Equilibrium and kinetic studies on folding of the authentic and recombinant forms of human alpha-lactalbumin by circular dichroism spectroscopy.


Abstract

The equilibrium and kinetics of the unfolding and refolding of authentic and recombinant human alpha-lactalbumin, the latter of which had an extra methionine residue at the N-terminus, were studied by circular dichroism spectroscopy, and the results were compared with the results for bovine and goat alpha-lactalbumins obtained in our previous studies. As observed in the bovine and goat proteins, the presence of the extra methionine residue in the recombinant protein remarkably destabilized the native state, and the destabilization was entirely ascribed to an increase in the rate of unfolding. The thermodynamic stability of the native state against the unfolded state was lower, and the thermodynamic stability of the molten globule state against the unfolded state was higher for the human protein than for the other alpha-lactalbumins previously studied. Thus, the population of the molten globule intermediate was higher during the equilibrium unfolding of human alpha-lactalbumin by guanidine hydrochloride. Unlike the molten globule states of the bovine and goat proteins, the human alpha-lactalbumin molten globule showed remarkably more intense circular dichroism ellipticity than the native state in the far-ultraviolet region below 225 nm. During refolding from the unfolded state, human alpha-lactalbumin thus exhibited overshoot kinetics, in which the alpha-helical peptide ellipticity exceeded the native value when the molten globule folding intermediate was formed in the burst phase. The subsequent folding involved reorganization of nonnative secondary structures. It should be noted that the rate constant of the major refolding phase was approximately the same among the three types of alpha-lactalbumin and that the rate constant of unfolding was accelerated 18-600 times in the human protein, and these results interpreted the lower thermodynamic stability of this protein. Study holds ProTherm entries: 10183, 10184, 10185, 10186, 10187, 10188, 10189 Extra Details: rate of unfolding; thermodynamic stability; molten globule state;,secondary structures

Submission Details

ID: sGH3MHGA3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:39 p.m.

Version: 1

Publication Details
Chaudhuri TK;Arai M;Terada TP;Ikura T;Kuwajima K,Biochemistry (2000) Equilibrium and kinetic studies on folding of the authentic and recombinant forms of human alpha-lactalbumin by circular dichroism spectroscopy. PMID:11112553
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
1CB3 1999-06-08 LOCAL INTERACTIONS DRIVE THE FORMATION OF NON-NATIVE STRUCTURE IN THE DENATURED STATE OF HUMAN ALPHA-LACTALBUMIN: A HIGH RESOLUTION STRUCTURAL CHARACTERIZATION OF A PEPTIDE MODEL IN AQUEOUS SOLUTION
1B9O 1999-03-31 1.15 HUMAN ALPHA-LACTALBUMIN, LOW TEMPERATURE FORM
3B0O 2012-06-13 1.61 Crystal structure of alpha-lactalbumin
1ALC 1989-10-15 1.7 REFINED STRUCTURE OF BABOON ALPHA-LACTALBUMIN AT 1.7 ANGSTROMS RESOLUTION. COMPARISON WITH C-TYPE LYSOZYME
1HML 1995-01-26 1.7 ALPHA_LACTALBUMIN POSSESSES A DISTINCT ZINC BINDING SITE
1A4V 1999-04-27 1.8 ALPHA-LACTALBUMIN
3B0I 2012-06-13 1.8 Crystal structure of recombinant human alpha lactalbumin
4L41 2013-10-02 2.7 Human Lactose synthase: A 2:1 complex between human alpha-lactalbumin and human beta1,4-galactosyltransferase

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.7 Alpha-lactalbumin P37154 LALBA_FELCA
94.3 Alpha-lactalbumin P12065 LALBA_PAPCY
100.0 Alpha-lactalbumin P00709 LALBA_HUMAN