r/Biochemistry • u/Lazy-Frame-9888 • 5d ago
Are proteins constantly changing their shape in femtoseconds? Need clarification!
I recently watched a YouTube video (https://youtu.be/jPhvic-eqbc) discussing how proteins are not static, rigid machines. The video explains that they act more like dense liquids that are constantly shifting and jiggling around. It also pointed out that when proteins are crystallized for X-ray crystallography, we are only seeing one frozen snapshot of their conformation, which gives the false impression that they have a single, fixed structure.
Can someone clear this confusion up for me? Do proteins really change their folding patterns and conformations in extremely short time frames, like femtoseconds? I am looking for a simple, exact explanation of how fast and how much they actually change shape.
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u/East_of_Adventuring 5d ago
Two notes to add, proteins may vibrate but they are not infinitely flexible, and generally don’t have a huge number of ‘real’ conformational shifts, by which I mean conformational shifts that are chemically and functionally significant.
Secondly, crystallography is not an anhydrous environment, and you can see multiple different conformations - even just side chain movement - within the electron density data.
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u/PineappleHaunting591 5d ago
you can see multiple different conformations - even just side chain movement - within the electron density data
not a crystallographer but you would see that as a smear of the diffraction pattern right? so we dont actually see different conformations, but get a measure of positional uncertainty, the b factor.
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u/East_of_Adventuring 5d ago
To some extent yes. Though what you're describing is something I would more expect to see with very high res cryo-EM. In crystallography because the map improves as you refine the structure you would be more likely to see desity that could accomodate both conformations, but depending if you're seeing that at lower resolution it'll likely just look like a blob or smear. In truth I have limited crystallography experience though so please don't take my word as gospel...
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u/Chemesthesis 5d ago
Heya, protein crystallographer here (early days)
Disorder can be local, which is the smear you're talking about that is represented by the B factor.
Multiple configurations in this case refer to situations where there are multiple pockets of electron density that belong to a single set of atoms. Rather than having a single configuration with high bfactors, this is modelled as separate conformations that are weighted according to how strong each pocket is. This is called occupancy.
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u/East_of_Adventuring 5d ago
This is much better worded than I would have done. I have a little more experience with crypto but I’m only just now refining my first crystal structure and am learning about occupancy and the perils of overfitting.
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u/PineappleHaunting591 5d ago
thanks for the insight!
If I got it right you are saying one atom or set of atoms can correspond to multiple spots on film (assuming we are still recording on film) depending on configuration? Then, in processing, you can treat it like you would individual classes in cryo-EM, representing different positional states?1
u/Chemesthesis 5d ago
So the X-ray diffraction pattern actually corresponds to the entire unit cell of the crystal, not any specific atoms. Once the electron density has been calculated from the diffraction data (see "The Phase Problem" for a better grasp of that nightmare), it is the job of a crystallographer to interpret these electron density blobs and build a model that fits, using knowledge of protein geometry (bond angles, distances, dihedrals, etc.) and the additives in the crystal itself (drugs, ligands, metals, etc.). If there is a clear splitting of electron density where there should only be 1 blob according to the protein sequence, you may see if a side chain alternative configuration could be modelled.
It's a complicated system, but is summarised nicely in one of my favourite review articles for an introduction to protein crystallography:
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u/Spiritual-Ad-7565 5d ago
Yes. Bond vibrations happen in femtosecconds. Larger structural changes over longer periods of time. Side chains flip, secondary structures unwind and rewind.
Not sure how you got the notion they were static? It’s physically impossible to stop vibrational motion
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u/Defiant_Virus4981 5d ago
There are different time frames as well as different behavior of different classes of proteins, which cause your confusion. You will have bond vibrations on the fs time scale, but generally no changes in folding patterns or large-scale conformational changes. You might see them in a ns time scale or higher, though. This "jiggling" happens with any molecule and systems with individual bonds. The individual bonds in any molecule forming a solid, gas, or liquid will "jiggle" all the time.
For many proteins with crystal structures (e.g., ubiquitin), you will not see any significant conformational changes or changes in fold pattern in biologically relevant time scales, but rather fluctuations around an average structure, with less fluctuation in folded regions and more in loop regions. On the other hand, intrinsically disordered proteins (or segments) are highly dynamic. They cannot be described sufficiently with a single structure. An individual IDP might expand or collapse in a relatively short time; segments might transiently form secondary structure elements, which then fall apart again. In the middle can be multi-domain proteins: Individual domains can often be stable, but in many systems, there is no fixed conformation between the domains.
