Synthesis of peptides and their study by magnetic resonance: New spin labels and novel protein structures.

摘要

Synthetic peptides are useful tools in the investigation of protein structure and function, which are fundamental and related aspects of developing molecular descriptions of living processes. As a result, the use and development of methods to synthesize novel peptides are widespread and evolving. Additionally, some of the most common, and powerful, techniques for the measurement of peptide structure involve magnetic resonance experiments. The execution and extension of peptide synthetic methods for use in magnetic resonance experiments is therefore useful in extending the available knowledge of protein structure.;Presented here are peptide studies using two different magnetic resonance techniques. Nuclear magnetic resonance is used to determine the structure of the C-terminal domain of the human Agouti-related protein (AGRP). The structure of this protein is novel in vertebrates, and interacts with hypothalamic receptors known to be critical in energy homeostasis. The results of this study motivate the synthesis described herein of the analogue of AGRP, the Agouti protein. The goals of the synthesis of the active Agouti domain are the determination of its structure, and possession of synthetic access to any chimeras of AGRP and Agouti which are useful for the elucidation of AGRP receptor function. Electron spin resonance (ESR) experiments were performed on a small, well characterized helical peptide system, the 3K peptide. One set of experiments investigated the folding behavior of 3K peptides using the rigidly attached nitroxide label, TOAC. While TOAC is shown herein to be an excellent probe of backbone conformation, its inclusion into sheet or coil structures is proscribed due to its strong helix-promoting behavior. Additionally, the common method of attaching nitroxides via a disulfide linkage (the Cys-SL approach) is chemically incompatible with a cysteine-rich domain such as AGRP. This motivates the development of the novel DAP-SL spin labeling strategy. This approach is shown to have a novel range of applicability compared to Cys-SL and TOAC, thus expanding the range of peptides that can be studied using spin-label experiments. This thesis then describes the use of magnetic resonance experiments, both new and old, in the determination of peptide structure and dynamics.