Microcalorimetric studies of conformational transitions of ferricytochrome c in acidic solution

Potekhin, Sergey A.; Pfeil, Wolfgang

doi:10.1016/0301-4622(89)80041-9

Cited by 72 publications

(68 citation statements)

References 17 publications

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“…The thermal unfolding of the molten globule state of cytochrome c and apomyoglobin involves a cooperative transition with an enthalpy change and heat capacity change of unfolding (Potekhin & Pfeil, 1989;Kuroda et al, 1992;Hagihara et al, 1994;Hamada et al, 1994;Nishii et al, 1994Nishii et al, , 1995. Cooperative thermal unfolding with distinct heat absorption has also been reported for the staphylococcal nuclease fragment (Gittis et al, 1993;Griko et al, 1994a).…”

Section: Formation Of the Molten Globulementioning

confidence: 85%

Structural characterization of the molten globule of α‐lactalbumin by solution X‐ray scattering

et al. 1997

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A compact denatured state is often observed under a mild denaturation condition for various proteins. A typical example is the a-lactalbumin molten globule. Although the molecular compactness and shape are the essential properties for defining the molten globule, there have been ambiguities of these properties for the molten globule of a-lactalbumin. Using solution X-ray scattering, we have examined the structural properties of two types of molten globule of a-lactalbumin, the apo-protein at neutral pH and the acid molten globule. The radius of gyration for the native holo-protein was 15.7 A, but the two different molten globules both had a radius of gyration of 17.2 A. The maximum dimension of the molecule was also increased from 50 8, for the native state to 60 8, for the molten globule. These values clearly indicate that the molten globule is not as compact as the native state. The increment in the radius of gyration was less than 10% for the a-lactalbumin molten globule, compared with up to 30% for the molten globules of other globular proteins. Intramolecular disulfide bonds restrict the molecular expansion of the molten globule. The distance distribution function of the a-lactalbumin molten globule is composed of a single peak suggesting a globular shape, which is simply swollen from the native state. The scattering profile in the high Q region of the molten globule indicates the presence of a significant amount of tertiary fold. Based on the structural properties obtained by solution X-ray scattering, general and conceptual structural images for the molten globules of various proteins are described and compared with the individual, detailed structural model obtained by nuclear magnetic resonance.

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Section: Formation Of the Molten Globulementioning

confidence: 85%

Structural characterization of the molten globule of α‐lactalbumin by solution X‐ray scattering

et al. 1997

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“…However, it is now becoming clear that this is not the case. Significant AC,,, and AH, values, although smaller than those of the native state, are well established for the molten globule state of cytochrome c (Potekhin & Pfeil, 1989;Hagihara et al, 1994). Nishii et al (1994) reported a small but significant AC,,, upon unfolding of the molten globule state of apomyoglobin.…”

Section: 1993;mentioning

confidence: 88%

“…As a typical example of the molten globule state, we will consider the acidic molten globule state of horse cytochrome c, for which a cooperative unfolding transition has been well established (Potekhin & Pfeil, 1989;Goto et al, 1993;Kataoka et al, 1993;Hagihara et al, 1994). Tables 1 and 3 compare several structural and thermodynamic properties of melittin tetramer in solution and the molten globule states of cytochrome c.…”

Section: Comparison With the Molten Globule State Of Cytochrome Cmentioning

confidence: 99%

Thermal unfolding of tetrameric melittin: Comparison with the molten globule state of cytochrome c

1994

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Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric a-helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion-and pH-dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid-denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far-UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2-state transition mechanism between the tetrameric a-helical state and the monomeric unfolded state. From the peptide or salt-concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)" K-l at 30 "C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)" K"), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins.

