 All right. Well, I would like to begin by thanking the organizers for inviting me here. And this has been a really neat experience because I am solidly on the biochemistry side. And so I don't think a lot or hear a lot about physics. And I think one of the best things that I call a good meeting is when you learn something. And I can clearly say I'm learning a whole lot here. And so I want to start with just, oh, that's not what I was supposed to show. That's my kids. That's what they do for fun. See, I told you my computer is having a little trouble. So while we're getting this sorted out, this is something that I've kind of been thinking about through these talks is, okay, in physics, people are asking, seem very straightforward questions. If we have a plane, well, then what happens if we deform that plane? You know, so for example, such as folding a piece of paper. And the way we're going to think about this, though, actually is fairly complex. You know, we may be using some sort of equations that look something one minus feet. For an equation I saw yesterday and I said, what the heck is that? But what the goal is is to, you know, have a very fundamental question. Think about this in a sophisticated way, but really, you know, get to, I think, back to something very straightforward, right? Where we can really say, hey, we understand this system. Now, in biochemistry, what we do is we're thinking about something a little bit more complicated. So for example, we may be saying, we've got a cell. Here is our little virus particle. It goes in and then out comes our little virus particle times 1000, right? This is the biochemistry, what they're thinking about. And how are we going to do this? We're just going to watch, essentially, right? We're going to watch and say, hey, what can we learn? And then the thing is, is we don't get out something really simple. What we may get out is a couple of different plots. We get out some histograms. We get out some pretty pictures of cells with viruses in them. And we have to really infer what's going on by essentially, you know, bringing all of this information together. Okay? So now what I want to try to do today with my talk is take the best of all the worlds, at least from my little perspective, which would be, okay, let's take the simple idea, combine it with just watching, and then come up with a nice little answer. So let's see if we can do that. It's going slow. Okay, so how are we going to do this? So here's what we'll call an energy landscape. And let's say we start with some protein subunits. And we've talked about this a lot, this meeting. And one of our really things that we're thinking about is, well, how do we take individual protein subunits? And then how do we actually make those into a virus? And we've thought about this in the context that there is some sort of delta G involved here, and that it's actually a negative delta G because this is a favorable process. This happens, right? We know that. And then viruses have an interesting problem. This can't be their global minimum state, right? Because if it was, that's the end of the virus, right? We have to have infection. So what the virus has to do is it has to get over here where we had our little, right? We know we've got some nucleic acids packed over there. And we have to get over here to a state where either we still have the intact capsid, but somehow our nucleic acids have got out. Or maybe our capsid has actually fallen apart and we're back to our triangles, right? So what we can see from this is essentially this means that a virus really has a thermodynamic balancing act that it has to go through, right? It has to be in a situation where inside of a cell it can essentially assemble, and this has to be a favorable process. And then essentially in a very similar environment, back in a cell, it has to then have a favorable process where it falls apart. So how does it occur? And so this is kind of what I want to bring up are some ways, the experiments that we've been doing looking at the stability and dynamics of proteins and virus particles. And so my talk, what I want to do is I want to start and focus on, let's just look at the proteins and think about these proteins as what are really the functional attributes of the proteins that are going to help them assemble into a particle. And so I'm going to do this by showing you some data from hepatitis B virus, which you've heard in a number of talks already. And then what I want to do is look at this process of how does the virus actually go through this process of assembly and maturation to where you have an infectious particle here. And so to look at this, I'm actually going to look at P22, so bacteriophage, which has the details have been worked out really nicely of looking at this. And then I want to finish up with actually looking at a whole bunch of different viruses. A library of virus particles where we can start to think about, well, what really is the difference here? What is this delta G? And I think really importantly, what is this activation energy? Because there's got to be a hill here because these particles have to be stable. They have to be able to get from the host cell to a new cell to infect. So there has to be some sort of barrier here, but can we think about that barrier and what might it look like? Okay, so I'll start with talking about hepatitis B virus. And so the reason we picked hepatitis B virus for this work is because essentially we can study the disassembled proteins, the capsid proteins, which are dimeric under the exact same solution conditions that we can study the assembled particle. And so that's really one of these unique systems. So Tully mentioned she could do this this morning also. And so this allows us to really understand what are the inherent properties of the capsid protein and what are the emergent properties of the virus particle. And so today I'm going to focus on the capsid protein. And so this is a well-studied system, the way HBV assembles from essentially a dimeric set of the capsid proteins. They fold into a dimer. And then