Molten Globule-Like State of Cytochrome <i>c</i> under Conditions Simulating Those Near the Membrane Surface

Bychkova, Valentina E.; Dujsekina, Alexandra E.; Klenin, S.I.; Tiktopulo, E. I.; Uversky, Vladimir N.; Ptitsyn, Oleg B.

doi:10.1021/bi9522460

Cited by 216 publications

(183 citation statements)

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“…The intermediate state in 50% DMSO resembles a highly ordered molten globule-like state, which has been observed in interleukin 4 (Redfield et al, 1994), apomyoglobin (Lin et al, 1994), and staphylococcal nuclease (Carra et al, 1994;Shortle, 1996). The present study, together with related reports (Dobson, 1992;Harding et al, 1991;Bychkova et al, 1996;Kamatari et al, 1996), points to the potential importance of organic solvents with contrasting structure-modulating properties in stabilizing a wide range of conformational states of proteins. The ability to characterize a range of states from disor- dered to highly ordered under equilibrium conditions will pennit a detailed dissection of the folding pathway of proteins.…”

Section: Discussionsupporting

confidence: 73%

“…It is noteworthy that NMR studies of a synthetic sequence corresponding to helix C of HEWL in aqueous TFE establish a high helical structure of this peptide in solution (Bolin et al, 1996). Recently, aqueous organic solvents have been used in generating partially folded states of some proteins (Harding et al, 1991;Fan et al ., 1993;Alexandrescu et al, 1994;Bychkova et al, 1996;Kamatari et al, 1996). A partially denatured state of hen lysozyme has been obtained in a 50% T-water mixture (Buck et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

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Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

Bhattacharjya

Balaram

1997

Protein Science

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A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced a f f i n i t y towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 'H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (250%). This transition is characterized by a dramatic loss of A N S binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. HID exchange experiments yield higher protection factors for many of the backbone amide protons from the four a-helices along with the C-terminal 3'0 helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel @-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for a-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.

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Section: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

Bhattacharjya

Balaram

1997

Protein Science

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“…Protein folding involves a discrete pathway with the formation of intermediate states between the native and denatured states (Kim and Baldwin, 1990). It is also argued that the equilibrium molten globule state, which is observable under artificial conditions, such as low pH and low denaturant concentrations, may also be involved in physiological processes in the living cell (Bychkova et al, 1996). Therefore, the identification and characterization of the intermediate states that are populated during the folding process has received considerable attention, and a lot of effort is being made in this direction.…”

Section: Introductionmentioning

confidence: 99%

Acid and Chemical Induced Conformational Changes of Ervatamin B. Presence of Partially Structured Multiple Intermediates

Sundd¹,

Kundu²,

Jagannadham³

2002

BMB Reports

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The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the α + β class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0-2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly a β-sheet conformation and shows a strong binding to 8-anilino-1-napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily.

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“…The appearance of a molten globule state in the methanol/water system is considered physiologically important because of the resemblance of the alcohol-rich environment to the environment of the membrane surface (Bychkova et al, 1996). However, whether the appearance of 1~ and the transition N(native), I,, H in proteins are general phenomena in many proteins is an interesting and important question, together with the generality of their characteristic structures.…”

mentioning

confidence: 99%

The methanol‐induced transition and the expanded helical conformation in hen lysozyme

et al. 1998

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Methanol-induced conformational transitions of hen egg white lysozyme were investigated with a combined use of farand near-UV CD and N M R spectroscopies, ANS binding and small-angle X-ray scattering. Addition of methanol induced no global change in the native conformation itself, but induced a transition from the native state to the denatured state which was highly cooperative, as shown by the coincidence of transition curves monitored by the far-and near-UV CD spectroscopy, by isodichroic points in the far-and near-UV CD spectra and by the concomitant disappearance of individual 'H N M R signals of the native state. The ANS binding experiments could detect no intermediate conformer similar to the molten globule state in the process of the methanol denaturation. However, at high concentration of methanol, e.g., 60% (v/v) methanol/water, a highly helical state (H) was realized. The H state had a helical content much higher than the native state, monitored by far-W CD spectroscopy, and had no specific tertiary structure, monitored both by near-UV CD and N M R spectroscopy. The radius of gyration in the H state, 24.9 A, was significantly larger than that in the native state (15.7 A). The Kratky plot for the H state did not show a clear peak and was quite similar to that for the urea-denatured state, indicating a complete lack of globularity. Thus we conclude that the H state has a considerably expanded, flexible broken rod-like conformation which is clearly distinguishable from the "molten globule" state. The stability of both N and H states depends on pH and methanol concentration. Thus a phase diagram involving N and H was constructed.

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Molten Globule-Like State of Cytochrome c under Conditions Simulating Those Near the Membrane Surface

Cited by 216 publications

References 42 publications

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

Acid and Chemical Induced Conformational Changes of Ervatamin B. Presence of Partially Structured Multiple Intermediates

The methanol‐induced transition and the expanded helical conformation in hen lysozyme

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