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Folding intermediate and folding nucleus for I-->N and U-->I-->N transitions in apomyoglobin: contributions by conserved and nonconserved residues.

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 phi-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.

SUBMITTER: Samatova EN

PROVIDER: S-EPMC2856158 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Folding intermediate and folding nucleus for I-->N and U-->I-->N transitions in apomyoglobin: contributions by conserved and nonconserved residues.

Samatova Ekaterina N EN Melnik Bogdan S BS Balobanov Vitaly A VA Katina Natalya S NS Dolgikh Dmitry A DA Semisotnov Gennady V GV Finkelstein Alexei V AV Bychkova Valentina E VE

Biophysical journal 20100401 8

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 phi-analysis for apomyoglobin. As shown, these mutations drastically destabilized th ...[more]

PMID: 20409491

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Project description:The structures of apomyoglobin folding intermediates have been widely analyzed using physical chemistry methods including fluorescence, circular dichroism, small angle X-ray scattering, NMR, mass spectrometry, and rapid mixing. So far, at least two intermediates (on sub-millisecond- and millisecond-scales) have been demonstrated for apomyoglobin folding. The combination of pH-pulse labeling and NMR is a useful tool for analyzing the kinetic intermediates at the atomic level. Its use has revealed that the latter-phase kinetic intermediate of apomyoglobin (6 ms) was composed of helices A, B, G and H, whereas the equilibrium intermediate, called the pH 4 molten-globule intermediate, was composed mainly of helices A, G and H. The improved strategy for the analysis of the kinetic intermediate was developed to include (1) the dimethyl sulfoxide method, (2) data processing with the various labeling times, and (3) a new in-house mixer. Particularly, the rapid mixing revealed that helices A and G were significantly more protected at the earlier stage (400 µs) of the intermediate (former-phase intermediate) than the other helices. Mutation studies, where each hydrophobic residue was replaced with an alanine in helices A, B, E, F, G and H, indicated that both non-native and native-like structures exist in the latter-phase folding intermediate. The N-terminal part of helix B is a weak point in the intermediate, and the docking of helix E residues to the core of the A, B, G and H helices was interrupted by a premature helix B, resulting in the accumulation of the intermediate composed of helices A, B, G and H. The prediction-based protein engineering produced important mutants: Helix F in a P88K/A90L/S92K/A94L mutant folded in the latter-phase intermediate, although helix F in the wild type does not fold even at the native state. Furthermore, in the L11G/W14G/A70L/G73W mutant, helix A did not fold but helix E did, which is similar to what was observed in the kinetic intermediate of apoleghemoglobin. Thus, this protein engineering resulted in a changed structure for the apomyoglobin folding intermediate.

Project description:In two-component signal transduction, response regulator proteins contain the catalytic machinery for their own covalent phosphorylation and can catalyze phosphotransfer from a partner sensor kinase or autophosphorylate using various small molecule phosphodonors. Although response regulator autophosphorylation is physiologically relevant and a powerful experimental tool, the kinetic determinants of the autophosphorylation reaction and how those determinants might vary for different response regulators and phosphodonors are largely unknown. We characterized the autophosphorylation kinetics of 21 variants of the model response regulator Escherichia coli CheY that contained substitutions primarily at nonconserved active site positions D + 2 (CheY residue 59) and T + 2 (CheY residue 89), two residues C-terminal to conserved D57 and T87, respectively. Overall, the CheY variants exhibited a >10(5)-fold range of rate constants (kphos/KS) for reaction with phosphoramidate, acetyl phosphate, or monophosphoimidazole, with the great majority of rates enhanced versus that of wild-type CheY. Although phosphodonor preference varied substantially, nearly all the CheY variants reacted faster with phosphoramidate than acetyl phosphate. Correlation between the increased positive charge of the D + 2 and T + 2 side chains and faster rates indicated electrostatic interactions are a kinetic determinant. Moreover, sensitivities of rate constants to ionic strength indicated that both long-range and localized electrostatic interactions influence autophosphorylation kinetics. The increased nonpolar surface area of the D + 2 and T + 2 side chains also correlated with an enhanced autophosphorylation rate, especially for reaction with phosphoramidate and monophosphoimidazole. Computer docking suggested that highly accelerated monophosphoimidazole autophosphorylation rates for CheY variants with a tyrosine at position T + 2 likely reflect structural mimicry of phosphotransfer from the sensor kinase histidyl phosphate.

Dataset Information

Folding intermediate and folding nucleus for I-->N and U-->I-->N transitions in apomyoglobin: contributions by conserved and nonconserved residues.

Publications

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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