Nick Pace

Hydrogen bonding stabilizes globular proteins

Biophysical Journal, Oct 1, 1996

It is clear that intramolecular hydrogen bonds are essential to the structure and stability of gl... more It is clear that intramolecular hydrogen bonds are essential to the structure and stability of globular proteins. It is not clear, however, whether they make a net favorable contribution to this stability. Experimental and theoretical studies are at odds over this important question. Measurements of the change in conformational stability, A(AG), for the mutation of a hydrogen bonded residue to one incapable of hydrogen bonding suggest a stabilization of 1.0 kcal/mol per hydrogen bond. If the A(AG) values are corrected for differences in side-chain hydrophobicity and conformational entropy, then the estimated stabilization becomes 2.2 kcal/mol per hydrogen bond. These and other experimental studies discussed here are consistent and compelling: hydrogen bonding stabilizes globular proteins. The principle of science, the definition, almost, is the fol- lowing: the test of all knowledge is experiment. Experiment is the sole judge of scientific "truth." -R. P. Feynman (1963)

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Ribonuclease T1 Complexed with 2'GMP/G23A Mutant

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

Protein Science, Oct 1, 1995

Denaturant m values, the dependence of the free energy of unfolding on denaturant concentration, ... more 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,,

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The disulphide folding pathway of ribonuclease T1

Journal of Molecular Biology, Apr 1, 1986

The pathway of unfolding and refolding that accompanies, respectively, the breakage and formation... more

The disulphide folding pathway of ribonuclease T

J Mol Biol, 1986

Protein Stability

eLS, Dec 15, 2009

Thermodynamics of ribonuclease T1 denaturation

Biochemistry, May 1, 1992

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation... more Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.

Protein conformations and their stability

Journal of the American Oil Chemists' Society, May 1, 1983

the secondary products of oxidation (2). Verma and Prabhakar (9) have shown that the peroxide val... more the secondary products of oxidation (2). Verma and Prabhakar (9) have shown that the peroxide values in oxidizing refined safflower oil are similar at low and high a w at each storage period. The unusual low peroxide values at high aw, therefore, can be attributed to some components like phospholipids present in the raw peanut oil (degummed peanut oil has been found to oxidize rapidly at high water activities). Reports on antioxidant and synergistic effects of phospholipids are conflicting (10,11) and the information on the role of phospholipids in oils at different aw is scanty. These studies indicate that it would be better to trade the raw oils at high a w in the international markets as the rate of peroxide formation is minimum at high aw.

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Single surface stabilizer

Nature Structural Biology

ABSTRACT

Increasing Enzyme Stability

Chemical Aspects of Enzyme Biotechnology, 1990

Enzymes are active only when their polypeptide chain is folded into a unique globular conformatio... more Enzymes are active only when their polypeptide chain is folded into a unique globular conformation with a functional active site. In most naturally occurring enzymes, this globular conformation is only 5 to 10 kcal/mole more stable than unfolded, biologically inactive conformations (1). The conformational stability of a protein is defined as the free energy change for the reaction: $$ \textup{folded}\;\textup{(native)}\; \;\textup{unfolded\;(denatured)} $$ (1) under ambient conditions, such as water at 25°C. The most convenient methods of estimating the conformational stability of a protein are urea (or guanidine hydrochloride (GdnHCl)) unfolding curves and thermal unfolding curves. Estimates of the conformational stability based on urea unfolding curves are designated ΔG(H2O), and estimates from thermal unfolding curves are designated ΔG(25°C). In the first part of this article, we describe how to measure ΔG(H2O) and ΔG(25°C).

Contribution of the hydrophobic effect to globular protein stability

Journal of Molecular Biology, 1992

The decrease in conformational stability, delta(delta G), has been measured for 72 aliphatic side... more The decrease in conformational stability, delta(delta G), has been measured for 72 aliphatic side-chain mutants from four proteins in which a larger side-chain is replaced by a smaller side-chain so that steric effects are minimal. When these delta(delta G) values are corrected to the same accessibility, namely 100% buried, then the following -delta(delta G) values per -CH2- group (in kcal/mol) are obtained: Ile----Val (1.26), Ala (1.26), Gly (1.26); Leu----Ala (1.16), Gly (1.21); Val----Ala (1.23), Gly (1.53). The average of these values is 1.27(+/- 0.07) kcal/mol. The 72 individual values range from 0 to 2.4 kcal/mol with an average value of 1.27(+/- 0.51) (standard deviation) kcal/mol. When the delta Gtr values from n-octanol to water are corrected for the difference in volume between the solutes and the solvents, the average value for the same substitutions is 1.25(+/- 0.05) kcal/mol. This suggests that proteins gain 1.3(+/- 0.5) kcal/mol in stability for each -CH2- group buried in folding, and, furthermore, that the volume corrected delta Gtr values for n-octanol for the amino acid side-chains provide good estimates of the contribution of the hydrophobic effect to globular protein stability.

