Dr. Lauren Porter — Molecular “Transformers:” Switching Form and Function

>> Diego (narration): One of the things I love most about science is that it’s always changing. The theories that we use to understand ourselves and the world around us, although they’re thoroughly tested and supported by mountains of evidence before they’re widely accepted, are just that—theories, reliable but maybe more importantly, receptive to change.

Modern science doesn’t claim to understand everything about everything, nor does it shy away from discoveries that challenge that status quo. Sometimes new information comes along that outright disproves some theories – RIP flat-earth theory – but most of the time it refines them and gives them nuance to better explain how things work. That usually starts when scientists encounter exceptions to the established rule. And it’s currently case with Dr. Lauren Porter who is helping redefine the way we understand how proteins behave.

>> Dr. Porter: For a long time, people have thought that proteins, when they fold, assume only one stable structure that performs a specific function.

>> Diego (narration): All proteins are made of amino acids strung together in a specific order coded for in our DNA. Many biochemistry books depict proteins like a string of pearls, because just as pearls are the building blocks of a necklace, amino acids serve as the building blocks for proteins.

The sequence of amino acids is unique to each protein and dictates how the string will arrange itself in three-dimensional space. The resulting structure, or conformation, is what then gives a protein its function inside the cell. Each protein normally folds into a shape that will make it the most thermodynamically stable.

>> Dr. Porter: There's kind of one very deep energy minimum and nothing else anywhere close.

>> Diego (narration): Which means most proteins only adopt one conformation. Well, that is, until they don’t.

>> Dr. Porter: It turns out that there is this emerging class of proteins called fold-switching proteins, which can actually change their folds and their functions in response to cellular stimuli.

>> Diego (narration): This would be as if your kitchen blender [sound of blender starts and stops] could all of the sudden turn into a toaster [sound of toast popping out of a toaster] with the flip of a switch [click of light switch].

>> Dr. Porter: The first time I saw that, I was like, “Whoa! There is some stuff we do not understand about proteins, because based on everything I’ve learned that should not be possible.”

>> Diego (narration): Fold-switching proteins are reminiscent of Transformers, you know, those huge alien robots from comic books and movies that can change from one machine into another at the drop of a dime.

[Clip from “Transfomers” movie: My name is Optimus Prime. We are autonomous robotic organisms from the planet Cybertron.]

>> Dr. Porter: The analogy with Optimus Prime is good, because in one case, he's a robot, and in another case, he's a car, and you would never know that the robot could become a car, or vice versa, just by looking at it. So, some of these proteins really can shift completely, and you would never know it just by looking at it.

>> Diego (interview): How different are these changes? I mean are we talking small differences like a car to like a truck or something more stark like a car to a helicopter?

>> Dr. Porter: We’re talking like a car to an elephant. It’s a completely different change.

>> Diego (narration): What’s even more astonishing is that sometimes the switch is reversible. In other words, the elephant can turn back into a car.

Of course, sometimes the changes aren’t as dramatic, but understanding how even the smallest switch occurs could help scientists understand the molecular basis of certain diseases like cancer, autoimmune disorders, and bacterial and viral infections.

For this episode, I talked with Dr. Porter about how she studies these molecular “transformers,” how they’re reshaping our classical understanding of proteins, and the potential they hold for transforming human health.

>> Diego (interview): Since fold-switching proteins can change how they look and what they do, I imagine it must be really difficult to pin them down in order to study them— it really seems like hitting a moving target. (pause) without getting too into the nitty-gritty how do you go about finding these guys?

>> Dr. Porter: Yeah, so right now, my work focuses on like the biggest changes, so things that really do change from like cars to elephants, you know, something that's just a very, very drastic change. When people think about proteins, the first thing they think of is the primary structure, which is the amino acid sequence of the protein. Then in the 1950s, Linus Pauling showed that there were kind of two local, repeating structures, that proteins could stably assume. These repetitive structures are alpha helices, which are shaped kind of like springs, and then also beta pleated sheets, which – they're depicted as kind of like flat arrows, and they often bond to other parts of the protein that are a little distant from them.

>>Diego (interview): Right, so the amino acids are the individual links in the chain that make up the protein—that’s the primary structure. And then the secondary structure is how parts of that chain arrange themselves, so either like coils if it’s alpha helices or flat strips if it’s beta sheets. And these characteristic secondary structures are important because they give proteins their shape but especially stability so that the chain isn’t just kind of wiggling around randomly.

>> Dr. Porter: Yeah, and what my lab is looking for right now are examples of proteins that switch from alpha helix to beta sheet, because those are like the most drastic changes one can imagine, and so they are probably the easiest to find. Right now we use machine learning-based methods that classify secondary structures of proteins, and what we look for are when we change things about the sequence, like we shorten it, or something like that, does the secondary structure prediction change or does it stay the same? And then when we see substantial changes to secondary structure by altering the context of the sequence, that to us is a strong indicator that this protein might switch folds.

>> Diego (interview): And what triggers those folds to switch from one state to the another? Is it something in the environment?

