FIBR: How Do Proteins Fold Into Their Native and Functional Structures In-Vitro and in The Physiological Milue of The Living cell?

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In all living cells, proteins self-assemble or fold into precise, three dimensional structures having unique functions from a linear polypeptide chain assembled by the ribosome (the enzyme responsible for protein synthesis). The precise folding of a protein is dictated by its DNA sequence but researchers have not yet deciphered the rules for encoding structure by sequence ("the protein folding problem"). Addressing this problem is crucial to understanding how gene sequence variation translates into variation in protein and cell function. The delicate balance of forces which controls and guides the structural dynamics of the folding process is highly sensitive to environmental conditions inside the cell. The goal of this project is to synergistically apply cutting-edge methodologies, including single molecule spectroscopy, ultra-fast microfluidics mixing, photo-induced electron transfer, non-natural amino-acid labeling, mitochondrial protein transport, chemical peptide synthesis and simulation modeling using distributed and super-computing systems, to the study of protein folding under conditions that mimic the natural folding environment inside the living cell. The consortium of researchers will study the unfolded state of three different proteins in simple solutions (in-vitro) under a variety of conditions, and while the proteins are being made directly on the ribosome itself. By comparing such studies to protein folding experiments conducted within the crowded environment of the mitochondrial matrix (a mimic for the intracellular folding environment), this project seeks to understand the major differences between in-vitro and in-vivo folding environments and the effects of such differences on protein folding mechanisms. This project will have broad impacts on the field of cellular biology through the development of novel tools and methods as well as a general approach for studying complex biological processes on the molecular level. An outreach program will target the dissemination of these research tools to faculty and students from underrepresented institutions, and the enhancement of scientific and technological knowledge at the secondary education level. This project represents an interdisciplinary collaboration of researchers led by Shimon Weiss, at the University of California-Los Angeles with subawards to Stanford University (Vijay Pande), Texas A&M University (Arthur Johnson), University of California-Davis (Olgica Bakajin), Michigan State University (Lisa Lapidus) and Scripps Research Institute (Jeff Kelly). A large number of students and postdoctoral fellows will receive advanced training in conceptual and technical aspects of research at the interface of chemistry, biophysics, and simulation.