Nov 272009
 
It surprises me how often I hear students, postdocs, and even professors talk about determining the structure of a protein. A singular structure has the advantage of being relatively easy to interpret, but the cost of this is often the loss of functional data. It’s easy to understand how this terminology emerges from the discipline of crystallography, which after all only works when the protein molecules adopt only a small number of conformations. Yet even when it comes to NMR, a technique that should be very sensitive to the fact of structural multiplicity, the language of researchers and the structural tools available to them are too often oriented towards the idea of a singular structure. But any representation of a protein as a single conformation is a simplification — every protein exists in multiple structural states.

Trivially, we are aware that a given polypeptide chain can adopt a number of different conformations — the “folded state” of any given polypeptide chain covers only a tiny sliver of the possible conformational space. A protein that is “unfolded” occupies not a single, well-defined state but a vast multiplicity of states, and this kind of statement is not controversial because we tend to imagine unfoldedness as a messy chaotic jumble of conformations. The reality is less cut-and-dried: although unfolded proteins may have no regular structure, many still have a propensity to form particular secondary structures or interactions. The reality of denatured proteins is that they have a complex and varied energy landscape, not an array of possible structures that all have roughly equivalent energy. The flipside of the popular view is that the a protein’s native state draws down to a sharp energy well, and this conception is also misguided.

The most dramatic counterexamples to the idea of a neat, punctate energy well come from proteins that adopt several different folds in the native state. One relevant case is lymphotactin, which freely interconverts between an α/β monomer and an all-β dimer under physiological conditions. Lymphotactin may be unusual, but the principal message from that study is one that ought be paid attention to in others, particularly when the protein in question has functional conformational diversity. Consider α-synuclein, a protein implicated in Parkinson’s disease. In the presence of some detergent micelles this protein is known to take on an α-helical hairpin structure, with two helices laying down on the charged surface of the lipid headgroup. In solution, however, it seems to take on a number of different forms, and may interact with true lipid bilayers in a completely different way than it interacts with micelles. For proteins that interconvert between several different physiologically-relevant folds, one is never pursuing the structure, but rather a structure.

Of course, we don’t expect most proteins or domains to regularly adopt alternate overall folds. However, reorientations of domains or monomers is a relatively common behavior, and one that poses a sticky challenge for structural biologists because incidental properties of a particular arrangement may bias our experiments towards observing it. A minor member of the ensemble, if it has favorable packing geometry, may exclusively populate a crystal. Similarly, NMR experiments to determine domain arrangement via residual dipolar couplings must always be undertaken with an eye to ensuring that interactions with the aligning media do not bias the results. No single structure of adenylate kinase can instruct us about its catalytic cycle, and structures of the unbound state do not capture the reality that the protein continues to open and close in the absence of ligand. Single structures do not capture motions of domains or monomers relative to each other and that often means an incomplete understanding of function.

Domain motions are also an overly dramatic example, because simpler rearrangements of the backbone take place in many proteins, even when regular secondary structures are evident. Fluctuations of the main chain play a functional role in several proteins — as, for instance, in the flaps of the HIV protease. Additionally, rearrangements of the backbone have a significant role in signaling, as in NtrC, which I’ll talk about more in two weeks. Proteins where the main chain rearranges in response to ligand binding or post-translational modification generally cannot be described by a single structure.

Even if the backbone is rigid, every protein will have flexibility in the side chains of its amino acids. One of course expects to see this kind of behavior in side chains on the surface of a protein, where it is usually dismissed as irrelevant. However, we also know that side chains can rotate and move in the core of a protein, and that on some protein surfaces they can undergo coherent rearrangements. I’ll talk a bit more about the functional relevance of side-chain motions next Thursday. For now, suffice to say that side chain rotations cannot be so easily ignored and sometimes have functional effects. Structural studies that do not capture these rotations may be missing something important.

My point here is not that single structures are stupid or useless. A structure can be very informative about about a protein’s function, and often has great power to explain the effects of mutations and ligands. However, we should not mislead ourselves into thinking that any single structure will have all the answers, or indeed any of them. Every protein is a constantly interconverting ensemble of structures, and there are many layers of structural diversity within that ensemble, reaching from whole fold rearrangements to “mere” side-chain adjustments. Determining the structure of a protein is not a coherent goal for a research program. The successful structural biology study will characterize the conformation and energy of key, functionally-relevant members of the protein’s structural ensemble and identify the pathways between them.

 Posted by at 9:00 PM

  4 Responses to “Don’t look for "the" structure”

  1. Looks that "determining the structure of a protein" is sort of a crystallographic jargon which leads to misunderstanding by the colleagues from other fields, and this is something one should avoid. For instance NMR people find misleading a concept of "forbidden transitions" so widely used in EPR. In fact those "forbidden" transitions are allowed in a sense of quantum mechanics, and it is only the wording that makes it sound contradictory.

  2. We should resist the temptation to blame crystallographers for the idea of the singular structure. It probably does start from there, because a singular structure is what's typically observed in those experiments, and optimization often involves limiting the structural ensemble in a crystal. However, this language is improperly embraced all the time, even by researchers using techniques sensitive to dynamics and variability such as NMR. The (fortunately dying) quest of NMR technicians to make their structures as "good" as crystal structures, and even the idea that crystal structures are made better by limiting the sampled ensemble, show how pervasive this idea has been.

  3. I'm anxiously waiting for your review of the following review:

    http://www3.interscience.wiley.com/journal/122679619/abstract

    At the dawn of the 21st century: Is dynamics the missing link for understanding enzyme catalysis?
    Shina C. L. Kamerlin, Arieh Warshel *
    Department of Chemistry, University of Southern California, Los Angeles, California 90089

    Proteins: Structure, Function, and Bioinformatics
    Early View (Articles online in advance of print)
    Published Online: 6 Nov 2009

  4. I think I've mentioned my opinion of Dr. Warshel's work before, in connection to his recent paper on adenylate kinase. That is, I basically agree that dynamics probably do not contribute directly to the chemical steps of catalysis, but that this isn't particularly surprising or relevant to most of the work in the field. I would be very surprised were it ever proven that equilibrium dynamics directly contributed to the chemical coordinate of catalysis, and I wouldn't expect such a contribution to be significant. The question is whether the chemical step is rate-limiting, and if it is not, what then dictates the apparent kcat. Equilibrium fluctuations are likely to be most significant in that regard.

Sorry, the comment form is closed at this time.