The objective of this project is to understand how enzymes tune their energy landscapes so as to perform their functions. It is well known that rather than existing as the static structures usually used to depict them, proteins are actually ensembles of conformational states. The fact that proteins undergo both structural and dynamic changes as part of the function indicates that the ensemble is critical to its function. The implication of this finding is that in addition to coding for the three dimensional structures, a protein's sequence also evolved to code for the entire energy landscape (i.e. the ensemble of conformations). It is of great import to know how this is done. Are there unifying principles that connect proteins with different functions? Here we leverage a unique discovery by our group during the previous grant period, which shows that the enzyme adenylate kinase (AK) from E. coli, uses local unfolding to modulate its enzymatic activity - in essence the energy landscape has unfolding within its functionally important repertoire. This mode of conformational change stands in stark contrast to the current accepted model, whereby an opening and closing reaction is believed to facilitate catalytic turnover. In the proposal, we leverage this unfolding reaction into a mutation strategy designed to investigate the coupling between the different regions of AK, and how that coupling produces an ensemble of states that constitutes the energy landscape of AK. Our approach is geared to understanding the role of conformational fluctuations in function, as well as reconciling the observations that, 1) dynamic changes in proteins can occur in the absence of structural changes;2) function can be correlated to the stability of different regions of the protein;3) binding can increase (rather than decrease) dynamics at many sites;and 4) structural and dynamic changes can propagate to distal parts of a protein structure in the absence of a pathway of structural or dynamic changes linking the two sites. We will perform binding and stability measurements using isothermal titration calorimetry (ITC), circular dichroism (CD) monitored thermal unfolding and hydrogen exchange (HX), and we will monitor the kinetics of the conformational and enzymatic processes using NMR 15N relaxation dispersion (CPMG) and steady state enzymatic analysis. Our studies are designed elucidate the states in the energy landscape of AK and to understand the role they play in driving the catalytic process.
of the proposed studies to understand the structural and dynamic basis of catalysis and function is not just academic. Development of a model that can quantitatively account for the changes in dynamics, thermodynamics and structure would represent the cornerstone of strategies targeted to the design of proteins with new or improved functions or to the design of therapeutic ligands that target those proteins. The studies proposed here are designed to fill the gap in knowledge about how conformationally heterogeneous ensembles can be tuned by Nature to facilitate function.
|Hoffmann, Jordan; Wrabl, James O; Hilser, Vincent J (2016) The role of negative selection in protein evolution revealed through the energetics of the native state ensemble. Proteins 84:435-47|
|Motlagh, Hesam N; Toptygin, Dmitri; Kaiser, Christian M et al. (2016) Single-Molecule Chemo-Mechanical Spectroscopy Provides Structural Identity of Folding Intermediates. Biophys J 110:1280-90|
|Toptygin, Dmitri; Chin, Alexander F; Hilser, Vincent J (2015) Effect of Diffusion on Resonance Energy Transfer Rate Distributions: Implications for Distance Measurements. J Phys Chem B 119:12603-22|
|Hilser, Vincent J; Anderson, Jeremy A; Motlagh, Hesam N (2015) Allostery vs. ""allokairy"". Proc Natl Acad Sci U S A 112:11430-1|
|Choi, Jay H; Laurent, Abigail H; Hilser, Vincent J et al. (2015) Design of protein switches based on an ensemble model of allostery. Nat Commun 6:6968|
|Hilser, Vincent J; Whitten, Steven T (2014) Using the COREX/BEST server to model the native-state ensemble. Methods Mol Biol 1084:255-69|
|Motlagh, Hesam N; Wrabl, James O; Li, Jing et al. (2014) The ensemble nature of allostery. Nature 508:331-9|
|Hegde, Muralidhar L; Tsutakawa, Susan E; Hegde, Pavana M et al. (2013) The disordered C-terminal domain of human DNA glycosylase NEIL1 contributes to its stability via intramolecular interactions. J Mol Biol 425:2359-71|
|Hadzipasic, Omar; Wrabl, James O; Hilser, Vincent J (2013) A horizontal alignment tool for numerical trend discovery in sequence data: application to protein hydropathy. PLoS Comput Biol 9:e1003247|
|Schrank, Travis P; Wrabl, James O; Hilser, Vincent J (2013) Conformational heterogeneity within the LID domain mediates substrate binding to Escherichia coli adenylate kinase: function follows fluctuations. Top Curr Chem 337:95-121|
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