there's been a lot of work particularly by Adam Slotnik and his group showing you that this is a, it appears to be an allosterically regulated process. So things like ionic strength, heat and divalent cations all will essentially drive assembly. And so some of the work that we've done in the past is essentially looking at well what could potentially this activated or assembly competence state look like and we've been able to show that it's more protected or more folded than what appears to be the assembly incompetent form. And so this gives us this kind of little model where we have some sort of activators that drive nucleation of a folded state leading to assembly of the particle. Now there's a really interesting mutant of hepatitis B capsid protein. And so a single immune acid mutation, a tyrosine at position 132 to an alanine essentially leads to an assembly inactive dimer. So this dimer, you can mix it together under any conditions you want and it will not assemble into capsids. And so in this case we're looking at the, you can see a nice view of this very helical dimeric assembly unit. Now Adam's Latinx group was actually able to determine the structure of this dimer in solution. It actually crystallized as a trimer of dimers. And so one of the first things they noticed is there were very subtle changes in the structure from the wild type protein to this Y132A mutant. As you might imagine it's a single mutation and the mutation is actually out on this little turn here. This is the C-terminus and this is the part that ends up inside the capsid in the assembled virus. And so the mutation is way out here. One of the biggest conformational changes in this is actually in this spike domain. You can see this helix actually gets bent. So that right there tells us something interesting about this protein. It tells us that you make a mutation out here. The big change is in the spike way over here. And this fits the functionality that this protein is really functionally designed and it's an allosteric protein. So things that happen at a far really propagate through the system. So if you take this mutant form of the protein and you run it through sides of the exclusion chromatography what you see is Y132A looks bigger than the wild type protein. And we believe that's because those C-termoral arms near the mutation the wings are kind of flapping. And so it has a bigger hydrodynamic radius than the wild type protein. So now what if we probe it biophysically a little bit? So if we do a thermal denaturation experiment shown here on the top what we see is that Y132A actually is less stable by about 12 degrees than the wild type protein. So again this is kind of interesting because that mutation was way out on the ends. You wouldn't think that would really have a direct impact on the hydrophobic core of this protein which is going to really be important for the stability yet it clearly does. Well how about chemical denaturation? So if we use urea what we see is that Y132A also denatures at almost a full molar unit less of urea than the wild type protein. So it's both thermal and chemical stability is lower. So now what about thinking about the dynamic properties? So one of the ways we look at the dynamic properties of a protein is using hydrogen deuterium exchange mass spectrometry. This is just a quick overview. So if we take our protein and it's going to be all the side chains and the protein backbone will be bound with hydrogens. If we drop that into D2O and allow it to sit for a while then hydrogens that essentially are exposed on the surface or are not involved in a stable hydrogen bond are going to exchange with the water. So these are your amide protons. These are your side chain protons. So then if we quench that then we have an intact protein that has some hydrogens and some deuterium. So we can either look at the intact protein using mass spec or we can digest it with an enzyme cut it up into pieces and look at the pieces and localize exactly where that exchange is occurring. So if we do this with our dimeric hepatitis B capsid protein what we actually see is that Y132A has less exchange. So what we're looking at here is over time we're monitoring the increase in deuterium uptake in these proteins and we can see that actually the mutant, I mean I'm sorry I'm pointing at the wild type CP149 has more exchange the mutant has less exchange. So this tells us the mutant actually seems to have a more stable hydrogen bonding network than the wild type protein yet we just saw that under conditions of heat and chemical stress it actually comes apart more easily. So it seems to be a little difference here depending on how you probe this system. Well that was a global look. So what if we look at the individual peptides. So I'm just showing you what the data actually looks like so we cut the protein into little peptides and then if we monitor over time so here is time zero you're this is a single peptide and we're looking at the C13 isotope ratio. As we add deuterium we shift to higher M over Z values mass to charge values as we pick up deuterium and so essentially we get an uptake curve. So we can look at these uptake curves and if we know which peptide we have we can essentially map those uptakes and so this is just showing you each of the this is the our CP149 protein and these are all the peptides that we have to do this mapping and so by combining all of this data the uptake curves from each of those we essentially can get near single amino acid resolution as far as our exchange and this is just showing you all of the peptides we have to go through this one little helix and what you can see is that depending on the you know this specific peptide we can see very different levels of exchange and so this allows us then to pinpoint exactly where that exchange is happening. So now we can do this between our wild type and mutant protein and we can say okay we know that overall the Y132a appeared more stable but where is it more stable or you know is it everywhere or is it just in places and so here's our dimer again so the little stars show you where