RUI Proposal: Learning the Rules that Govern the Folding and Stability of Coiled Coils

Haverford College, Haverford PA

Investigators

Abstract

The objective of this project is to explore the relationship between sequence and structure for very long coiled coils, such as found in myosin, using both protein folding and design approaches. Three-pronged approach will be used to accomplish the goal. First, synthetic genes that encode copolymers of 14-amino acid blocks will be constructed, whose sequences are based on de novo, minimalist-design principles. These genes will be cloned into expression vectors to make dimeric coiled coils that range from 70 residues to greater than 1,000 residues per helix. Specifically, this system will be used to test the role of intermediates in the assembly of long coiled coils, such as monomeric helix formation and nucleation of specific helix pairing interactions to dictate proper phasing of helices. After expressing and purifying these designed proteins, their structures will be characterized using circular dichroism, analytical ultracentrifugation, and single molecule techniques such as atomic force microscopy and laser tweezing. Second, to complement these design studies, a myosin coiled-coil rod domain will be used as a model system for folding studies involving segment swapping between designed and natural sequences. In addition, the myosin coiled coil will be used to help develop biophysical protocols for studying designed coiled coils, using the instruments described above. Finally, these synthetic peptide blocks will be used to make long copolymers for the study of other coiled coil topologies and higher order assembly to form fibrils. Long copolymers are generated by forming staggered helical structures that act as templates for their own head-to-tail self-assembly. Peptides will be synthesized and purified in the laboratory and then characterized using the same biophysical techniques described above. The overall goal of this research is to understand the basic relationship between protein sequence and structure. It is still not possible to predict protein structure and function from first principles, mainly because it is still not understood how proteins balance the major chemical forces in attaining their three dimensional shape. Two approaches have been applied to study this problem, defining the fields of protein folding and design. The two questions are the inverse of one another: scientists in the field of protein folding ask, "Can we predict the structure of a protein given its amino acid sequence?" and those who work on protein design ask, "Can we predict what sequence of a protein will result in a target structure?" Both of these strategies will be used in the study of coiled coils. These structural motifs, predicted to be in 1/3 of all proteins, involve the interaction between two or more alpha-helices. The modular design of the experiments will allow students to make significant achievements over the course of a summer experience and an academic year working towards a senior thesis project. This cohesive program in design, along with a strong modular component, should provide a rewarding experience for students interested generally in interdisciplinary sciences, including elements of biochemistry and biophysics.