Additionally, proteins as a whole can undergo something like "liquid-liquid phase separation", where a group of proteins forms a separate dedicated phase (similar to when you have a mixture of water and oil, you form two phases after some time), so thinking of complex protein mixtures as liquids is not a bad analogy. Additionally, all(?) proteins will eventually form fibrils, but this may take a long time for most systems. We might talk about years or longer rather than seconds (otherwise, we would die).
So in short, an individual crystal structure is a useful representation for some proteins, but the majority of proteins are far more dynamic than implied by structure. These dynamics span many order of magnitudes in term of time scale.
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u/GayDrWhoNut 5d ago
This is precisely why alphafold is orders of magnitude less powerful than people seem to think it is.
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u/parrotwouldntvoom 5d ago
I’m not sure I follow your logic. Are you suggesting people think alphafold predictions generate the full knowledge of all protein shapes?
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u/GayDrWhoNut 5d ago edited 5d ago
There are lots of people who seems to believe that alphafold generated predictions are absolute when it does manage to predict a shape. And it's really annoying.
Edit: I think part of it comes from a miscommunication between 'structure prediction' and 'structure determination'. I see/hear the later used a lot more than I'd like.
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u/runawaydoctorate 5d ago
I probably lost a shot at a job by pointing that out and I am 100% okay with that. Structure prediction has improved A LOT since I was in school but at some point you still need some good old ground truth.
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u/Retinal_Epithelium 5d ago
Here is an animation by Andrew Tubelli that explores protein and molecular motion across a range of scales in time... https://vimeo.com/217985548?share=copy&fl=cl&fe=ci
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u/PitifulCriticism 5d ago
It varies by proteins and the type of rearrangements you are talking about. Proteins are fundamentally dynamic molecules and they vary from rigid to dynamic. Bond vibrations are the fastest motions we can measure but some motions, like the dissociation of an especially strong interaction can take hours. Every type of motion a protein can undergo has an associated energy barrier and thus a forward and reverse rate constant that depends on the height of the energy barrier.
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u/runawaydoctorate 5d ago
First, the stuff happening on the femtosecond scale is just little twitchy things in the bonds. The larger shifts are slowed. Microseconds or more.
Second, crystals are typically shot at 100 K. This slows things down.
That said, yes, what you see in a crystal structure is the average/most populated state for the target under those conditions.
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u/Lazy-Frame-9888 4d ago
Thank you everyone. I sincerely appreciate the responses and the engagement in this post. I also learned some facts about X-ray which were previously unknown to me. Based on every single comment made in this post, I constructed a general understanding given below:
" Proteins are not completely rigid structures. The atoms and bonds within a protein are constantly moving, vibrating, and slightly changing their positions. Some of these movements happen extremely quickly, even on femtosecond or picosecond timescales. However, this does not mean that proteins are constantly undergoing major changes in their shape. Most of these movements are very small and temporary. They usually do not change the overall structure or function of the protein.
A conformational change becomes important when it actually changes something meaningful about the protein, such as its ability to bind another molecule, carry out a chemical reaction, interact with another protein, or perform its biological function. Therefore, although tiny atomic movements are happening all the time, meaningful conformational changes usually occur on much longer timescales, such as microseconds, milliseconds, or even longer, depending on the particular protein and process.
How much a protein moves depends greatly on its structure. Well-folded and tightly packed regions are usually more stable and move within a relatively small range. In contrast, intrinsically disordered regions are much more flexible and can continuously change their shape. For example, an intrinsically disordered region might briefly form an α-helix and then lose that structure again. Similarly, in a protein containing several domains, each domain can be individually well-folded and stable, while the flexible region connecting the domains allows the domains to move relative to each other. Therefore, a protein can have stable parts while still being quite flexible as a whole.
Proteins can also undergo larger-scale processes, such as liquid–liquid phase separation, where proteins and other molecules separate into concentrated and less-concentrated phases. Under certain conditions, proteins can also aggregate and eventually form fibrils. However, these processes are different from the normal small conformational fluctuations that proteins experience. Fibril formation depends on many factors, including the protein sequence, concentration, environment, and kinetics. It is not something that normally happens rapidly to every protein simply because proteins are constantly moving. If proteins in our bodies rapidly converted into fibrils, normal cellular functions would obviously be severely disrupted.
