Sequence Dependence of Polyproline II Helix Formation

The overall goal of this project is to determine the effects of surrounding sequence upon the propensity for protein residues to adopt the polyproline II (PII) helical conformation. It has been hypothesized that protein unfolded states possess significant PII helix content. The experiments outlined in this proposal are designed to test this hypothesis and to provide data concerning the formation of other secondary structures. This will be achieved through two aims: i) determining the effects of surrounding blocks of alanine, glutamine, serine, leucine and valine residues upon the conformational properties of central guest residues, and ii) the role of electrostatics in PII helix formation. Data will be collected using both circular dichroism (CD) spectroscopy and NMR spectrometry. CD data will be analyzed using singular value decomposition, which will provide information on PII helix content and the presence of other secondary structures in each of the peptides studied. NMR will be used to determine average conformational properties of guest residues in some of the peptides to be examined. The use of lanthanide chemical shift reagents will be explored in order to alleviate resonance overlap in the NMR experiments. At all stages data collected will be compared to the calculations of Dr. Rohit Pappu at the Washington University in St. Louis. Intellectual Merit It is widely acknowledged that a lack of understanding of protein unfolded states is the single biggest impediment to understanding the protein folding process. Should the hypothesis that unfolded states possess significant PII helix content prove true, then protein unfolded states are significantly more structured than commonly believed. Data collected in the execution of this project will either support or refute this hypothesis. In addition, data will be obtained on the presence of other secondary structure types in protein unfolded states. These data will provide the beginnings of a comprehensive picture of the ensemble of protein unfolded states. In addition, data collected will prove useful in the understanding of biomolecular interactions mediated by PII helices. The development of protocols for the use of chemical shift reagents in NMR will make the study of small, weakly-structured peptides more tractable and will prove to be of wide general use. Broader Impacts It is planned to have both a postdoctoral scholar and several undergraduate students work on aspects of this project. Where possible, undergraduates will be recruited from underrepresented groups. The postdoctoral scholar will receive advanced training in spectroscopy and NMR spectrometry. The undergraduate students will receive basic laboratory training, training in spectroscopy, scientific ethics, and in the analysis and presentation of data. All trainees will participate in the dissemination of knowledge generated. The knowledge generated in this project will be incorporated into a graduate-level course on structural biology. In addition, data will be published in peer-reviewed journals, and presented at scientific meetings and research institutions.