Effects of a helix substitution on the folding mechanism of bovine alpha-lactalbumin.


The structure, stability, and unfolding-refolding kinetics of a chimeric protein, in which the amino acid sequence of the flexible loop region (residues 105-110) comes from equine lysozyme and the remainder of the sequence comes from bovine alpha-lactalbumin were studied by circular dichroism spectroscopy and stopped-flow measurements, and the results were compared with those of bovine alpha-lactalbumin. The substitution of the flexible loop in bovine alpha-lactalbumin with the helix D of equine lysozyme destabilizes the molten globule state, although the native state is significantly stabilized by substitution of the flexible loop region. The kinetic refolding and unfolding experiments showed that the chimeric protein refolds significantly faster and unfolds substantially slower than bovine alpha-lactalbumin. To characterize the transition state between the molten globule and the native states, we investigated the guanidine hydrochloride concentration dependence of the rate constants of refolding and unfolding. Despite the significant differences in the stabilities of both the molten globule and native states between the chimeric protein and bovine alpha-lactalbumin, the free energy level of the transition state is not affected by the amino acid substitution in the flexible loop region. Our results suggest that the destabilization in the molten globule state of the chimeric protein is caused by the disruption of the non-native interaction in the flexible loop region and that the disruption of the non-native interaction reduces the free energy barrier of refolding. We conclude that the non-native interaction in the molten globule state may act as a kinetic trap for the folding of alpha-lactalbumin. Study holds ProTherm entries: 17564, 17565, 17566 Extra Details: Native -> Intermediate non-native interaction; chimera; protein folding; molten globule; alpha-lactalbumin

Submission Details

ID: j2w2GiWA4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:49 p.m.

Version: 1

Publication Details
Mizuguchi M;Kobashigawa Y;Kumaki Y;Demura M;Kawano K;Nitta K,Proteins (2002) Effects of a helix substitution on the folding mechanism of bovine alpha-lactalbumin. PMID:12211019
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 P00711 LALBA_BOVIN
99.3 Alpha-lactalbumin Q9TSR4 LALBA_BOSMU
98.6 Alpha-lactalbumin Q9TSN6 LALBA_BUBBU
97.2 Alpha-lactalbumin P09462 LALBA_SHEEP
95.1 Alpha-lactalbumin P00712 LALBA_CAPHI