Protein folding from molten globules to miniature proteins

The knowledge of protein folding mechanism is a key to understanding the relationship between folded proteins, misfolded proteins, and diseases. Thus studies of protein folding are an important and attractive topic of structural biology. In this dissertation, three proteins, human alpha-lactalbumin (alphaLA), the N-terminal domain of the ribosomal protein L9 (NTL9), and the C-terminal domain of the ribosomal protein L9 (CTL9), are used as model systems for folding studies. alphaLA is a 123 residue Ca2+-binding protein containing four disulfides and forms a molten globule under a range of conditions including acidic pH (A-state) and neutral pH in the absence of Ca2+ with modest amounts of denaturant. Studies of the pH dependent properties of molten globules have shown that the 6-120 disulfide and the beta-subdomain play contrasting roles in the molten globule state at low and neutral pH. The 6-120 disulfide is more important than the beta-subdomain to the molten globule state at pH 2, but is less important at pH 7. This indicates that the A-state of alphaLA may not be a rigorous mimic of the folding intermediate populated at pH 7.;NTL9 is a 56 residue and two-state folding protein. A set of peptide fragments derived from NTL9 were prepared and characterized to explore the minimum folding motif of NTL9. The first 39 residues of NTL9 (NTL91--39 ) have been shown to fold independently and adopt the same structure as this region in the intact protein as determined by CD and NMR spectroscopy. Thermodynamic and kinetic studies of NTL91--39 also show that its folding is two-state and cooperative, and it folds as fast as intact NTL9. NTL91--39 is the first example of a folded isolated split beta-alpha-beta motif with naturally occurring sequences of fewer than 40 residues. A thermostable variant of NTL91--39 was made by mutating K12 to M and was characterized and found to have a higher Tm and a faster folding rate than NTL91--39. Two fluorinated NTL9 variants were prepared by substituting V3 or V21 with trifluorovaline. The incorporation of trifluorovalines significantly stabilizes the protein and increases folding rates without inducing significant strain. phi-value analysis of the fluorinated NTL9 shows similar results to those from classical mutation studies.;CTL9 contains 92 residues and folds in two-state fashion. CTL9 folding is strongly pH dependent due to three histidines, H106, H134, and H144. Studies of pH dependent folding on a set of His to Gln mutants have been performed to investigate the denatured state and the transition state. The results show that there is no significant structure in the denatured state in the region of these three histidines and H134 is the most critical histidine in the transition state. The linkage analysis between pH-dependent stability and folding kinetics provides a novel way to study the transition state.