>> Dr. Porter: So, it depends. Certain fold switching proteins actually sample both confirmations at the same time, but there are other proteins that do need to be triggered, and there are a number of different triggers; so changes in pH, binding a protein, changes in redox potential, any of those things and many more things can trigger the fold switching.

>> Diego (interview): Is it usually a physical slash chemical trigger or can it happen spontaneously?

>> Dr. Porter: Again, like there are different mechanisms. So, some of them really need a trigger, but there is a protein called KaiB, which helps regulate the circadian clock of cyanobacteria, and it actually will spontaneously switch between one-fold and another. And the key to this protein's function is that it switches very, very slowly. So, Andy LiWang's lab has shown that if they speed up the rate of fold switching, it changes the periodicity of the clock. And getting rid of the fold switching abolishes the clock completely. And so sometimes the rate actually limits a reaction, and that's an integral part of its functions.

>> Diego (interview): Fascinating. So, there are proteins that can switch with just light?

>> Dr. Porter: Yes, so for example, bacterial phytochromes sense light in bacteria. They have this region called the tongue, which in one case is a beta sheet, so it's just one beta sheet, but when it's exposed to light, the tongue refolds into an alpha helix, and that brings the two domains of the phytochrome together, so that's in its light-activated form. And so those domains maintain their fold, but their orientation and like distance is regulated by the small fold switch that responds to light.

>> Diego (interview): Hmm, a literal light switch. Well, how pervasive would you say fold-switch proteins are? Or are they very much still the exception to the rule?

>> Dr. Porter: Personally, I think that fold switching proteins are going to be the exception to the rule, and yet, we've recently submitted a paper for publication – it's under review – where we've shown that in a universally conserved family of transcription factors, 25% of the proteins look like they switch folds and that's a much larger number than we expected, or anybody expected previously. It's also important to say that, right now, if you look at like the database of non-redundant protein sequences that are available, there are about 220 million of them. And in terms of protein structures that have been solved, there are only 58,000. So, 58,000 is still an enormous amount of work, but compared to 220 million, it's really hard to know how much fold switching is out there and where it's occurring.

So, from everything we can see, fold switching is a rarity, but it's also true that fold switching proteins often resist structural characterization, as you can imagine, if they assume two different folds. Or sometimes it could be that solving the structure of a protein will give one fold, and there may be another fold accessible that just nobody has found. So, I think that the single folding paradigm where proteins assume one fold with like a low energy, is the dominant state, but fold switching could be a considerable subset of the remaining number of proteins out there, and how much remains to be seen. Hopefully, we'll know more as the years go on.

>> Diego (interview): Got it. Well now I’m wondering, are there proteins out there that take on, you know, more than two forms?

>> Dr. Porter: Well, for sure, there are proteins called intrinsically disordered proteins, which are different than fold switching proteins, because they're just naturally unstable, and then their binding partners will determine their confirmations. Whereas for fold switching proteins, they have stable structures on their own, so they're different. I don't know yet of any proteins that can switch between multiple – like more than two different folds. That doesn't mean they don't exist. It just means we don't know about them yet.

>> Diego (interview): So, I guess we’ll have to see if the elephant turns into something else.

Well, once you’ve identified a potential fold switching protein using your computational methods, I understand you validate those predictions in the lab. So, does that mean you physically produce and isolate the protein in its two separate forms?

>> Dr. Porter: Yes. Well, sometimes we try to be smart, and at least start with like proteins where we can actually assess fold switching with just one form. And it's possible to do that, because often their dominant ground state differs from the dominant ground state of other members of their families that don't switch folds. So, we can leverage that in the lab and use comparisons and like lower resolution approaches at the beginning. And then as time goes on, yes, we can use nuclear magnetic resonance to get information about the chemical environments of the protein under different sets of conditions. And that gives us a pretty good sense of what's going on. And as time has been going on now, we're trying to move in the direction of actually getting, you know, crystal structures or NMR structures to give very firm validation for what we're seeing.

>> Diego (interview): Yeah. Well, I have to tell you, I spent quite a bit of time in my undergrad in an X-ray crystallography lab trying to find the structure of a very specific protein domain, and unfortunately, it was to no avail, so I can really appreciate how laborious and expensive it is to work with proteins. They're very finicky little things, and each one is so unique. You know, it's funny that we used to think that once the structure was solved, the protein could kind of be, you know, like, crossed off the list, but fold switching-proteins really add a whole layer of complexity. It kind of feels like epigenetics to genes or post-transcriptional modifications to RNA.

>> Dr. Porter: Oh, it's hard for me to say that about my own work.

>> Diego: Yeah. No, but still it’s changing the way we think about something that has been thought to be fully understood for so long.

>> Dr. Porter: For sure. I’m definitely comfortable saying that fold switching proteins show that there's some stuff we don't understand, and it's important to understand it, especially because of how relevant towards integral biological processes. We recently wrote a paper basically showing that fold switching seems like a specific mechanism of regulation, and being able to respond to the environment quickly is a much more effective way of regulation than, for example, conditions change, and now a whole new protein needs to be expressed. The timescale on just switching a fold is a lot faster than on transcribing and translating a new protein.

