Understanding how proteins spontaneously fold to their unique native structures is one of the premier topics of structural biology. This thesis presents the results of experimental studies designed to investigate an intermediate state involved in the folding of the 123 residue protein α-lactalbumin (αLA).; To understand the importance of local interactions in the denatured state of αLA that may be important for the formation of structure in the molten globule state, several peptide models derived from the crucial α-subdomain of the protein were synthesized using solid phase peptide methods and purified using reverse phase HPLC. It was determined from circular dichroism (CD) experiments, 1 and 2-dimensional 1H nuclear magnetic resonance (NMR) experiments and computation that residues from the D and C-terminal 310 helix regions have a propensity to form locally-stabilized, non-native structures in isolation. Disruption of these non-native structures likely represents small barriers for the correct folding of the protein.; Combination of two peptides encompassing the native A, B, D and C-terminal 310 helix regions of αLA via the native 28 to 111 disulfide bond leads to a peptide construct that displays all the major properties of the molten globule state as judged by near and far UV CD, fluorescence, ANS binding and urea denaturation measurements. Removal of any one native helical element results in a peptide construct that is primarily unfolded.; The structurally homologous protein hen egg white lysozyme folds in a very different fashion than αLA. A peptide construct of the A, B, D and C-terminal 310 helices of hen lysozyme was found to be primarily unstructured in solution as judged from CD and fluorescence measurements. Experiments with chimeric αLA/lysozyme constructs indicate that any peptide construct containing the A and B helices of αLA has the ability to fold given that it can make a significant number of non-specific interactions with other peptide regions. The inability of the isolated α-subdomain of hen lysozyme to form structure in isolation may account for the fact that hen lysozyme does not form an equilibrium stable molten globule state.; The disulfide bonds in the α-domain are susceptible to reduction by dithiotreitol (DTT). A three disulfide variant of αLA with the 6-120 disulfide broken and a two disulfide variant of αLA with both the 6-120 and 28-111 disulfide bonds broken were prepared. Both variants are capable of folding to the native structure. The results of urea denaturation experiments demonstrate that the region in the immediate vicinity of the 6-120 disulfide bond is likely unstructured in the molten globule state. Reduction of the 28-111 disulfide only marginally destabilizes both the native and molten globule states of the protein. These results indicate that the region near the 28-111 disulfide bond may already have a native-like topology in the unfolded state of the protein.
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