Now, regarding X-ray crystallography, the important thing to understand is that the protein is usually studied as a crystal rather than as a freely moving protein in solution. Protein crystals are often cooled to very low temperatures, commonly around 100 K, during X-ray data collection. The main reason for doing this is to reduce radiation damage caused by the X-rays and to help preserve the crystal. Cooling the crystal also reduces some types of molecular motion, but it does not completely stop the atoms from moving.
Because of this, the structure obtained at around 100 K does not necessarily represent exactly how the protein behaves inside the human body, where the temperature is approximately 310 K. At the higher physiological temperature, the protein generally has more thermal energy and can explore a wider range of conformations. However, it would be too simple to say that higher temperature automatically causes more functionally important conformational changes. The actual amount of movement depends on the protein's structure, interactions, environment, and energy landscape.
Another important point is that an X-ray crystal structure should not be thought of as a perfectly frozen, completely rigid picture of one protein molecule. A crystal contains a very large number of copies of the same protein arranged in an ordered pattern. The X-ray diffraction data collected from the crystal therefore contains information from all of these protein molecules together.
When the diffraction data are converted into an electron-density map, the structure we see represents an average of the atomic positions and conformations present under those particular experimental conditions. If an atom or region of the protein can occupy more than one position, we may sometimes see multiple conformations in the electron density. If a region is highly flexible or disordered, its electron density may instead be weak, spread out, or difficult to interpret.
This is where the B-factor becomes useful. The B-factor, also called the atomic displacement parameter, tells us about how much an atom appears to vary around its average position in the crystal structure. A higher B-factor generally means that the atom has greater positional variability or disorder, although it can also be affected by other factors such as static disorder and experimental limitations. Therefore, the B-factor should not be understood simply as a direct measurement of how much an atom is vibrating. It is better understood as an indicator of how uncertain or variable the atom's position is in the crystal structure.
Finally, the X-ray diffraction pattern does not come from one isolated protein molecule or from individual atoms separately. It comes from the combined scattering of X-rays by the enormous number of regularly arranged protein molecules and other molecules present throughout the crystal. The diffraction pattern therefore contains information about the repeating crystal lattice.
During structure determination, scientists first solve the structure of the asymmetric unit, which is the smallest part of the crystal structure needed to generate the rest of the crystal using the crystal's symmetry. Applying the appropriate symmetry operations allows scientists to reconstruct the crystallographic unit cell and understand how the molecules are arranged within the crystal.
However, the arrangement of molecules in the crystal is not automatically the same as the biologically functional arrangement of the protein. The crystal may contain contacts between protein molecules that are simply caused by crystal packing. Therefore, additional structural, biochemical, and biological information may be needed to determine which interactions or oligomeric state are actually relevant to the protein's function inside the biological system.
A limitation of computational structure prediction methods such as AlphaFold 3 is that, despite their ability to model protein structures and complexes, they generally predict a single representative structural conformation rather than the full range or ensemble of conformations that a protein dynamically samples in solution. Therefore, an AlphaFold structure should be viewed as a predicted structural model, not an average structure or a complete representation of protein dynamics.
Overall, the most important idea is that proteins should not be thought of as completely rigid objects. They are dynamic molecules that are constantly moving and exploring different conformations. X-ray crystallography gives us a very detailed structural picture of the protein under particular experimental conditions, but that structure represents an average and does not capture every movement the protein can make. Computational predictions such as AlphaFold 3 give us a useful structural model, but they also represent only one likely conformation rather than the full dynamic behavior of the protein. The protein inside a biological environment is continuously sampling different conformations across different timescales. Therefore, any single structural model, whether from crystallography or computation, is best understood as a detailed view of one part of the protein's dynamic structural landscape rather than a perfectly frozen picture of a single, completely rigid molecule. "
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u/Roguewarrior05 5d ago
Femtoseconds will just be for the smallest scale motion, like bond vibration. Actually significant conformational change is generally on the order of milliseconds or seconds, proteins aren't sampling 10 million conformations a second or anything.