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.

Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II

“…Approximation of the complicated chevron plot requires a special explanation and mathematical calculations as described in detail previously. 2,7,17 For the current purposes, it is sufficient to have a general form of the chevron plot obtained from experiment without its mathematical analysis. However, it is noteworthy that the minimum of the chevron plot corresponds to the concentration of the denaturing agent when the folding of the protein turns into its unfolding.…”

Loops linking secondary structure elements affect the stability of molten globule intermediate state of apomyoglobin

“…Based on a plasmid containing a wild-type apomyoglobin gene, plasmids containing mutant apomyoglobin gene variants with selected substitutions were derived. Recombinant proteins were isolated and purified according to methods described in our earlier works [2,[6][7][8][9]. The effect of mutations on the structure of the native state of apomyoglobin was estimated by means of circular dichroism (CD) spectra in far ultraviolet (UV) area.…”

“…In previous works of our laboratory, systematic investigations were conducted in order to reveal the effect of various mutations on apomyoglobin energy profile [2,[6][7][8][9]. Circular dichroism and fluorescence measurement showed that none of 20 studied single amino acid substitutions in the core or on the myoglobin surface did alter the stability of molten globule intermediate state [6,8,9].…”

“…All the measurements were carried out at 11˚C in 20 mM sodium phosphate buffer, pH = 6.2.Denaturation/renaturation kinetic curves at different urea concentrations (0-5.5M) were obtained for each protein variant. The technique of the measurements was described in detail in our previous works [2,[6][7][8][9].…”

How membrane surface affects protein structure

“…In the Laboratory of Protein Physics (Institute of Protein Research, Russian Academy of Sciences) a detailed study of apoMb folding was done. The object of the investigation was the kinetics of folding of mutant proteins with substitutions both in the conserved and non conserved positions important for folding [52,53].…”