Unfolding and refolding pathways of a major kinetic trap in the oxidative folding of alpha-lactalbumin.


Abstract

Alpha-lactalbumin (alphaLA)-IIIA is a major kinetic intermediate present along the pathways of reductive unfolding and oxidative folding of bovine alpha-lactalbumin (alphaLA). It is a three-disulfide variant of native alphaLA lacking Cys(6)-Cys(120) at the alpha-helical domain. Stability and the unfolding/refolding mechanism of carboxymethylated alphaLA-IIIA have been investigated previously by stop-flow circular dichroism (CD) and fluorescence spectroscopy. A stable intermediate compatible with molten globule was shown to exist along the pathways of unfolding-refolding of alphaLA-IIIA [Ikeguchi et al. (1992) Biochemistry 31, 16695-12700; Horng et al. (2003) Proteins 52, 193-202]. We investigate here the unfolding-refolding pathways and conformational stability of alphaLA-IIIA using the method of disulfide scrambling with the following specific aims: (a) to isolate and characterize the observed stable molten globule, (b) to analyze the heterogeneity of folding-unfolding intermediates, (c) to elucidate the disulfide structure of extensively unfolded isomer of alphaLA-IIIA, and (d) to clarify the relative conformational stability between alphaLA-IIIA and alphaLA. Two scrambled isomers, designated as X-alphaLA-IIIA-c and X-alphaLA-IIIA-a (X stands for scrambled), were isolated under mild and strong denaturing conditions. Their disulfide structures, CD spectra, and manners of refolding to form the native alphaLA-IIIA were analyzed in this report. The results are consistent with the notion that X-alphaLA-IIIA-c and X-alphaLA-IIIA-a represent a partially unfolded and an extensively unfolded isomers of native alphaLA-IIIA, respectively. The unfolding-refolding pathways of alphaLA-IIIA are elaborated and compared with that of intact alphaLA. These results display new insight into one of the most extensively studied molecules in the field of protein folding and unfolding. Study holds ProTherm entries: 18637, 18638, 18639, 18640 Extra Details: kinetic intermediate; conformational stability; molten globule; disulfide

Submission Details

ID: R4ePXALr

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:50 p.m.

Version: 1

Publication Details
Salamanca S;Chang JY,Biochemistry (2005) Unfolding and refolding pathways of a major kinetic trap in the oxidative folding of alpha-lactalbumin. PMID:15641801
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
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