Date of Award
Doctor of Philosophy
Gary L Emmert
Richard L Petersen
This research is focused on appplying mass spectrometry based methods to understand the conformational changes of bioactive peptides in solution. In this dissertation, electrospray ionization mass spectrometry (ESI-MS) coupled with hydrogen/deuterium exchange (HDX) has been employed to probe the secondary structure of selected peptides in membrane-mimetic enviroments. The membrane-mimetic systems used in our study include water and trifluoroethanol (TFE). HDX of the amide protons in native proteins depends on the solvent accessibility and spatial arrangement of amino acid residues, thus providing information about the secondary structure of a protein. A total of six biologically active peptides have been chosen for this study. The dissertation consists of six chapters; the first two are introductory in nature and both of which discuss the background information about the peptides, basic concepts involved in ESI-MS, HDX, and MS instrumentation. The remaining three chapters provide discussion on structural investigation of the peptides containing similar sequences. Dynorphin A (1-8) and bovine adrenal medualla dodecpeptide 12P (BAM 12P) with a similar message domain (YGGF), are the two opioid-related peptides whose structures have been investigated and discussed in chapter 3. The solution structure of these peptides was probed using mass spectrometry and circular dichroism (CD) spectrometry techniques. While CD data gave an idea about the type of probable secondary structure; data obtained by tandem mass spectrometry helped in identifying the elements involved in the secondary structure formation of the target peptides. In water, dynorphin shows open conformation whereas; BAM 12P exhibits ordered structure to some extent which is further evident from the exchange numbers obtained from HDX-MS experiments. Both dynorphin and BAM 12P assume a ß-turn conformation in TFE. Chapter 4 deals with the similarity in the conformational analysis of the peptides bombesin (BBS) and neuromedin B (NMB). A common character of these peptides is the C-terminal sequence G-H-X-M-NH2. Both BBS and NMB are known to act on the G-protein-coupled receptors with a varied physiological response. The HDX-MS experiments revealed that both the peptides show dis-ordered conformation in water and exhibit helical conformation in TFE. Chapter 5 discusses the conformational structures of substance P (SP) and kassinin. These two peptides belong to the family of tachykinins and share a common C-terminal sequence Phe-X-Gly-Leu-Met-NH2 and a varied N-terminal sequence. The biological actions of these peptides are also mediated through specific G-protein-coupled neurokinin receptors. The structural analysis of SP and kassinin using HDX-MS in TFE revealed that; SP shows two ß-turns followed by an extended turn in the C-terminal, kassinin exhibits ß-turn in the N-terminal followed by a helical conformation in the remaining portion of the peptide. In water, both the peptides assume random coil conformation. Lastly, chapter 6 summarizes the objectives obtianed in the current research.
dissertation or thesis originally submitted to the local University of Memphis Electronic Theses & dissertation (ETD) Repository.
Kunapuram, Deepa, "Solution Conformation of Biologically Active Peptides as Determined by Hydrogen/Deuterium Exchange and Mass Spectrometry" (2011). Electronic Theses and Dissertations. 398.