Studies in protein folding: Understanding helix backbone interactions and the structural characterization of a compact domain

The protein folding problem is still one of the most important intellectual challenges in biological science. An enormous amount of work has been done to study this problem. In this thesis, the protein folding problem is attacked at two different levels. At the secondary structural level, model helices are manipulated to try to reveal some of the basic interactions that stabilize the helix. At the tertiary structural level, the protein structure is studied in an attempt to isolate and characterize a small subdomain.; Helical peptides, acetyl-WGG(EAAAR){dollar}sb4{dollar}A-amide and acetyl-WGG(RAAAA)4R-amide, have been chosen as model peptides to study helix backbone interactions. Since the hydrogen bond between amides and carbonyls along the peptide backbone is an important feature of the {dollar}alpha{dollar}-helix, experiments were designed to interfere the backbone hydrogen bonds. The backbone hydrogen bonds of model helices have been disrupted in two ways. In the first, the hydrogen bond donor ability of specific peptide bonds has been removed by replacing the NH group with a NCH{dollar}sb3{dollar} group. In the second modification, the hydrogen bond accepting ability of a residue is eliminated by replacing the CO functional group with a CH{dollar}sb2{dollar} group. These modifications have been placed into the model peptides so that the same hydrogen bond is removed. CD has been used to follow the helix-coil transition of the model peptides and derivative peptides. Both the N-methyl modification and the reduced carbonyl modification decrease the helicity of peptides; however, the amount of destabilization is more than expected. The thermodynamic properties of the disruption can be analyzed using different approaches: The Two-State model, Lifson-Roig theory, and the more complicated Dichroic model. Different backbone-backbone interactions may be addressed by comparing the two different backbone modifications.; Understanding the properties that identify domains can also help us study the protein folding problem. The compact unit theory, an algorithm that identifies domains based on their compactness, has been chosen for this study. When applied to staphylococcal nuclease, several compact units were located. The most compact unit, consisting of residues 12-36 of the nuclease sequence, has been synthesized and studied. If this compact unit is a domain, it should retain its native {dollar}beta-beta-beta{dollar} structure when isolated from the intact protein. CD and NMR were chosen to study the structural properties for this peptide, and many solvent conditions were used to try to find the structure. However, conditions where the peptide could form its native structure have not been found.