the mutation is this is where that conversion from tyrosine to alanine is well we can see blue here so blue means increased exchange in the mutant so less stable hydrogen bonding so this region the end is less stable just as we expected those wings are kind of flapping that's what our size exclusion data told us but if we look kind of into the hydrophobic core of this protein it's actually more stable and then in the spike region we see one of the helixes in each of the dimers and each of the monomers actually shows significantly increased exchange and so this is really interesting because again we make a mutation here and what we see is essentially we see an effect here but we see bigger effects essentially distal from that mutation this protein is really allosterically tuned and being regulated by this and so I think this really serves to show us kind of how complicated this system is now that data I showed you was at one time point that was one hour but we have a whole trajectory of time points so if we start thinking about well let's look at different time points because those tell us about events that happen quickly or slowly which can tell us about dynamics and stability so this plot is a little complicated but what we're looking at is essentially our amino acid sequence and we're looking at our percent change of exchange so whether there's more solvent accessibility in the wild type or more solvent accessibility in the mutant and then we're looking at a half minute two minute ten minute and sixty and so what we can see is that the wild type actually is much more dynamic in the early time points but as time goes along they get closer together except for certain regions so here's that spike region which we saw in the wild type in the mutant was really dynamic and then here's that C-terminal region where again it becomes more dynamic whereas the hydrophobic core of the protein is less dynamic and so if we think of exchange as just occurring as a two state model then we can use this information to essentially get out some proxy free energy information but what we're really looking at is our delta delta G so our change in the change in free energy for these mutations and so we can look at this on the fast time scale and see that our mutant rather has lower free energy but when we get closer to sixty minutes what we're seeing is a little bit different situation with this change in the change in free energy where our wild type protein actually now has regions that have favorable delta G energies and then if we just look overall we see that the Y132A does have more favorable delta delta Gs and we knew this because our original overall data told us this was more stable but our dynamics data says this is more stable but as you remember our chemical and thermal denaturation said it was not more stable that that form of the protein actually denatured more easily so Y132A seems to have a globally stabilized hydrogen bonding network and the impact of this mutation seems to stabilize the hydrophobic core while increasing dynamics in other regions of the protein so I mean this sets us up to I think understand that this is not a simple process going from unassembled to assembled proteins and lots of things regulate this but this is also critical because these same sort of allosteric regulators are involved with taking the virus from here to allowing us to get to here okay so now I want to shift to from looking at the individual subunits but how do we get to a particle that's an infectious system and so I want to look at we're going to use P22 so this is a bacteria phase and we've heard about this system already so this is a T7 bacteria phase and this is a very nice structure from Gabe Lander and this is a really good model for understanding the maturation process and a lot of work has been done by John King, Peter Privilege, Trevor Douglas and Carol Teschke on looking at this system during maturation it's also used for nanomaterials as well okay so just a quick little background of P22 assembly so this virus assembles from a set of coat proteins it has a portal protein that goes in so the part that Peter was just talking about actually is the ejection portal but this is involved with packaging the DNA as well and it has a scaffold protein so then the scaffold protein gets ejected once the DNA starts getting pumped into this system and then there's an expansion that occurs and you go from a spherical particle, a procapsid to an icosahedral capsid our hypothesis when entering this project we think that this particle maturation and adoption of icosahedral symmetry are really going to increase the stability of these particles and essentially decrease protein dynamics and this maturation process proceeds through a series of potentially local energy minima or that each of these steps from procapsid to capsid essentially could be thought of as a metastable confirmation and so of course we're going to do this in vitro to keep with our idea we got to keep things simple there and so you can take a procapsid so this is an empty procapsid and you can treat it with half molar guanidine HCl for example and you can remove the scaffold protein because this particle will not assemble without the scaffold alternatively and then it's possible to take the empty shell and move to our expanded shell which has the icosahedral structure or you can go directly from the procapsid through heating and get your expanded shell and if you heat even further from your expanded shell you get this interesting particle that essentially has blown out the pentamers you get what's called the wiffle ball form of this particle and so a lot of nanomaterials work has been done with this wiffle ball form where you've lost your pentamers and so here's a little diagram showing you the procapsid empty shell expanded shell and wiffle ball and so this will be on all the slides so you can kind of yes well in vitro we can do this with heat heat also also from here and from here so this is what's really cool and I'll show you on the next slide that's a great point because it really tells us