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
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:50 p.m.
|Number of data points||12|
|Proteins||Alpha-lactalbumin ; Alpha-lactalbumin|
|Assays/Quantities/Protocols||Experimental Assay: Cm ionic:CaCl2: 5 mM ; Experimental Assay: m ionic:CaCl2: 5 mM ; Experimental Assay: dG_H2O ionic:CaCl2: 5 mM ; Experimental Assay: Cm ionic:: ; Experimental Assay: m ionic:: ; Experimental Assay: dG_H2O ionic::|
|Libraries||Mutations for sequence MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL|