Folding Intermediate and Folding Nucleus for I→N and U→I→N Transitions in Apomyoglobin: Contributions by Conserved and Nonconserved Residues |
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Authors: | Ekaterina N Samatova Vitaly A Balobanov Dmitry A Dolgikh Alexei V Finkelstein |
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Institution: | † Institute of Protein Research, Russian Academy of Sciences, Moscow, Russian Federation ‡ M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation |
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Abstract: | Kinetic investigation on the wild-type apomyoglobin and its 12 mutants with substitutions of hydrophobic residues by Ala was performed using stopped-flow fluorescence. Characteristics of the kinetic intermediate I and the folding nucleus were derived solely from kinetic data, namely, the slow-phase folding rate constants and the burst-phase amplitudes of Trp fluorescence intensity. This allowed us to pioneer the ?-analysis for apomyoglobin. As shown, these mutations drastically destabilized the native state N and produced minor (for conserved residues of G, H helices) or even negligible (for nonconserved residues of B, C, D, E helices) destabilizing effect on the state I. On the other hand, conserved residues of A, G, H helices made a smaller contribution to stability of the folding nucleus at the rate-limiting I→N transition than nonconserved residues of B, D, E helices. Thus, conserved side chains of the A-, G-, H-residues become involved in the folding nucleus before crossing the main barrier, whereas nonconserved side chains of the B-, D-, E-residues join the nucleus in the course of the I→N transition. |
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