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

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FairmanMCB-9817188:Coiled coils are motifs which involve the interaction of sets of a-helicesand are stabilized largely through hydrophobic amino acids that line theinterface of the structure. This study focuses on a natural four-chaincoiled coil model motif from Lac repressor to quantitate sequence-structurerelationships in a systematic fashion. A mutational approach is used tostudy the effects of different amino acid sidechains at specific positionsin the Lac repressor coiled coil. Experimental strategies involvesynthesis of peptides by solid-phase synthetic methods, purification byreversed-phase chromatography, and biophysical characterization. Coiledcoil stability is measured by the oligomeric states during temperature orchemical denaturation, peptide concentration titrations using circulardichroism spectropolarimetry and analytical ultracentrifugation. Usingsuch approaches, a series of experiments will systematically explore therelationship between coiled coil stability and several definingcharacteristics of coiled coils. Sequences chosen for study will test therelationship between helix length and stability, the role of hydrophobicamino acids on stability and oligomerization, the importance of side-chaininteractions at other heptad positions on tetramer stability, and theability to design simplified sequences based on rules so learned.With an understanding of the determinants that define coiled coilstructure, it will be possible to design novel structures that may be ofuse in nano-engineering, particularly for the synthesis of long"nanoropes", and with appropriate modifications, may even be converted intoconducting materials such as "nanowires." This project is well-suited foran undergraduate laboratory since the synthesis and characterization ofeach peptide described herein is a project unto itself and can beaccomplished easily within the constraints of a one-year senior researchproject. Important skills that will be learned by undergraduate studentsinclude rigorous quantitative methods in protein biochemistry usingbiophysical methods and cross-cutting interdisciplinary methods that spanfrom biology and chemistry to physics and mathematics.