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Localized Nature of the Transition-state Structure in Goat α-Lactalbumin Folding

Journal of Molecular Biology
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  • Biology


To investigate whether the structure partially formed in the molten globule folding intermediate of goat α-lactalbumin is further organized in the transition state of folding, we constructed a number of mutant proteins and performed Φ-value analysis on them. For this purpose, we measured the equilibrium unfolding transitions and kinetic refolding and unfolding reactions of the mutants using equilibrium and stopped-flow kinetic circular dichroism techniques. The results show that the mutants with mutations located in the A-helix (V8A, L12A), the B-helix (V27A), the β-domain (L52A, W60A), the C-helix (K93A, L96A), the C–D loop (Y103F), the D-helix (L105A, L110A), and the C-terminal 310-helix (W118F), have low Φ-values, less than 0.2. On the other hand, D87N, which is located on the Ca2+-binding site, has a high Φ-value, 0.91, indicating that tight packing of the side-chain around Asp87 occurs in the transition state. One β-domain mutant (I55V) and three C-helix mutants (I89V, V90A, and I95V) demonstrated intermediate Φ-values, between 0.4 and 0.7. These results indicate that the folding nucleus in the transition state of goat α-LA is not extensively distributed over the α-domain of the protein, but very localized in a region that contains the Ca2+-binding site and the interface between the C-helix and the β-domain. This is apparently in contrast with the fact that the molten globule state of α-lactalbumin has a partially formed structure inside the α-domain. It is concluded that the specific docking of the α and β-domains at a domain interface is necessary for this protein to organize its native structure from the molten globule intermediate.

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