The shapes that proteins take on greatly affect their characteristics and activities, and so studying how they contort themselves is naturally a big part of life science research. Yet, identifying the shape of a large molecule remains a challenge, since X-ray crystallography, the most commonly used technique, has its limitations.
Now a team of researchers from Lawrence Berkeley National Lab and Scripps Research Institute showed that small-angle X-ray scattering (SAXS) has the resolution necessary to visualize the varying shapes that flexible molecules take on. Moreover, the technique does not require crystallization, which many proteins are not subject to, and can be performed in many types of molecular environments.
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“With SAXS, there are relatively few restraints on conditions, construction, concentration or solution chemistry,” Hura says. “However, analytical methods have not kept pace with the hardware. While there are many factors that may induce a protein to undergo structural changes, these factors are difficult to predict. Our structural comparison map technique gives us a high-throughput screening capability. The combination of SAXS and our maps allows us to highlight those factors that make the biggest difference in structural conformations. We’re also able to track trends and identify intermediate states and other factors that shift equilibrium from one structure to another.”
The data in a structural comparison map is presented in the form of a color-coded checkerboard with similarity scores displayed as gradients moving from red, indicating high, to white, indicating low, and various shades of orange and yellow in between.
“With structural comparison maps, I can immediately see which structures under which conditions are the same and which are not,” says McMurray. “The maps provide both structural and chemical information and enable us to identify those conformations we should be looking at.”
To test the structural conformation map technique, co-author Budworth, a member of McMurray’s research group, prepared samples of a protein known as MutSß, an inviting chemotherapeutic target because of its ability to remove problematic DNA that can lead to cancer and other genetic mutations.
“MutSß is a heterodimer whose two macromolecules undergo an ordered series of nucleotide-dependent steps to initiate DNA repair,” Budworth says. “Each discrete nucleotide-bound state is a conformational state decision point that primes the next pathway step. A mechanistic understanding of these steps is crucial to learning how cells avoid mutation.”
Says McMurray, “Initially this was a very big puzzle because MutSß had no crystal structure, nor could we take a look at any one conformational state and say this is good or this is bad. The structural conformation maps allowed us to characterize the different conformational states individually and then compare them to one another. We discovered that DNA has surprisingly little impact on MutSß conformational structures, a fact that was not evident from biochemical measurements, but obvious when examining the maps.”
From the SAXS imaging and structural conformation map analysis, McMurray and her group believe that DNA is sculpted to the protein conformation and that nucleotide-binding drives MutSß conformational changes. This, they say, holds implications for future cancer therapies.
Lawrence Berkeley National Lab: Comparing Proteins at a Glance
Study in Nature Methods: Comprehensive macromolecular conformations mapped by quantitative SAXS analyses
