To test the occurrence of local particularities during the unfolding of Ca2+-loaded goat alpha-lactalbumin (GLA) we replaced Trp60 and -118, either one or both, by Phe. In contrast with alternative studies, our recombinant alpha-lactalbumins are expressed in Pichia pastoris and do not contain the extra N-terminal methionine. The substitution of Trp60 leads to a reduction of the global stability. The effect of the Trp118Phe substitution on the conformation and stability of the mutant, however, is negligible. Comparison of the fluorescence spectra of these mutants makes clear that Trp60 and -118 are strongly quenched in the native state. They both contribute to the quenching of Trp26 and -104 emission. By the interplay of these quenching effects, the fluorescence intensity changes upon thermal unfolding of the mutants behave very differently. This is the reason for a discrepancy of the apparent transition temperatures derived from the shift of the emission maxima (Tm,Fl lambda) and those derived from DSC (Tm,DSC). However, the transition temperatures derived from fluorescence intensity (Tm,Fl int) and from DSC (Tm,DSC), respectively, are quite similar, and thus, no local rearrangements are observed upon heat-induced unfolding. At room temperature, the occurrence of specific local rearrangements upon GdnHCl-induced denaturation of the different mutants is deduced from the apparent free energies of their transition state obtained from stopped-flow fluorescence measurements. By phi-value analysis it appears that, while the surroundings of Trp118 are exposed in the kinetic transition state, the surroundings of Trp60 remain native. Study holds ProTherm entries: 19259, 19260, 19261, 19262, 19263, 19264, 19265, 19266, 19267, 19268, 19269, 19270, 19271, 19272, 19273, 19274, 19275 Extra Details: Goat alpha lactalbumin prepared from fresh milk whey; unfolding temperature is deduced from the shifts of the fluorescence wavelength Trp-Phe mutants; protein folding; thermal unfolding; chemical unfolding; circular dichroism; fluorescence; stopped-flow kinetics; differential scanning calorimetry
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:51 p.m.
|Number of data points||27|
|Proteins||Alpha-lactalbumin ; Alpha-lactalbumin|
|Assays/Quantities/Protocols||Experimental Assay: Tm prot_conc:30 microM ; Experimental Assay: Tm prot_conc:5 microM ; Derived Quantity: dTm prot_conc:30 microM ; Derived Quantity: dTm prot_conc:5 microM|
|Libraries||Mutations for sequence EQLTKCEVFQKLKDLKDYGGVSLPEWVCTAFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSRNICNISCDKFLDDDLTDDIVCAKKILDKVGINYWLAHKALCSEKLDQWLCEKL|