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C N Pace

Publications and source records attributed to C N Pace.

At least 37 records · Page 2Linked to original sources
PubMed1998

A helix propensity scale based on experimental studies of peptides and proteins.

The average globular protein contains 30% alpha-helix, the most common type of secondary structure. Some amino acids occur more frequently in alpha-helices than others; this tendency is known as helix propensity. Here we derive a helix propensity scale for solvent-exposed residues in the middle positions of alpha-helices. The scale is based on measurements of helix propensity in 11 systems, including both proteins and peptides. Alanine has the highest helix propensity, and, excluding proline, glycine has the lowest, approximately 1 kcal/mol less favorable than alanine. Based on our analysis, the helix propensities of the amino acids are as follows (kcal/mol): Ala = 0, Leu = 0.21, Arg = 0.21, Met = 0.24, Lys = 0.26, Gln = 0.39, Glu = 0.40, Ile = 0.41, Trp = 0.49, Ser = 0.50, Tyr = 0. 53, Phe = 0.54, Val = 0.61, His = 0.61, Asn = 0.65, Thr = 0.66, Cys = 0.68, Asp = 0.69, and Gly = 1.

Amino Acids↗
PubMed1997

Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric intermediate.

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a homodimeric enzyme that catalyzes the hydrolysis of organophosphorus pesticides and nerve agents. We have analyzed the urea- and guanidinium chloride-induced equilibrium unfolding of OPH as monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence. These spectral methods, which monitor primarily the disruption of protein secondary structure and tertiary structure, respectively, reveal biphasic unfolding transitions with evidence for an intermediate form of OPH. By investigating the protein concentration dependence of the unfolding curves, it is clear that the second transition involves dissociation of the monomeric polypeptide chains and that the intermediate is clearly dimeric. The dimeric intermediate form of OPH is devoid of enzymatic activity, yet clearly behaves as a partially folded, dimeric protein by gel filtration. Therefore, we propose an unfolding mechanism in which the native dimer converts to an inactive, well-populated dimeric intermediate which finally dissociates and completely unfolds to individual monomeric polypeptides. The denaturant-induced unfolding data are described well by a three-state mechanism with delta G for the interconversion between the native homodimer (N2) and the inactive dimeric intermediate (I2) of 4.3 kcal/mol while the overall standard state stability of the native homodimer relative to the unfolded monomers (2U) is more than 40 kcal/mol. Thus, OPH is a remarkably stable protein that folds through an inactive, dimeric intermediate and will serve as a good model system for investigating the energetics of protein association and folding in a system where we can clearly resolve these two steps.

Aryldialkylphosphatase↗
PubMed1997

Helix propensities are identical in proteins and peptides.

Our understanding of the factors stabilizing alpha-helical structure has been greatly enhanced by the study of model alpha-helical peptides. However, the relationship of these results to the folding of helices in intact proteins is not well characterized. Helix propensities measured in model peptides are not in good agreement with those from proteins. In order to address these questions, we have measured helix propensities in the alpha-helix of ribonuclease T1 and a helical peptide of identical sequence. We have previously demonstrated excellent agreement between peptide and protein for the nonpolar amino acids [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2833-2837]. Most other amino acids also show good agreement, although certain polar amino acids are exceptions. Helix propensities measured in the ribonuclease T1 peptide/protein are compared with those measured in other systems. Reasonable agreement is found between most systems; however, our propensities differ substantially from those measured in several model peptide systems. Alanine-based peptides overestimate the propensity differences by a factor of 2, and host/guest experiments underestimate them by a factor of 2-3.

Amino Acid Sequence↗
PubMed1997

A direct comparison of helix propensity in proteins and peptides.

alpha-Helical secondary structure occurs widely in globular proteins and its formation is a key step in their folding. As a consequence, understanding the energetics of helix formation is crucial to understanding protein folding and stability. We have measured the helix propensities of the nonpolar amino acids for an alpha-helix in an intact protein, ribonuclease T1, and for a 17-residue peptide with a sequence identical to that of the alpha-helix in the protein. The helix propensities are in excellent agreement. This shows that when compared in the same sequence context, the helix propensities of the nonpolar amino acids are identical in helical peptides and intact proteins, and that conclusions based on studies of the helix-to-coil transitions of peptides may, in favorable cases, be directly applicable to proteins. Our helix propensities based on ribonuclease T1 are in good agreement with those from similar studies of barnase and T4 lysozyme. In contrast, our helix propensities differ substantially from those derived from studies of alanine-stabilized or salt bridge-stabilized model alpha-helical peptides.

Amino Acid Sequence↗
PubMed1997

Conformational stability of ribonuclease T1 determined by hydrogen-deuterium exchange.

