The equilibrium unfolding and the kinetic folding and unfolding of goat alpha-lactalbumin (GLA) were studied by near- and far-ultraviolet circular dichroism (CD) and by stopped-flow fluorescence spectroscopy. Specifically, the influence of environmental conditions such as pH and Ca2+ binding was examined. Compared to the apo-form, the Ca2+-bound form was found to be strongly stabilized in equilibrium conditions at pH 7.5 and 25 degrees C. The kinetics of the refolding of apo-GLA show a major change of fluorescence intensity during the experimental dead-time, but this unresolved effect is strongly diminished in holo-GLA. In both cases, however, the chevron plots can adequately be fitted to a three-state model. Moreover, double-mix stopped-flow experiments showed that the native state (N) is reached through one major pathway without the occurrence of alternative tracks. In contrast to the homologous bovine alpha-lactalbumin (BLA), the compactness of GLA is strongly influenced by the presence of Ca2+ ions. Unlike the two-state transition observed in guanidine hydrochloride (GdnHCl)-induced equilibrium denaturation experiments at higher pH, an equilibrium intermediate state (I) is involved in denaturation at pH 4.5. In the latter case, analysis of the kinetic data makes clear that the intermediate and the unfolded states (U) show practically no Gibbs free energy difference and that they are in rapid equilibrium with each other. A possible explanation for these variations in stability and in folding characteristics with pH could be the degree of protonation of His107 that directly influences non-native interactions. Variation of environmental conditions and even small differences in sequence, therefore, can result in important effects on thermodynamic and folding parameters. Study holds ProTherm entries: 18215, 18216, 18217, 18218, 18219, 18220 Extra Details: 2mM EDTA was added in the experiment lactalbumin; stopped-flow fluorescence spectroscopy; kinetics; protein folding; intermediate state; circular dichroism; lysozyme
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:50 p.m.
|Number of data points||12|
|Proteins||Alpha-lactalbumin ; Alpha-lactalbumin|
|Assays/Quantities/Protocols||Experimental Assay: m pH:4.5 ; Experimental Assay: dG_H2O pH:4.5 ; Experimental Assay: m pH:5.5 ; Experimental Assay: dG_H2O pH:5.5 ; Experimental Assay: m pH:6.5 ; Experimental Assay: dG_H2O pH:6.5 ; Experimental Assay: m pH:7.5 ; Experimental Assay: dG_H2O pH:7.5|
|Libraries||Mutations for sequence EQLTKCEVFQKLKDLKDYGGVSLPEWVCTAFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSRNICNISCDKFLDDDLTDDIVCAKKILDKVGINYWLAHKALCSEKLDQWLCEKL|