Thermodynamic analysis of the chemotactic protein from Escherichia coli, CheY

Filimonov, Vladimir V.; Prieto, Jesús; Martínez, José C.; Bruix, Marta; Mateo, Pedro L.; Serrano, Luís

doi:10.1021/bi00210a045

Cited by 64 publications

(88 citation statements)

References 53 publications

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“…a Swint and Robertson, 1993;' O'Neil et al, 1995;Hurle et al, 1990;Viguera et al, 1994; e Jackson et al, 1993;'Akke and Forsen, 1990;Khorasanizadeh et al, 1993;Wintrode et al, 1994;Scholtz, 1995;J Agashe and Udgaonkar, 1995;Lim et al, 1992; ' Marqusee and Sauer, 1994;McLendon and Smith, 1978;Hagihara et al, 1994; O Privalov and Gill, 1988;P Pace et al, 1990;Shirley et al, 1992;Yu et al, 1994; ' Bowie and Sauer, 1989; ' Egan et al, 1993;Ramdas et al, 1986;"Bryant et al, 1985; " Cohen and Pielak, 1994;x Kelley et al, 1987;Santor0 and Bolen, 1992;'Clarke and Fersht, 1993; aa Pace et al, 1992;" Griko et al, 1994; cc Greene and Pace, 1974;dd Munson et al, 1994;Filimonov et al, 1993; f f Saito and Wada, 1983; gg Ahmad and Bigelow, 1982; hh Taniyama et al, 1992; I' Herning et al, 1992;'' Ropson et al, 1990; kk Shortle and Meeker, 1986;"Carra et al, 1994; mm Craig et al, 1987; "" Makhatadze et al, 1994;O0 De Young et al, 1993...…”

Section: Aasamentioning

confidence: 99%

Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding

1995

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Denaturant m values, the dependence of the free energy of unfolding on denaturant concentration, have been collected for a large set of proteins. The m value correlates very strongly with the amount of protein surface exposed to solvent upon unfolding, with linear correlation coefficients of R = 0.84 for urea and R = 0.87 for guanidine hydrochloride. These correlations improve to R = 0.90 when the effect of disulfide bonds on the accessible area of the unfolded protein is included. A similar dependence on accessible surface area has been found previously for the heat capacity change (AC,), which is confirmed here for our set of proteins. Denaturant m values and heat capacity changes also correlate well with each other. For proteins that undergo a simple two-state unfolding mechanism, the amount of surface exposed to solvent upon unfolding is a main structural determinant for both m values and AC,, Keywords: denaturation; guanidine hydrochloride; heat capacity changes; m values; protein folding; protein stability; solvent-accessible surface area; urea It has been known for many years that proteins can be unfolded in aqueous solution by high concentrations of certain reagents such as guanidine hydrochloride or urea. Denaturation with these chemicals is one of the primary ways of measuring the conformational stability of proteins and comparing the stabilities of mutant proteins. The use of these two denaturants is extremely widespread (Pace, 1986), even though the exact nature of the molecular interaction of denaturant molecules with protein surfaces is not well understood. It is known from solubility and transfer experiments with model compounds that the interaction of urea and Gdn HCI with the constituent groups of proteins is more favorable than the interaction of those groups with water (Tanford, 1970). These denaturants alter the equi-~"

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Section: Aasamentioning

confidence: 99%

Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding

1995

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“…Flavodoxins may appear in three different redox forms of presumably different stability. From a structural point of view, flavodoxins are archetypal examples of a/@ proteins with an open 0-sheet, a class of proteins for which only a few detailed stability studies are available (Filimonov et al, 1993;. We have chosen the flavodoxin from the cyanobacteria Anabcena PCC 7119 (Fig.…”

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confidence: 99%

Conformational stability of apoflavodoxin

et al. 1996

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Flavodoxins are ar/p proteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin from A n a b~n a PCC 71 19 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 1 1 and pH 6, the apoprotein is folded properly as judged from near-ultraviolet (UV) circular dichroism (CD) and high-field 'H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two-state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5-2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far-UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two-state process at acidic pH. Apoflavodoxin constitutes a prornising system for the analysis of the stability and folding of cr/0 proteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors.

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“…DSC studies of proteins composed of independent domains, or of subunits that unfold independently, even at the same temperature, may display more than one calorimetric peak and/or a cooperativity parameter (AHcu,/AHvH) larger than one (Privalov, 1982). However, cases have also been reported in which multisubunit proteins display one peak and a cooperativity parameter approaching unity (Gitelson et al, 1991;Filimonov et al, 1993;Tamura et al, 19941, suggesting that they unfold as a single cooperative unit. The shape of the single calorimetric peak, as well the cooperativity parameter, indicate that MG,,,, although much larger, behaves similarly.…”

Section: Discussionmentioning

confidence: 98%

Two partially unfolded states oftorpedo californicaacetylcholinesterase

et al. 1996

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Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue CysZ3' in each subunit of a disulfide-linked dimer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1-2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physicochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long-lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thioldisulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on extrapolation to zero guanidine hydrochloride, a high free energy barrier (ca. 23.8 kcal/mol) separating these two flexible, partially unfolded states.

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Thermodynamic analysis of the chemotactic protein from Escherichia coli, CheY

Cited by 64 publications

References 53 publications

Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding

Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding

Conformational stability of apoflavodoxin

Two partially unfolded states oftorpedo californicaacetylcholinesterase

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