Localized nature of the transition-state structure in goat alpha-lactalbumin folding.
To investigate whether the structure partially formed in the molten globule folding intermediate of goat alpha-lactalbumin is further organized in the transition state of folding, we constructed a number of mutant proteins and performed Phi-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 beta-domain (L52A, W60A), the C-helix (K93A, L96A), the C-D loop (Y103F), the D-helix (L105A, L110A), and the C-terminal 3(10)-helix (W118F), have low Phi-values, less than 0.2. On the other hand, D87N, which is located on the Ca(2+)-binding site, has a high Phi-value, 0.91, indicating that tight packing of the side-chain around Asp87 occurs in the transition state. One beta-domain mutant (I55V) and three C-helix mutants (I89V, V90A, and I95V) demonstrated intermediate Phi-values, between 0.4 and 0.7. These results indicate that the folding nucleus in the transition state of goat alpha-LA is not extensively distributed over the alpha-domain of the protein, but very localized in a region that contains the Ca(2+)-binding site and the interface between the C-helix and the beta-domain. This is apparently in contrast with the fact that the molten globule state of alpha-lactalbumin has a partially formed structure inside the alpha-domain. It is concluded that the specific docking of the alpha and beta-domains at a domain interface is necessary for this protein to organize its native structure from the molten globule intermediate.
Department of Physics, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Kimiko
K
Saeki
Munehito
M
Arai
Takao
T
Yoda
Masaharu
M
Nakao
Kunihiro
K
Kuwajima
D000818:Animals; D002942:Circular Dichroism; D006041:Goats; D007768:Lactalbumin / Q000737:chemistry*; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding
D007768:Lactalbumin