>> Diego (interview): Yeah, definitely and that leads pretty perfectly into my next question, because it seems like it's a very streamlined strategy for nature to adopt. I mean, like you said, a protein can take on double duty, it saves the cell from expending, you know, energy or resources to make two specialized proteins. So, in terms of evolution, this ability to switch, doesn't seem like an accident or an artifact. It provides a benefit, so therefore, it’s been conserved, right?

>> Dr. Porter: Well, we have definitely, at this point, pretty solid data showing that yes, it is conserved, and our recent work showed that if you do something called coevolutionary analysis where you look at pairs of amino acids that are in contact with each other, if you do this for a single folding protein, you'll get pairs from one fold, showing that the amino acids to amino acids evolved together to maintain stabilizing contacts for one fold. What's really cool that we've recently found is that for fold switching proteins, you actually see pairs of amino acids evolving to maintain stabilizing contacts for two folds, so that seems like pretty strong evidence of what you're saying, that yes, both folds are conserved evolutionarily.

>> Diego (interview): Interesting. Well, kind of switching back from the past to the present: beyond understanding the dynamics of these fold-switching proteins, what is the goal when it comes to implications in human health? I know they're kind of integral in some diseases.

>> Dr. Porter: Yeah, so I think the first thing is we got to understand the biophysics of these proteins, like the dynamics and how they work. If we can get a better handle on that, my hope is down the road, therapeutics could be developed that target fold switching proteins and force them to stay in one conformation or favor one conformation. So, like, for example, the protein we study called RfaH, regulates the expression of virulence genes in proteins like E. coli. On a basic level, what that means is RfaH plays an integral role of giving people food poisoning. And so if it were possible to force RfaH to not be able to switch between its two folds, that could dramatically reduce the amount of virulence proteins that bacteria could produce.

>> Diego (interview): And effectively lower the likelihood of getting food poisoning.

>> Dr. Porter: Yeah. So, it may be possible down the road to produce drugs that target these. On the flip side, a kind of tantalizing study in the Journal of Molecular Biology came out a couple years ago showing that an amino acid change associated with a certain form of human cancer switched a secondary structure element in a human protein from an alpha helix to a beta sheet. And so it could be possible perhaps to design some sort of therapeutic that targets that protein, you know, that has that amino acid change, so that maybe it could be restored to its native form, and then maybe that would help, you know, decrease the number of cancer cells that could grow or something. So, I think those are kind of the two ways that I imagine, you know, decades down the road, this research being useful in the clinic, and I hope to see that in my lifetime.

>> Diego (interview): Well, is it possible, do you think, to maybe in the future, design, you know, fold switching proteins themselves and use those in biomedicine and, you know, other applications?

>> Dr. Porter: It's possible. I've toyed around with the idea of using them as sensors within cells, or perhaps to regulate transcription in response to some sort of environmental trigger. And my lab is interested in engineering fold switching proteins for those kind of applications down the road, so maybe that's in the future.

>> Diego (interview): Yeah. Well, that’s what I think is so great about basic science, yes, there’s the thrill of discovering something new and the curiosity to really understand it at a fundamental level, but I think it also feeds into like the scientific imagination, you know what I mean. It kind of opens the mind to a new world of possibilities, especially ones that can make an impact on a lot of people’s lives.

>> Dr. Porter: Absolutely. Yeah. And, and that's – I don't think I could have pursued this, without knowing that this really could make an impact downstream. When I was a junior in college, my dad got diagnosed with stage four cancer, and seeing how much he had to suffer to survive – like he had to have, you know, multiple rounds of chemo, he had a bone marrow transplant, and it was really, really hard for him and for us as a family watch. And so, I remember thinking like, well, you know, I would love to do something so that, you know, down the road, someone like me doesn't have to watch, you know, their parent suffer and their parent doesn't have to though the suffering, and so that really helps me stay motivated.

>> Diego (interview): That’s very admirable. And again, it speaks to the power of basic science in making an impact on a large scale. Well before I let you go, where do you see the field of fold-switching proteins headed? What’s are your plans for your lab?

>> Dr. Porter: Yeah. So right now, my lab has developed sort of binary classifiers to be able to predict fold switching from genomic sequences. We are currently developing methods now that can show not only that a protein switches folds, but what folds it assumes. So that is a big jump, and what we'd like to do with that, is run it on entire genomes and predict which proteins in this genome are switching folds. And then functionally, why would that be important, and then look at that in the lab. So that's a big question I would like to address. Like in the next five years, I would love to pull a sequence out of a genome, having said it switched folds and show experimentally that it does.

I think in terms of the field in general, we really need better methods, especially experimental methods for assaying fold switching, because with crystallography, for example, it kind of locks the protein into one conformation, usually, and so you can't see what else the protein could be doing. It would be great to develop high-throughput approaches to screen proteins under many different conditions to look for fold switching. So our lab has been talking about that, potentially down the road using mass spec to look for fold switching, but I think that's something other labs could also work on, and a lot of proteins or fold switching proteins are kind of stumbled upon by chance, so I'm hoping that, you know, our methods will help elucidate them more quickly, but I imagine other labs will continue to stumble upon them, and that learning more about those specific examples will also help to advance the field.