Apomyoglobin stability as dependent on urea concentration and temperature at two pH values

Baryshnikova, E N; Шарапов, М. Г.; Kashparov, I. A.; Ilyina, Nelly B.; Bychkova, Valentina E.

doi:10.1007/s11008-005-0041-9

Cited by 12 publications

(15 citation statements)

References 24 publications

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“…All fluorescence and far ultraviolet (UV) circular dichroism (CD) measurements were carried out at 11 C; 0.01 M sodium acetate, pH 6.2, was used as a buffer system (40). Equilibrium protein fluorescence measurements were taken on a model No.…”

Section: Equilibrium Studies Of Urea-induced Denaturing Transitionsmentioning

confidence: 99%

Folding Intermediate and Folding Nucleus for I→N and U→I→N Transitions in Apomyoglobin: Contributions by Conserved and Nonconserved Residues

Samatova

Melnik

Balobanov

et al. 2010

Biophysical Journal

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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.

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Section: Equilibrium Studies Of Urea-induced Denaturing Transitionsmentioning

confidence: 99%

Folding Intermediate and Folding Nucleus for I→N and U→I→N Transitions in Apomyoglobin: Contributions by Conserved and Nonconserved Residues

Samatova

Melnik

Balobanov

et al. 2010

Biophysical Journal

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“…Because apomyoglobin folds quite rapidly at room temperature, a lower temperature should be used to slow down the reaction rate. On the other hand, the possibility of cold denaturation of this protein previously reported by Griko et al (1988) made us choose 11 C for our experiments (Baryshnikova et al 2005).…”

Section: Tryptophan (Trp) Fluorescencementioning

confidence: 99%

Three‐state protein folding: Experimental determination of free‐energy profile

et al. 2005

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When considering protein folding with a transient intermediate, a difficulty arises as to determination of the rates of separate transitions. Here we overcome this problem, using the kinetic studies of the unfolding/refolding reactions of the three-state protein apomyoglobin as a model. Amplitudes of the protein refolding kinetic burst phase corresponding to the transition from the unfolded (U) to intermediate (I) state, that occurs prior to the native state (N) formation, allow us to estimate relative populations of the rapidly converting states at various final urea concentrations. On the basis of these proportions, a complicated experimental chevron plot has been deconvolved into the urea-dependent rates of the I$N and U$N transitions to give the dependence of free energies of the main transition state and of all three (N, I, and U) stable states on urea concentration.Keywords: protein folding; folding intermediates; tryptophan fluorescence; chevron plot; stoppedflow; apomyoglobin Apomyoglobin is a good object for protein folding studies because its thermodynamic (Griko et al. 1988;Hughson et al. 1990;Jennings and Wright 1993;Jamin et al. 2000) and structural (Barrick and Baldwin 1993a,b;Eliezer and Wright 1996;Eliezer et al. 1998;Jamin and Baldwin 1998;Jamin et al. 1999;Lecomte et al. 1999;Tcherkasskaya and Ptitsyn 1999;Tsui et al. 1999;Tcherkasskaya et al. 2000) properties in both the native and intermediate conformational states are well elucidated. At neutral pH, apomyoglobin structure is ''native'' globular, with seven of eight helices of holomyoglobin tightly packed (A-E and G, H; while F, involved in the heme binding, is disordered in apoprotein) (Eliezer and Wright 1996). At pH 4.2, the native structure undergoes the transition into a thermodynamically stable ''molten globule' ' (Dolgikh et al. 1981; Ptitsyn 1995) intermediate state (Griko et al. 1988) that contains three helices (Hughson et al. 1990), A, G, and H. This intermediate has two sub-states (stable at pH 4.2 and pH 3.9), which convert one into the other within a millisecond time range (Jamin and Baldwin 1998;Jamin et al. 1999). It was shown using the stopped-flow and quenchflow techniques that urea-induced apomyoglobin refolding goes via a kinetic intermediate that forms within 6 msec and is structurally similar to the equilibrium molten globule intermediate observed at pH 4.2 (Jennings and Wright 1993). Subsequent kinetic studies suggested that this intermediate is on-pathway (Jamin and Baldwin 1998). Quenchflow amide proton exchange combined with mass-spectrometry confirmed that apomyoglobin folds by a single pathway and that the intermediate is obligatory (Tsui et al. 1999). NMR analysis of mutant apomyoglobins also showed that some point mutation may change the folding pathway of apomyoglobin (Garcia et al. 2000).The presence of a kinetic intermediate on the apomyoglobin folding pathway makes this protein attractive for studies of three-state folding/unfolding reactions. Protein folding involves a transition state and, for the m...

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“…In the case of myoglobin, folding of which is described by the two-stage model, an additional parameter F I , occupancy of intermediate state [3], must be introduced into the formula (2) to take into account the influence of fast folding phase onto the subsequent slow phase:…”

Section: Resultsmentioning

confidence: 99%

“…where A f , B f , A u , and B u are the parameters of linear functions depicting real rate constants of slow phase of apomyoglobin folding/unfolding; X i and S i are parameters of S-shaped curves depicting occupancy of the intermediate state: X i is the center of the sigmoid, and S i is the slope of sigmoid [3]. If the folding pathway of apomyoglobin is not affected by mutations, then the chevron plots for the wild-type protein and its mutations should have the same slope of folding branches (A f ), the same slope of unfolding branches (A u ), and the same occupancy parameters for the intermediate state, X i and S i [3]. Only the position of the unfolding and refolding branches, that is., parameters B f and B u , should change, but they can be quite accurately determined experimentally at low and high urea concentrations ('weights' of the points at the borders of the plot are higher than in the middle).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2020

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Apomyoglobin is a widely used model for studying the molecular mechanisms of globular protein folding. This work aimed to analyze the effects of rigidity and length of loops linking protein secondary structure elements on the stability of the molten globule intermediate state. For this purpose, we studied folding/unfolding of mutant apomyoglobin forms with substitutions of looplocated proline residues to glycine and with loop extension by three or six glycine residues. The kinetic and equilibrium experiments performed gave an opportunity to calculate free energies of different apomyoglobin states. Our analysis revealed that the mutations introduced into the apomyoglobin loops have a noticeable effect on the stability of the intermediate state compared to the unfolded state.

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Apomyoglobin stability as dependent on urea concentration and temperature at two pH values

Cited by 12 publications

References 24 publications

Folding Intermediate and Folding Nucleus for I→N and U→I→N Transitions in Apomyoglobin: Contributions by Conserved and Nonconserved Residues

Folding Intermediate and Folding Nucleus for I→N and U→I→N Transitions in Apomyoglobin: Contributions by Conserved and Nonconserved Residues

Three‐state protein folding: Experimental determination of free‐energy profile

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

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