pH-dependent stability of the human alpha-lactalbumin molten globule state: contrasting roles of the 6 - 120 disulfide and the beta-subdomain at low and neutral pH.
Many proteins are capable of populating partially folded states known as molten globule states. Alpha-lactalbumin forms a molten globule under a range of conditions including low pH (the A-state) and at neutral pH in the absence of Ca(2+) with modest amounts of denaturant. The A-state is the most thoroughly characterized and thought to mimic a kinetic intermediate populated during refolding at neutral pH. We demonstrate that the properties and interactions that stabilize the A-state and the pH 7 molten globule of human alpha-lactalbumin differ. The unfolding of the wild-type protein is compared to the unfolding of a variant that lacks the 6 - 120 disulfide bond and to an autonomously folded peptide construct that we have previously shown represents the minimum core structure of the A-state of human alpha-lactalbumin. Studies conducted at pH 2 and 7 show that the disulfide makes little contribution to the stability of the molten globule at pH 7 but is important at pH 2. In contrast, the beta-subdomain of the protein is less important at pH 2 than at pH 7. The role of helix propensity in stabilizing the different forms of the molten globule state is examined and it is shown that it cannot account for the differences. The strikingly different behavior observed at pH 2 and 7 indicates that the A-state may not be a rigorous mimic of the folding intermediate populated at pH 7.
Horng
Jia-Cherng
JC
Department of Chemistry, State University of New York at Stony Brook, 11794-3400, USA.
Demarest
Stephen J
SJ
Raleigh
Daniel P
DP
D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007768:Lactalbumin / Q000737:chemistry*; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D004220:Disulfides; D007768:Lactalbumin