transition states is the same thing as saying you're going to catalyze the reaction you're lowering energy that's required for the reaction to take place and here we're seeing a dramatic example of this by buying into this transition state you're Europe forcing the substrate to get its carbons in the right location for this reaction to take place and if you don't do this them reaction there this stuff is just going on all over the place and on its all over the place is finding lots of other stuff to do with its time in the reaction never takes place I have
so much more when it tell you about when we come back on Thursday I have something really cool and saving up for a while I want to talk to you about how it is that enzymes work and not just in terms of binding transition state by the way this is kind of the classical secular biochemistry it's not wrong it's actually totally writer also would say to you but I wanted to talk to you about the other aspect of enzymes which is enzymes also had motion they actually are going to be physically forcing together these these on transition states to lower the transition state energy and in turn accelerate reactions and to me that's really 1 of the most extraordinary aspects of enzymatic tells us so let's start here we pick it up on Thursday will be talking about motion as a mode for accelerating chemical reaction
Computer animation
Computer animation
Computer animation
Computer animation
Computer animation
Computer animation
Computer animation
Computer animation
Computer animation
Computer animation


  482 ms - page object


AV-Portal 3.8.0 (dec2fe8b0ce2e718d55d6f23ab68f0b2424a1f3f)