[14]Determination and analysis of urea and guanidine hydrochloride denaturation curves

Cn, Pace

doi:10.1016/0076-6879(86)31045-0

Cited by 2,439 publications

(2,396 citation statements)

References 34 publications

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“…The relative concentrations of F and U can be easily determined from studies where spectral probes are used to monitor the conformational state of the protein (see Pace, 1986). Naturally, Equation 1 only works if the protein molecules are in either one of two conformational states (a two-state unfolding reaction), which seems to be true for most small, globular proteins.…”

Section: ~"mentioning

confidence: 99%

“…In this method, the parameters AGH*O and An are somewhat sensitive to the choice of k (Pace, 1986). Binding constants for the denaturation of proteins and peptide helices and from the study of denaturant interaction with model compounds are not in exact agreement, so the proper binding constants to use for urea and Gdn HCI are not clear (Pace, 1986;Makhatadze & Privalov, 1992;Scholtz et al, 1995, and references therein). Evidence for specific binding of denaturant molecules to protein is not very strong in any case.…”

Section: ~"mentioning

confidence: 99%

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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: ~"mentioning

confidence: 99%

Section: ~"mentioning

confidence: 99%

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

1995

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“…Concentrations of urea and GdmCl solutions were calculated from the refractive index as described by Pace (1986). Unfolding of the protein was monitored using CD or fluorescence spectroscopies.…”

Section: Solvent Denaturation Experimentsmentioning

confidence: 99%

Anion binding to the ubiquitin molecule

et al. 1998

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Effects of different salts (NaCl, MgC12, CaC12, GdmC1, NaBr, NaC104, NaH2PO4, Na2S04) on the stability of the ubiquitin molecule at pH 2.0 have been studied by differential scanning calorimetry, circular dichroism, and Tyr fluorescence spectroscopies. It is shown that all of the salts studied significantly increase the thermostability of the ubiquitin molecule, and that this stabilization can be interpreted in terms of anion binding. Estimated thermodynamic parameters of binding for C1-show that this binding is relatively weak (Kd = 0.15 M) and is characterized by a negative enthalpy of -15 kJ/mol per site. Particularly surprising was the observed stabilizing effect of GdmCl through the entire concentration range studied (0.01-2 M), however, to a lesser extent than stabilization by NaCl. This stabilizing effect of GdmCl appears to arise from the binding of C1-ions. Analysis of the observed changes in the stability of the ubiquitin molecule in the presence of GdmCl can be adequately described by combining the thermodynamic model of denaturant binding with C1-binding effects.

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“…Ultra-pure GdnHCl was used without further purification to make 8 M stock solutions containing NaCl and sodium cacodylate. The concentration of GdnHCl in the stock solution was verified by refractive index (Pace, 1986) AC, = AGE -mC, where C is the concentration of GdnHCI, m is the slope of the transition; y is the observed molar ellipticity, and y N and y , are the native and denatured baseline linear functions, respectively (Pace, 1986). Reversibility of unfolding was tested by measuring both molar ellipticity and RNA affinity of proteins used in stability studies.…”

Section: Protein Unfoldingmentioning

confidence: 99%

Contribution of the tyrosines to the structure and function of the human U1A N‐terminal RNA binding domain

Kranz

Hall

1996

Protein Science

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RNA binding domains (RBDs) are members of a large family of proteins that share minimal sequence conservation but adopt an a0 sandwich global fold. Defining the contributions of specific amino acids to RBD structure and RNA binding is critical to understanding the functions of these proteins. In these experiments with the human UlA N-terminal RNA binding domain (RBDl), the contributions from each of its four tyrosines to protein structure, stability, and RNA binding were measured. Each tyrosine was substituted with phenylalanine and one other selected residue, and the resulting proteins were characterized by chemical denaturation to measure their unfolding free energy, by binding free energies to the wild-type RNA hairpin, and by I9F NMR to probe for structural changes. Features of the protein identified in these experiments include a possible tyrosine/lysine contact in an a-helix, which may be an example of an energetically favorable aromatidamin0 side chain interaction. One long loop of the protein, which shows unusual I5N backbone and tyrosine side-chain dynamics, is implicated in protein:protein association. The diverse interactions of the four tyrosine residues in the organization of RBDl illustrate how each member of this family of proteins will have unique molecular details that contribute to function.

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[14]Determination and analysis of urea and guanidine hydrochloride denaturation curves

Cited by 2,439 publications

References 34 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

Anion binding to the ubiquitin molecule

Contribution of the tyrosines to the structure and function of the human U1A N‐terminal RNA binding domain

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