that each of these are thermodynamically distinct and independent and you can actually move through them all together so the questions we're going to ask are you on the properties we want to look at are stability and conformational flexibility at each of these different forms and so the first technique we're going to use is differential scanning fluorimetry and so essentially this is a really straightforward technique where we take a small fluorescent die molecule that's quenched in polar environments if it binds to a non-polar environment such as a pocket and a protein as it's unfolding your fluorescence intensity increases and what's really cool about this is you can actually do this in a qPCR machine so you can run these in multiplex so you can do 100 samples at once and it takes about 3 microliters per reaction so it's a great way to do a lot of screening experiments and so if we look at our different forms of p22 using this DSF technique let's start here at the top where we're looking at procapsid so as we increase temperature we're looking at fluorescence intensity and we've just stacked these plots we see a big broad transition then as we get closer to 80 degrees we see a second transition and then at just over 90 degrees we see a third transition for procapsid now if we take particles that are purified that we've already gone through the first step we've already removed the scaffolding protein all of a sudden we lose that first transition we see the second and the third and they're just the same as in the procapsid form so the second and third transitions haven't changed if we go to the expanded form so our icosahedral version we don't see that first transition we still see the second and third transitions now if we go to the wiffle ball we've lost the pentamers we take purified wiffle ball we now don't have the first or the second transition we only have our third transition so then this allows us to essentially assign what are those transitions we're looking at and to put them into the scheme and so transition one then is really that's associated with the release of our scaffold essentially we're watching the scaffold come out and this is a really broad event as you can see transition two is pentamer release and transition three essentially this is when our virus completely deforms so then this table just reconfirms this what this really tells us is we have big differences in the thermal stability of each of these forms of the particle as we move through this maturation at least through the expanded shell we see increasing TMs and the wiffle ball actually has the highest TM that kind of suggests that when you pop out those pentamers you essentially improve your thermal stability with that which fits with some of what we've heard about the pentamers being defects in these particles so now let's look at that was at pH 7 well what's the effect of pH and we can just look really quickly at this because if we concentrate ignore the black trace so this is procapsid at pH 4 to 9 essentially there's no change pH has no effect as long as it's between 4 to 9 if you drop it down to pH 2.8 you see that the transition start to change so very low pH is affect this empty shell we see the same thing pH 4 to 9 has no change and the same with the other form so really this particle is independent of pH why did we do this experiment? well for those of you who study viruses in cells we know that with eukaryotic systems a lot of viruses are very pH sensitive because they enter the cell through endocytosis this is a bacteria phase it doesn't do that right it doesn't have that process it injects its nucleic acid into the cell but we needed to check that so here's our conclusions from thermal stability really pH independent and each we have these very distinct transitions between each form what about chemical stability so now let's look at guanidinium hydrochloride and so in each of these plots we're looking at so this is at a half molar guanidinium hydrochloride and we're looking at procapsid and we see essentially the procapsid trace we now see transition 2 and 3 have changed so even at a half molar guanidinium hydrochloride procapsid has changed ES has also changed but the expanded shell and the wiffle ball have not so the expanded shell and the wiffle ball are more stable to chemical denaturation so once we've made this transition from a procapsid a spherical structure to adopting our icosahedral geometries the capsid is more stable and we just do this with increasing concentrations just to show you so at one molar our wiffle ball is still fine but now our expanded capsid is starting to show a little change and the procapsid and the empty shell are definitely changing so this again helps us look at the stability of these and how the pronounced destabilization of the spherical forms and less of the expanded and the wiffle ball now this process of maturation we wanted to understand if the role of protein folding how are there changes in secondary structure so we use circular dichroism which allows us to look for changes in secondary structure and really the story is we can detect no changes in secondary structure so essentially this is our heating isotherms of procapsid, empty shell expanded and wiffle ball and what you can see is from 20 to 85 degrees essentially are all right here we see no change in our CD signal but essentially when we get to 90, 95 all of these spectra change so what that tells us essentially is that remember that's our transition where our wiffle ball thermally denatures so essentially there's no change up until you get to the wiffle ball this thing denatures and just precipitates so we completely lose our CD signal so secondary structural changes and folding is not really involved in this process okay structural rigidity how am I doing on time okay so five minutes to finish we can have questions structural rigidity well we're going to look at AFM and we've had some nice introductions to AFM so I will skip over that but I just want to show you that actually this is work that we did in collaboration with Pedro and one of