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“…Both of these amino acids are beta-branched and such amino acids are expected to lead to slight expansion of a random coil denatured state (Tanford, 1968;Miller & Goebel, 1968). Expansion of the denatured state of a protein to a more An initial point to clarify since we are monitoring thermal denaturations down to reasonably low pH is that we are not crossing over into a region where a molten globule state of cytochrome c might be stabilized (Potekhin & Pfeil, 1989;Kuroda et al, 1992;Fink et al, 1994). We do not expect molten globule states to be involved at either end point of our low pH denaturations as these states appear to be primarily driven by anion binding and the concentration of anion should be lowest (<lo mM) in our low pH acetate buffers.…”

Section: Discussionmentioning

confidence: 99%

“…In the pH range 3 to 5, buffer and protein ionization enthalpy effects appear to cancel each other (Pfeil & Privalov, 1976b, Potekhin & Pfeil, 1989. A plot of AH,,, versus T, thus can be made using Equation 8 (see Materials and methods) to obtain the heat capacity increment, AC,,, for protein unfolding.…”

Section: Lm Herrmann and Be Bowlermentioning

confidence: 99%

Thermal denaturation of iso-1-cytochromecvariants: Comparison with solvent denaturation

Herrmann

Bowler

1997

Protein Science

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Thermal denaturation studies as a function of pH were carried out on wild-type iso-I-cytochrome c and three variants of this protein at the solvent-exposed position 73 of the sequence. By examining the enthalpy and T, at various pH values, the heat capacity increment (AC,,), which is dominated by the degree of change in nonpolar hydration upon protein unfolding, was found for the wild type where lysine 73 is normally present and for three variants. Both the m-value and AC,, are related to the change in solvent exposure upon unfolding and other investigators have shown a correlation exists between these two parameters. However, for this subset of variants of iso-I-cytochrome c, a lack of correlation exists which implies that there may be basic differences between the guanidine hydrochloride and thermal denaturations of this protein. Spectroscopic data are consistent with different denatured states for thermal and guanidine hydrochloride unfolding. The different response of rn-values and ACp for these variants will be discussed in this context. Keywords: circular dichroism; guanidine hydrochloride denaturation; heat capacity increment; iso-1 -cytochrome c; m-values; protein stability; thermal denaturation Understanding the factors that control the equilibrium between the native and denatured state of a protein is of great importance to elucidating the protein folding problem (Dill, 1990;Matthews, 1993; Shortle, 1995). Early work from Tanford's lab (Tanford, 1968) suggested that most denatured states approach a random coil, and therefore much research has focused on how native state interactions stabilize a folded protein (Privalov & Gill, 1988;Matthews, 1993). More recently it has become apparent that denatured states of proteins may be more complex than originally suspected Reprint requests to: Bruce E. Bowler, Department of Chemistry and Biochemistry, University of Denver, 2050 E. Iliff Street, Denver, Colorado 80208-2436; e-mail: bbowler@du.edu.'Present address: NIAID, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana 59840.Abbreviations: gdnHC1, guanidine hydrochloride; AG." "*O, free energy of unfolding in the absence of denaturant; rn-value, the rate of change of AG." as a function of guanidine hydrochloride concentration; ACp, heat capacity increment of protein unfolding; AASA, change in accessible surface area upon unfolding; WT, wild type; T,,,, midpoint temperature of unfolding; AH,,,, enthalpy of unfolding at T,; AS, , entropy of unfolding at T , ; AGD,T, free energy of unfolding at temperature, T; AH,.,, enthalpy of unfolding at temperature, T; ASDJ, entropy of unfolding at temperature, T.

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Microcalorimetric studies of conformational transitions of ferricytochrome c in acidic solution

Cited by 72 publications

References 17 publications

Structural characterization of the molten globule of α‐lactalbumin by solution X‐ray scattering

Structural characterization of the molten globule of α‐lactalbumin by solution X‐ray scattering

Thermal unfolding of tetrameric melittin: Comparison with the molten globule state of cytochrome c

Thermal denaturation of iso-1-cytochromecvariants: Comparison with solvent denaturation

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