Contribution of Thr29 to the thermodynamic stability of goat alpha-lactalbumin as determined by experimental and theoretical approaches.


Abstract

The Thr29 residue in the hydrophobic core of goat alpha-lactalbumin (alpha-LA) was substituted with Val (Thr29Val) and Ile (Thr29Ile) to investigate the contribution of Thr29 to the thermodynamic stability of the protein. We carried out protein stability measurements, X-ray crystallographic analyses, and free energy calculations based on molecular dynamics simulation. The equilibrium unfolding transitions induced by guanidine hydrochloride demonstrated that the Thr29Val and Thr29Ile mutants were, respectively, 1.9 and 3.2 kcal/mol more stable than the wild-type protein (WT). The overall structures of the mutants were almost identical to that of WT, in spite of the disruption of the hydrogen bonding between the side-chain O-H group of Thr29 and the main-chain C=O group of Glu25. To analyze the stabilization mechanism of the mutants, we performed free energy calculations. The calculated free energy differences were in good agreement with the experimental values. The stabilization of the mutants was mainly caused by solvation loss in the denatured state. Furthermore, the O-H group of Thr29 favorably interacts with the C=O group of Glu25 to form hydrogen bonds and, simultaneously, unfavorably interacts electrostatically with the main-chain C=O group of Thr29. The difference in the free energy profile of the unfolding path between WT and the Thr29Ile mutant is discussed in light of our experimental and theoretical results. Study holds ProTherm entries: 11616, 11617 Extra Details: protein stability; X-ray crystallography; free energy calculation;,molecular dynamics simulation

Submission Details

ID: ipuTCwrj3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:42 p.m.

Version: 1

Publication Details
Horii K;Saito M;Yoda T;Tsumoto K;Matsushima M;Kuwajima K;Kumagai I,Proteins (2001) Contribution of Thr29 to the thermodynamic stability of goat alpha-lactalbumin as determined by experimental and theoretical approaches. PMID:11536356
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 PDB Entries

Structure ID Release Date Resolution Structure Title
3B0K 2012-06-13 1.6 Crystal structure of alpha-lactalbumin
1FKQ 2001-02-14 1.8 RECOMBINANT GOAT ALPHA-LACTALBUMIN T29V
6IP9 2019-02-20 1.85 Crystal Structure of Lanthanum ion (La3+) bound bovine alpha-lactalbumin
1HMK 1999-11-26 2.0 RECOMBINANT GOAT ALPHA-LACTALBUMIN
1FKV 2001-02-14 2.0 RECOMBINANT GOAT ALPHA-LACTALBUMIN T29I
1F6S 2000-12-13 2.2 CRYSTAL STRUCTURE OF BOVINE ALPHA-LACTALBUMIN
1F6R 2000-12-13 2.2 CRYSTAL STRUCTURE OF APO-BOVINE ALPHA-LACTALBUMIN
2G4N 2007-02-20 2.3 Anomalous substructure of alpha-lactalbumin
1HFZ 1997-07-29 2.3 ALPHA-LACTALBUMIN
1HFY 1997-07-07 2.3 ALPHA-LACTALBUMIN

Relevant UniProtKB Entries

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