Thermodynamics of ribonuclease T1 denaturation

Biochemistry, 1992

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation... more Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.

Conformational stability of mixed disulfide derivatives of .beta.-lactoglobulin B

Biochemistry, 1983

... Snyder, GH, Rowan, R., Karplus, S., &amp;amp; Sykes, BD (1975) Snyder, GH, Rowan, R., &am... more ... Snyder, GH, Rowan, R., Karplus, S., &amp;amp; Sykes, BD (1975) Snyder, GH, Rowan, R., &amp;amp; Sykes, BD (1976) Biochem-Sternlicht, H., &amp;amp; Wilson, D. (1967) Biochemistry 6, ... Conformational Stability of Mixed Disulfide Derivatives of ,&amp;amp;Lactoglobulin Bt James F. Cupof and C. Nick Pace* ...

Steady-state kinetic studies of arginase with an improved direct spectrophotometric assay

Analytical Biochemistry, 1980

... 109, 261265 (1980) SteadyState Kinetic Studies of Arginase with an Improved Direct Spectropho... more

Buried, Charged, Non-Ion-Paired Aspartic Acid 76 Contributes Favorably to the Conformational Stability of Ribonuclease T1

Biochemistry, 1999

The side-chain carboxyl of Asp 76 in ribonuclease T1 (RNase T1) is buried, charged, non-ion-paire... more The side-chain carboxyl of Asp 76 in ribonuclease T1 (RNase T1) is buried, charged, non-ion-paired, and forms three good intramolecular hydrogen bonds (2.63, 2.69, and 2.89 A) and a 2.66 A hydrogen bond to a buried, conserved water molecule. When Asp 76 was replaced by Asn, Ser, and Ala, the conformational stability of the protein decreased by 3.1, 3.2, and 3.7 kcal/mol, respectively. The stability was measured as a function of pH for wild-type RNase T1 and the D76N mutant and showed that the pH dependence below pH 3 was almost entirely due to Asp 76. The pK of Asp 76 is 0.5 in the native state and 3.7 in the denatured state. Thus, the hydrogen bonding of the carboxyl group of Asp 76 contributes more than half of the net stability of RNase T1 at pH 7. In addition, the charged carboxyl of Asp 76 stabilizes structure in the denatured states of RNase T1 that is not present in D76N, D76S, and D76A.

How Site‐Directed Mutagenesis Alters the Solubility and Stability of RNase Sa

The FASEB Journal, 2006

How to determine the molar absorption coefficient of a protein

[24] Evaluating contribution of hydrogen bonding and hydrophobic bonding to protein folding

Methods in Enzymology, 1995

Publisher Summary This chapter evaluates contribution of hydrogen bonding and hydrophobic bonding... more Publisher Summary This chapter evaluates contribution of hydrogen bonding and hydrophobic bonding to protein folding. The primary goal of studying hydrophobic mutants is to learn how much stability is gained when a protein buries a nonpolar side chain in folding. The goal of studying hyrogen-bonding mutants is to learn how much stability is gained when a polar group that is hydrogen bonded to water in the unfolded state is dehydrated in protein folding to form an intramolecular hydrogen bond. The interplay between experimental studies of the type described and theoretical studies will eventually lead to a good understanding of the forces that contribute to protein stability. Hydrogen bonding and hydrophobic bonding are thought to be the major forces contributing to the conformational stability of proteins. The techniques most often used to measure the conformational stability of a protein are solvent denaturation curves, which are generally urea or guanidine hydrochloride (GdnHC1), thermal denaturation curves, and differential scanning calorimetry (DSC). The chapter discusses the methods of determining denaturation curves and analyzing the results.

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

Methods in Enzymology, 1986

Mutants of RNase Sa

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