Denaturation of Bovine Carbonic Anhydrase B by Guanidine Hydrochloride

Wong, K.P.; Tanford, Charles

doi:10.1016/s0021-9258(19)43163-3

Cited by 188 publications

(50 citation statements)

References 33 publications

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“…The lower midpoint for the difference UV transition compared to that for the CD transition in the L28R/E139K double mutant shows that the tertiary structure is disrupted before the secondary structure. This observation is similar to those for other multistate unfolding transitions (Wong & Tanford, 1973;Robson & Pain, 1976) and is consistent with the framework model for folding (Kim & Baldwin, 1982). This conclusion is also supported by the results of Meeker and Shortle (1986) on a triple mutant in staphylococcal nuclease.…”

Section: Kisupporting

confidence: 90%

Long-range electrostatic interactions can influence the folding, stability, and cooperativity of dihydrofolate reductase

Km¹,

Jj²,

Ms³

et al. 1989

Biochemistry

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To test the possibility that long-range interactions might influence the folding and stability of dihydrofolate reductase, a series of single and double mutations at positions 28 and 139 were constructed and their urea-induced unfolding reactions studied by absorbance and circular dichroism spectroscopy. The alpha carbons of the two side chains are separated by 15 A in the native conformation. The replacement of Leu 28 by Arg and of Glu 139 by Gln resulted in additive effects on both kinetic and equilibrium properties of the reversible unfolding transition; no evidence for interaction was obtained. In contrast, the Arg 28/Lys 139 double replacement changed the equilibrium folding model from two state to multistate and showed evidence for interaction in one of the two kinetic phases detected in both unfolding and refolding reactions. The results can be explained in terms of a long-range, repulsive electrostatic interaction between the cationic side chains at these two positions.

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Section: Kisupporting

confidence: 90%

Long-range electrostatic interactions can influence the folding, stability, and cooperativity of dihydrofolate reductase

Km¹,

Jj²,

Ms³

et al. 1989

Biochemistry

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“…Of interest, the A-state even at 85 °C is not fully unfolded, but still maintains significant residual secondary structure (see Figure 7). This has been previously observed also in thermally denatured proteins (Tanford, 1968;Wong & Tanford, 1973), e.g., ribonuclease A (Robertson & Baldwin, 1991) and lysozyme (Evans et al, 1991), and in the heat-induced denaturation of apo-R-lactalbumin molten globule (Griko et al, 1994). It has been proposed that the residual structure of these proteins at high temperature, as well as of the protein molten globules, is stabilized primarily by nonspecific hydrophobic interactions (Griko et al, 1994;Khurana & Udgaonkar, 1994) and that thermal denaturation leads to an ensemble of conformations possessing different degrees of residual structure that progressively unfold upon further heating (Freire, 1995).…”

Section: Discussionsupporting

confidence: 77%

Acid-Induced Molten Globule State of a Fully Active Mutant of Human Interleukin-6

et al. 1996

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Interleukin-6 (IL-6), a four-helix bundle protein, is a multifunctional cytokine which plays an important role in the regulation of the immune system, hematopoiesis, and inflammatory response, as well as in the pathogenesis of multiple myeloma. We have previously shown that a single-disulfide variant of human IL-6, lacking 22 N-terminal amino acids and the disulfide bond connecting Cys-45 and Cys-51 in the 185-residue chain of the wild-type protein, fully retains the conformational, stability, and functional properties of the full-length human IL-6 [Breton et al. (1995) Eur. J. Biochem. 227, 573-581]. In this study, we have investigated the conformational and stability properties of mutant IL-6 at acidic pH (A-state). Using far- and near-ultraviolet (UV) circular dichroism (CD), fluorescence emission, and second-derivative absorption spectroscopy, we have established that mutant IL-6 at pH 2.0 fully retains the helical secondary structure of the native protein at pH 7.5, while the tertiary interactions are much weaker. At variance from the native species, mutant IL-6 in the A-state binds 1-anilinonaphthalene-8-sulfonic acid (ANS), a property considered most typical of a protein in the molten globule state. The pH-induced conformational change from the native to the A-state, monitored either by near-UV CD or by ANS-binding measurements, shows a transition midpoint at pH approximately 4.5, thus indicating that the partial unfolding of the protein is mediated by the titration of glutamic and/or aspartic acid residues. At pH 2.0, the thermal denaturation of mutant IL-6 occurs as a broad process of low cooperativity with a transition at 50-60 degrees C, whereas at pH 7.5 the thermal unfolding is cooperative and characterized by a transition midpoint at 65 degrees C. Of interest, the unfolding of the A-state is not complete even up to approximately 85 degrees C. The urea-mediated unfolding profile of mutant IL-6, measured by far-UV CD, is essentially identical at both pH 7.5 and 2.0, with a midpoint of the cooperative unfolding transition at 5.5 +/- 0.1 M denaturant. Both thermal and urea denaturations of the A-state are complex and cannot fit to a two-state model for unfolding. The unusual stability of mutant IL-6 in acid is also reflected by the resistance to proteolysis at pH 3.6-4.0 by Staphylococcus aureus V8 protease or cathepsin D, an acid protease released by machrophages upon inflammatory stimulation. It is suggested that the molten globule state of IL-6 at acidic pH can play a role in the biological activity of this cytokine, which can exert its activity also at mildly acidic pH, as in inflammation sites.

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“…Assuming 1+1 complexation, this is equivalent to a molecular weight of 3440 g mol 21 . This molecular weight is consistent with previously determined values of 29000 16 and 31000 17 and confirms that our assumption of 1+1 stoichiometry of the Zn-CA complex in sea-water was reasonable.…”

Section: Titrations At Various Concentrations Of Casupporting

confidence: 92%

Determination of the complex stability of zinc with carbonic anhydrase in sea-water

Sarthou

Berg

Riebesell

2001

Analyst

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Carbonic anhydrase (CA) is inactive unless associated with zinc, with possible substitution by cobalt. In this work, the complexation of zinc by CA was determined in sea-water using cathodic stripping voltammetry (CSV) with ligand competition. The zinc was found to be released from the CA over a period of 3 h when equilibrated with a competing complexing ligand and the complex was re-formed with the CA when zinc was added. A value of 8.90 ± 0.27 was found for logKA ZnCA where KA ZnCA is the conditional stability constant for the complex of Zn 2+ with CA in pH 8 sea-water. A value for the molecular weight of CA was calculated from its equivalent ligand concentration (in nM) obtained by titrations with zinc at various CA concentrations (1-4 mg l 21 ). The value found (34740 g mol 21 ) for the molecular weight is consistent with values found previously by other methods (29000-31000 g mol 21 ) confirming that the stoichiometry of the complex between zinc and CA is 1+1. This work confirms that the zinc-CA complex is reversible and that the interaction between zinc and CA can be determined using CSV with ligand competition.

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Denaturation of Bovine Carbonic Anhydrase B by Guanidine Hydrochloride

Cited by 188 publications

References 33 publications

Long-range electrostatic interactions can influence the folding, stability, and cooperativity of dihydrofolate reductase

Long-range electrostatic interactions can influence the folding, stability, and cooperativity of dihydrofolate reductase

Acid-Induced Molten Globule State of a Fully Active Mutant of Human Interleukin-6

Determination of the complex stability of zinc with carbonic anhydrase in sea-water

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