Influence of Trp mutation on native, intermediate, and transition states of goat alpha-lactalbumin: an equilibrium and kinetic study.


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

Submission Details

ID: XQJsyXi9

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:52 p.m.

Version: 1

Publication Details
Chedad A;Van Dael H;Vanhooren A;Hanssens I,Biochemistry (2005) Influence of Trp mutation on native, intermediate, and transition states of goat alpha-lactalbumin: an equilibrium and kinetic study. PMID:16285716
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Alpha-lactalbumin P00712 LALBA_CAPHI
97.9 Alpha-lactalbumin P09462 LALBA_SHEEP
96.5 Alpha-lactalbumin Q9TSN6 LALBA_BUBBU
95.1 Alpha-lactalbumin P00711 LALBA_BOVIN
94.4 Alpha-lactalbumin Q9TSR4 LALBA_BOSMU