Proteins perform a variety of biological functions, including muscle contraction and cell division. They are also increasingly used in diverse applications in the biotechnology industry ranging from oil spill cleanup to vaccine delivery. The structure and conformational dynamics (i.e. how their structure changes) of proteins are important properties that govern their function. A critical step in altering or designing proteins with specific novel and desirable properties requires a detailed understanding of how the amino acid sequence of a protein codes for its structural and dynamical properties. This project will test the idea that the patterning and separation of hydrophobic (water repelling) and electrically charged amino acids in the protein plays a significant role in modulating these properties. A variety of spectroscopic techniques (using e.g. light and nuclear magnetic resonance) will be used to determine the changes in structure and stability of these proteins and variants of them that have been specially designed. The experimental studies will be complemented by computer simulations to provide insights at the atomic level into the interplay between hydrophobic and electrical charge interactions. The project aims to substantially advance our understanding of protein folding, one of the most important and complex problems in biology. A new venture into specially designed proteins will test the applicability of principles derived from studies of natural proteins and, it is hoped, will lead to new criteria for the design of proteins with novel and useful properties. This project will involve teaching and training of young scientists, which will be enhanced by active participation in the Protein Folding Consortium.
The goal of this project is to test the contributions of local-in-sequence and long-range interactions, both in hydrophobic clusters of branched aliphatic side chains and between charged side chains in natural and designed beta/alpha-repeat proteins, in modulating the energy landscape of the proteins. Available evidence on CheY, a naturally occurring protein, suggests that locally connected clusters of isoleucine, leucine and valine side chains rapidly collapse via subdomains that can enhance or impede subsequent folding reactions leading to the native conformation. Recent work on Di-III_14, a de novo designed beta/alpha-repeat protein, suggests that long range electrostatic interactions with high charge segregation are responsible for the formation of very structured intermediates that interconvert extremely slowly with each other and with the native state. The project will extend current knowledge of CheY and Di-III_14 proteins with a battery of spectroscopic methods, at equilibrium and interfaced to ultra-rapid mixing systems, to probe the size, shape and pair-wise distances in the chemically denatured state and in partially folded states that appear in the microsecond time range after dilution to native-favoring conditions. Native-state hydrogen exchange experiments will explore the relationships between partially-folded states and sequence in Di-III_14 and other designed constructs. Mutational analysis will test the role of local and nonlocal ILV clusters and specific electrostatic interactions in the structures formed in high energy states of these proteins, and collaborative single molecule pulling experiments will be used to probe the unique energy surface of Di-III_14 in water. The experimental data will be used to validate collaborative high-resolution molecular dynamics simulations of the folding reactions of CheY and Di-III_14, and new design efforts by collaborators will test the role of charge and charge segregation in molding the free energy surface of beta/alpha-repeat proteins. It is anticipated that the combined application of experimental, computational and design methods to the same targets will substantially enhance our understanding of how sequence determines folding and stability in one of the most common folds in proteins.
This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate of Mathematical and Physical Sciences.
