Equilibrium circular dichroism and kinetic stopped-flow fluorescence studies on the stability and the folding kinetics of a set of Trp to Phe mutants of goat alpha-lactalbumin (GLA) were used to characterize the native, intermediate, and transition states of these constructs. GLA contains four tryptophan residues, three of which, Trp26, Trp104, and Trp118, are located in the alpha-domain, while the fourth, Trp60, is located in the beta-domain. Trp26, Trp60, and Trp104 are part of a hydrophobic cluster, whereas Trp118 is situated in a more flexible region near the C-terminal end of the protein. In each case, the mutation leads to a reduction in the overall stability, but only for W26F and W60F is an equilibrium intermediate observed in guanidine hydrochloride-induced unfolding experiments. In kinetic refolding experiments, however, for all samples a burst phase is observed, the amplitude of which depends on the specific mutation. Refolding and unfolding kinetics can adequately be described by a sequential three-state mechanism. phi value analysis showed that the local structure around Trp26, Trp60, and Trp104 is formed in the intermediate and in the transition state of the folding reaction, while around Trp118 no persistent native contacts are observed. From these findings, we conclude that, although hydrophobicity is a major driving force for folding, minor steric changes induced by point mutation can considerably influence the overall stability and the folding process of the protein. Study holds ProTherm entries: 19739, 19740, 19741, 19742, 19743, 19744, 19745, 19746, 19747 Extra Details: hydrophobic cluster; flexible; kinetics; three-state mechanism; steric changes
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:52 p.m.
|Number of data points||22|
|Proteins||Alpha-lactalbumin ; Alpha-lactalbumin|
|Assays/Quantities/Protocols||Experimental Assay: ddG_H2O ; Experimental Assay: m ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence EQLTKCEVFQKLKDLKDYGGVSLPEWVCTAFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSRNICNISCDKFLDDDLTDDIVCAKKILDKVGINYWLAHKALCSEKLDQWLCEKL|