Energetics of the alpha-lactalbumin states: a calorimetric and statistical thermodynamic study.


Abstract

The temperature dependence of the heat capacity function of holo and apo alpha-lactalbumin has been studied by high sensitivity differential scanning microcalorimetry. The heat capacities of the holo and apo forms in the native state were found to be close to, but somewhat higher than, that of lysozyme, which has a similar structure. At pH values higher than 5, the heat-denatured state and the unfolded state are indistinguishable. At lower pH values, the heat capacity of the state obtained by heat or acid denaturation is lower than what is expected for the completely unfolded polypeptide chain, but it approaches that value at higher temperatures. The heat capacity increment of the denatured state correlates well with the amount of residual structure measured by ellipticity (i.e., the lower the residual structure, the higher the heat capacity). The extent of residual structure in the denatured state, which is exceptionally high in alpha-lactalbumin, decreases upon increasing temperature and at approximately 110 degrees C becomes close to that observed in 6 M GdmCl. Above 110 degrees C, the denatured state of alpha-lactalbumin is practically indistinguishable in heat capacity and ellipticity from the fully unfolded state. The calorimetric data have been analyzed quantitatively using a statistically thermodynamic formalism. This analysis indicates that the long-range or global cooperativity of the protein is lost after heat denaturation of the native state, causing the remaining elements of residual structure to behave in a more or less independent fashion. At pH values close to neutral, heat denaturation occurs at high temperature and yields a totally unfolded polypeptide with no measurable population of partly folded intermediates. At lower pH values, denaturation occurs at lower temperatures and a progressively higher population of intermediates is observed. At pH 4.2, about 50% of the molecules is in compact intermediate states immediately after heat denaturation; however, at pH 3.5, this percentage is close to 80% and at pH 3.0 it reaches about 100% of the protein molecules. Upon heating, the unfolded state progressively becomes the predominant species. The analysis of the heat capacity data for alpha-lactalbumin indicates that the best model to account for the observed behavior is one in which the denatured state is represented as a distribution of substates with varying degrees of residual structure. At low temperatures, the distribution is centered around rather compact substates with significant residual structure. At higher temperatures, the distribution shifts toward states with less residual structure and eventually to the completely unfolded state. Study holds ProTherm entries: 4659, 4660, 4661, 4662, 4663, 4664, 4665, 4666, 4667, 4668 Extra Details: heat capacity; long-range; global cooperativity;,partly folded intermediates

Submission Details

ID: Js4sb9pU

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Griko YV;Freire E;Privalov PL,Biochemistry (1994) Energetics of the alpha-lactalbumin states: a calorimetric and statistical thermodynamic study. PMID:8110793
Additional Information

Study Summary

Number of data points 30
Proteins Alpha-lactalbumin ; Alpha-lactalbumin
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dCp temp:44.3 C, pH:3.0 ; Experimental Assay: dHcal temp:44.3 C, pH:3.0 ; Experimental Assay: dCp temp:29.8 C, pH:3.2 ; Experimental Assay: dHcal temp:29.8 C, pH:3.2 ; Experimental Assay: dCp pH:3.5, temp:41.0 C ; Experimental Assay: dHcal pH:3.5, temp:41.0 C ; Experimental Assay: dCp pH:4.2, temp:57.0 C ; Experimental Assay: dHcal pH:4.2, temp:57.0 C ; Experimental Assay: dCp temp:76.0 C, pH:5.2 ; Experimental Assay: dHcal temp:76.0 C, pH:5.2 ; Experimental Assay: dCp buffers:Tris-HCl: 5 mM, pH:8.0 ; Experimental Assay: dHcal buffers:Tris-HCl: 5 mM, pH:8.0 ; Experimental Assay: Tm buffers:Tris-HCl: 5 mM, pH:8.0 ; Experimental Assay: dHvH buffers:Tris-HCl: 5 mM, pH:8.0 ; Experimental Assay: dCp pH:3.2 ; Experimental Assay: dHcal pH:3.2 ; Experimental Assay: Tm buffers:acetate: 10 mM, pH:3.2 ; Experimental Assay: dHvH buffers:acetate: 10 mM, pH:3.2 ; Experimental Assay: dCp pH:3.5 ; Experimental Assay: dHcal pH:3.5 ; Experimental Assay: Tm buffers:acetate: 10 mM, pH:3.5 ; Experimental Assay: dHvH buffers:acetate: 10 mM, pH:3.5 ; Experimental Assay: dCp pH:4.2 ; Experimental Assay: dHcal pH:4.2 ; Experimental Assay: Tm pH:4.2, buffers:acetate: 10 mM ; Experimental Assay: dHvH pH:4.2, buffers:acetate: 10 mM ; Experimental Assay: dCp pH:5.2 ; Experimental Assay: dHcal pH:5.2 ; Experimental Assay: Tm buffers:acetate: 10 mM, pH:5.2 ; Experimental Assay: dHvH buffers:acetate: 10 mM, pH:5.2
Libraries Mutations for sequence MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL

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
95.1 Alpha-lactalbumin P00712 LALBA_CAPHI
97.2 Alpha-lactalbumin P09462 LALBA_SHEEP
98.6 Alpha-lactalbumin Q9TSN6 LALBA_BUBBU
99.3 Alpha-lactalbumin Q9TSR4 LALBA_BOSMU
100.0 Alpha-lactalbumin P00711 LALBA_BOVIN