High Resolution Single Molecule Analysis of Fast Folding and its Coupling to Binding

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Proteins are the cellular nanomachines in charge of most biological functions, including energy production, DNA replication and transcription, enzymatic catalysis, signaling, cellular scaffolding and defense. But proteins are also inherently flexible polymers that must fold into the complex native 3D structures corresponding to their biologically functional states in a self-assembly process determined by the chemical blueprints encoded in their amino acid sequence. Thus the mechanisms by which proteins fold and function are a critical component of almost every aspect of molecular and cell biology. Understanding the intertwined mechanisms of folding and function also brings about the opportunity to predict, engineer, and design biological function "a la carte", thus conveying unparalleled transformative impact to Society. Moreover, because proteins are at the lowest echelon of biological complexity where Biology effectively meets Physics, Chemistry, and Engineering, their study constitutes an ideal arena for training the new generations of multidisciplinary researchers, preparing them for the emerging fields of Quantitative and Synthetic Biology. Activities are designed around a team-based structure aimed at facilitating integration of members at various levels of education ranging from postdoctoral fellows and graduate students to undergraduate and high school students/teachers. An equally important element of this project is the strong commitment to participation of underrepresented groups in research. The PI will actively recruit project members from several existing research mentoring programs for underrepresented minorities in STEM fields to participate in the research activities of this project.A major drive for modern protein research has been to develop experimental methods to resolve the myriads of pathways and complex mechanisms that are predicted by advanced theory and atomistic molecular simulations. Detecting such inherent kinetic complexity in experiments has remained elusive, even with modern methods that exhibit improved time, structural, or single-molecule resolution. The overall objective of this project is to bridge this gap by experimentally monitoring the transition paths of individual protein molecules as they fold. Advanced single-molecule fluorescence methods will be used in conjunction with theoretical and computational analyses to measure transition paths and folding mechanisms of fast-folding protein domains. Fast-folding domains are optimal targets because their marginal cooperativity ensures significant populations of "excited" states and, somewhat counterintuitively, slower transition paths over their broad, shallow folding barriers. Moreover, the microsecond folding of these domains facilitates direct comparison with modern atomistic simulations. To reach the required resolution, we rely on approaches we recently developed for achieving microsecond resolution single-molecule fluorescence detection, such as better photoprotection systems and procedures for maximum likelihood analysis of photon arrival times, together with implementation of 2-color and 3-color FRET schemes to measure multiple distances. Through the realization of these experiments and computational analyses on select fast-folding domains we will investigate the structural, sequence, and environmental determinants of the mechanisms for folding. This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences and the Physics of Living Systems Program in the Division of Physics.