Proteins need fold to perform their biological function. Thus, understanding how proteins fold could be the key to understanding life. In the first study, the stability and structure of several !-hairpin peptide variants derived from the C-terminus of the B1 domain of protein G (PGB1) were investigated by a number of experimental and computational techniques. Our analysis shows that the structure and stability of this hairpin can be greatly affected by one or a few simple mutations. For example, removing an unfavorable charge near the N-terminus of the peptide (Glu42 to Gln or Thr) or optimization of the N-terminal charge-charge interactions (Gly41 to Lys) both stabilize the peptide, even in water. Furthermore, a simple replacement of a charged residue in the turn (Asp47 to Ala) changes the !-turn conformation. Our results indicate that the structure and stability of this !?hairpin peptide can be modulated in numerous ways and thus contributes towards a more complete understanding of this important model !-hairpin as well as to the folding and stability of larger peptides and proteins. The second study revealed that PGB1 and its variants can form amyloid fibrils in vitro under certain conditions and these fibrils resemble those from other proteins that have been implicated in diseases. To gain a further understanding of molecular mechanism of PGB1 amyloid formation, we designed a set of variants with mutations that change the local secondary structure propensity in PGB1, but have similar global conformational stability. The kinetics of amyloid formation of all these variants have been studied and compared. Our results show that different locations of even a single mutation can have a dramatic effect on PGB1 amyloid formation, which is in sharp contrast with a previous report. Our results also suggest that the "-helix in PGB1 plays an important role in the amyloid formation process of PGB1. In the final study, we investigate the forces that contribute to protein stability in a very general manner. Based on what we have learned about the major forces that contribute to the stability of globular proteins, protein stability should increase as the size of the protein increases. This is not observed: the conformational stability of globular proteins is independent of protein size. In an effort to understand why large proteins are not more stable than small proteins, twenty single-domain globular proteins ranging in size from 35 to 470 residues have been analyzed. Our study shows that nature buries more charged groups and more non-hydrogen-bonded polar groups to destabilize large proteins.

The initial steps involved in the assembly of normally soluble proteins into amyloid fibrils remain unclear, yet over 20 human diseases are associated with proteins that aggregate in this manner. Protein surface modification is a potential means of mapping the interaction sites in early oligomers that precede amyloid formation. This dissertation focuses on the use of covalent labeling combined with mass spectrometry to elucidate the structural features of Cu(II)-induced β-2-microglobulin (β2m) amyloid formation. An improved covalent modification and MS-based approach for protein surface mapping has been developed to address the need for a reliable approach that ensures protein structural integrity during labeling experiments and provides readily detectable modifications. This approach involves measuring the kinetics of the modification reactions and allows any local perturbations caused by the covalent label to be readily identified and avoided. This MS-based method has been used to study human β2m, a monomeric protein that has been shown to aggregate into amyloid fibrils in dialysis patients leading to dialysis-related amyloidosis. Under conditions that lead to β2m amyloid formation, reactions of β2m with three complementary covalent labels have been used to identify the Cu(II) binding site, metal-induced conformational changes, and the oligomeric interfaces. Results confirm that Cu(II) binds to His31 and the N-terminal amine. Binding to these residues causes several structural changes in the N-terminal region and ABED β-sheet which likely enables formation of oligomeric intermediates. The covalent labeling data indicate that the pre-amyloid β2m dimer has an interface that involves the antiparallel arrangement of ABED sheets from two monomers. Moreover, our covalent labeling data allowed us to develop a model for the tetramer in which the interface is mediated by interactions between D strands of one dimer unit and the G strands of another dimer unit. Lastly, the selective covalent modification approach has been used to delineate the structural changes in β2m after interaction with Cu(II), Ni(II), and Zn(II) and their effect on its aggregation. Our covalent labeling data indicates that the unique effect of Cu(II) appears to be caused by the site at which the metal binds the protein and the conformational changes it induces.

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