The goal of this project is to perform a comparative analysis of the folding mechanisms of two members of the alpha/beta/alpha sandwich motif, one of the most common in biology. Previous NSF-supported studies of the mechanism of folding of one sub-class of this motif, dihydrofolate reductase (DHFR), have shown that this small two-domain protein folds via a sequential set of on-pathway partially-folded states in four parallel channels. By contrast, two members of the flavodoxin fold family, another sub-class of this motif, initially misfold to an off-pathway intermediate that must at least partially unfold to access the productive transition state leading to the native conformation. The molecular basis for this misfolding reaction will be probed by a combination of experimental and computational methods that will focus on determining the structural features and energetics of these intermediates for CheY, NtrC and Spo0F, all members of the response regulator sub-class of the flavodoxin fold. Continuous-flow (CF) small-angle x-ray scattering (SAXS), time-resolved Forster resonance energy transfer (trFRET) and far-UV circular dichroism (CD) measurements of microsecond folding reactions will provide quantitative information on dimensional properties and global secondary structure. Pulse-quench hydrogen exchange methodology and mass spectrometric analysis of mass-labeled peptides will enhance the circular dichroism data by identifying the segments that define the cores of stability. Mutational analysis will explore the roles of individual side chains in the folding and misfolding of CheY, with a particular focus on large nonpolar side chains in the pair of hydrophobic cores on either side of the central beta-sheet. The role of chain entropy in the CheY misfolding reaction will be tested by creating permutations that vary the chain connectivity while preserving 3D structure. Collaborative native-centric simulations of response regulator folding mechanisms will provide detailed structural insights into the misfolded species and the process by which they backtrack to the native conformations. Comparisons with the results for the on-pathway sub-millisecond folding reaction in DHFR will provide additional insights into the contrast with response regulators. The results of this multi-dimensional comparative analysis are expected to enhance the understanding of early folding reactions in alpha/beta/alpha sandwich proteins and the basis for the misfolding reactions in the flavodoxin fold.
The broader impact of this project has training, educational and technology development components. Undergraduate, graduate and postdoctoral fellows will receive training in molecular biophysics, including spectroscopic methods, thermodynamics and kinetics, sophisticated data analysis, protein engineering, and the protein folding problem. Several of the methods employed, including circular dichroism and fluorescence spectroscopy, and data analysis algorithms developed will form a part of a new advanced topics course in Biophysical Methods for graduate students in the PI's Department. The analytical tools developed during the course of this research on protein folding mechanisms are currently being used in over a dozen research labs around the world, and will be made more accessible via a website sponsored by the Biochemistry and Molecular Pharmacology Department. The continuous-flow mixing technology developed during the previous grant period will continue to be optimized and available to all users by collaboration (for CF-trFRET and CF-CD) or through the BioCAT beamline at the Advanced Photon Source at Argonne National Laboratory (for CF-SAXS).