his talented students Aida where we did imaging they imaged the different forms and actually then we did indentation experiments but the first was just to look at the particle height just to confirm that the AFM was telling us what we expected and it was we see the expanded shell being tallest and the wiffle ball appears lower because it's missing these pentamers so on average it's going to get a little bit lower on the mica but they did force indentation curves as we heard Pedro describe the other day in vooder that where we're looking essentially at our displacement and force and then we can use those to essentially determine spring constants for these particles and see that the expanded and the wiffle ball have a much higher spring constant so these are much essentially more rigid particles if we look at critical strain so essentially what sort of deformation can these particles withstand what we see is that actually the expanded shell is the most brittle and the wiffle ball now is actually appears to be less brittle that once we lost those pentamers the wiffle ball actually becomes a little bit more deformable before it fails the last technique I'm going to bring in is our quartz crystal micro balance so this is essentially a technique where it allows us to essentially absorb a material on a surface and we can measure the amount of material but we can also essentially so this is a vibrating quartz crystal we're using the piezoelectric effect and we turn off the potential and we essentially measure how fast it decays so very rigid material are very coupled to the crystal and so they dissipate very slowly whereas a soft material is uncoupled and so you see a really larger dissipation and so we can look at this f and d ratio and say something about the rigidity of a material and so when we essentially bind our virus to our quartz crystal we see a decrease in frequency this tells us about the mass we're depositing and then we see an increase in dissipation which tells us about the essentially something about the rigidity or the modulus of our material and so if we do these measurements we see that from PC to ES there's not really a change but once we get to our expanded shell and wiffle ball there is an increase in rigidity in these particles and we see that the wiffle ball now appears to be more rigid when we're looking at this global method compared with AFM when we were looking at more of a point source on this ok and so we've done HD exchange on these and what we can essentially see is our intact protein HD exchange shows that we get less exchange as these particles maturation occurs ok and that now one other quick analysis is we wanted to look at one of the structural changes involved and so it turns out there are a number of cryoem structures of the p22 capsid looking at procapsid and expanded shell in one case from christen parent's lab and procapsid and wiffle ball from wachu's lab and so we use these to essentially see if we could look at the difference in the solvent excluded surface area in each of these cases to get an idea of how much more protein-protein interaction there potentially is in these cases and what we really were able to see to make a complicated story is that we see a lot of increase in interaction at the quasi interfaces so these interfaces that are specific to the formation of an icosahedron are important for that's where we're seeing a lot more protein-protein interactions with this and overall there was a small overall increase in the effective core and so then you know really putting all of these different techniques together and trying to say well what do we learn well one maturation involves the formation of these quasi equivalent sub unit interactions scaffolding protein interestingly it does not impact the chemical or thermal stability of these particles or the AFM whether scaffold protein is there or not we can't tell by these techniques which I find very interesting with that icosahedral interactions resulted in the increased stability of EX and wiffle ball and the loss of these penton leads to a less brittle and more chemically stable particle and so then this is just our final trying to put this all together back to where I started our talk into an energy landscape and thinking about we have these distinct phases of maturation or forms of the particle and each of these you know preliminary are a metastable state down to our wiffle ball and remember this really isn't biologically important but also we can start to think about something about the width of these wells which is partially based on how dynamic or the rigidity of these particles something that's less rigid is going to have more conformational freedoms you're going to get a wider essentially energy well unless we've tried to show is EX which is very rigid we have a narrow energy well whereas wiffle ball and ES are more dynamic and so they have essentially more conformational freedom and what we don't know about is yet is really how high are these activation barriers between these different metastable states so and I'll just end here with this is we've looked at a whole library of particles using this quartz crystal micro balance technique and what we're looking at here is our rigidity our frequency dissipation ratio we're seeing our different T numbers of all the particles that we've looked at and how this relates to on this axis we're looking at diameter and so this is one of these giant algal viruses shown here pbcv1 and so what we can see essentially is if we graph this and well it's not our not a perfect fit we're seeing at least in this one measure of global stability or of these particles or rigidity we're seeing you know some sort of semi exponential fit here to try to tie it all back together to getting the simple answer and so with that I'd like to thank people in my group who help with this project Naveed, Ravi, Vomsi who you heard earlier from please a group and Angela have done most of this work my collaborators specifically Adam, Trevor, Pedro and Mavis and oh there's Ravi and with that I would be happy to take questions