The hydrogen-deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25, 40, 45, and 50 degrees C at pD 5.6 and at 40 and 45 degrees C at pD 6.6. The hydrogen exchange rate constants of the hydrogen-bonded residues varied over eight orders of magnitude at 25 degrees C with 13 residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in strands 2-4 of the central beta-pleated sheet. The residues located in the alpha-helix and the remaining strands of the beta-sheet exhibited exchange behaviors consistent with exchange occurring due to local structural fluctuations. For several residues at 25 degrees C, the global free energy change calculated from the hydrogen exchange data was over 2 kcal/mol greater than the free energy of unfolding determined from urea denaturation experiments. The number of residues showing this unexpected behavior was found to increase with temperature. This apparent inconsistency can be explained quantitatively if the cis-trans isomerization of the two cis prolines, Pro-39 and Pro-55, is taken into account. The cis-trans isomerization equilibrium calculated from kinetic data indicates the free energy of the unfolded state will be 2.6 kcal/mol higher at 25 degrees C when the two prolines are cis rather than trans (Mayr LM, Odefey CO, Schutkowski M, Schmid FX. 1996. Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique. Biochemistry 35: 5550-5561). The hydrogen exchange results are consistent with the most slowly exchanging hydrogens exchanging from a globally higher free energy unfolded state in which Pro-55 and Pro-39 are still predominantly in the cis conformation. When the conformational stabilities determined by hydrogen exchange are corrected for the proline isomerization equilibrium, the results are in excellent agreement with those from an analysis of urea denaturation curves.

Deuterium↗
PubMed1997

Purification of ribonucleases Sa, Sa2, and Sa3 after expression in Escherichia coli.

The genes for three small ribonucleases from different strains of Streptomyces aureofaciens have been cloned and expressed in Escherichia coli. The purification of these ribonucleases from the periplasmic space is described. The yields range from 10 to 50 mg of protein per liter of culture medium. The molar absorption coefficients, isoelectric pH values, and pH of optimum activity are reported.

Amino Acid Sequence↗
PubMed1996

The alpha-helix of ribonuclease T1 as an independent stability unit: direct comparison of peptide and protein stability.

Measurements of the change in conformational stability, delta(delta G), upon mutation of two acidic residues at the C terminus of the helix of ribonuclease T1 have recently been reported. Here, we investigate peptides based on the sequence of the helix with the same mutations: Glu28 replaced with Gln, Asp29 replaced with Asn, and the double mutant. In addition, the mutant Lys25 to Gln was studied. Changes in helix content of the peptides with pH confirm the conclusion found in the intact protein, that the charged forms of the acidic residues destabilize the protein by destabilizing the helix. The pH-dependence of the change in conformational free energy for the peptides and mutant proteins show fair correspondence for D29N and the double mutant. The mutants E28Q and K25Q, on the other hand, give striking agreement between the protein and peptide systems. This agreement suggests that the helix of ribonuclease T1 behaves as an independently stabilized structural unit of the intact protein and that stabilization of the helical form of the peptide is mirrored in the protein.

Amino Acid Sequence↗
PubMed1996

Hydrogen bonding stabilizes globular proteins.

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, delta (delta G), 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 delta (delta G) 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.

Bacteriophage T4↗
PubMed1996

Forces contributing to the conformational stability of proteins.

For 35 years, the prevailing view has been that the hydrophobic effect is the dominant force in protein folding. The importance of hydrogen bonding was always clear, but whether it made a net favorable contribution to protein stability was not. Studies of mutant proteins have improved our understanding of the forces stabilizing proteins. They suggest that hydrogen bonding and the hydrophobic effect make large but comparable contributions to the stability of globular proteins.

Hydrogen Bonding↗
PubMed1995

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

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 (delta Cp), 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 delta Cp.

Animals↗
PubMed1995

How to measure and predict the molar absorption coefficient of a protein.

The molar absorption coefficient, epsilon, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring epsilon for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319-326] and is based on data from Edelhoch [1967, Biochemistry 6:1948-1954]). The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average epsilon values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the epsilon values in water, propanol, 6 M guanidine hydrochloride (GdnHCl), and 8 M urea have been measured. For Trp, the average epsilon values for the proteins are less than the epsilon values measured in any of the solvents. For Tyr, the average epsilon values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured epsilon values for 80 proteins, the epsilon at 280 nm of a folded protein in water, epsilon (280), can best be predicted with this equation: epsilon (280) (M-1 cm-1) = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125) These epsilon (280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict epsilon.

1-Propanol↗
PubMed1994

Energetics of ribonuclease T1 structure.

The energetics of thermal denaturation of two isoforms of ribonuclease T1 (Gln25 and Lys25) in various solvents have been studied by differential scanning calorimetry. It has been shown that the thermal transition of both forms of RNase T1 is strongly affected by slow kinetics, which cause an apparent deviation of the transition from a simple two-state model. By decreasing the heating rate or increasing the transition temperature, the denaturation of RNase approaches an equilibrium two-state transition. This permits determination of the thermodynamic parameters characterizing unfolding of the native structure. These thermodynamic parameters were correlated with the structural features of protein. Analysis of different contributions to the stability of RNase T1 shows that van der Waals interactions and hydrogen bonding are the major contributors to the conformational stability of the protein.

Aspergillus oryzae↗
PubMed1993

Investigation of ribonuclease T1 folding intermediates by hydrogen-deuterium amide exchange-two-dimensional NMR spectroscopy.

The rate of hydrogen bond formation at individual amino acid residues in ribonuclease T1 (RNase T1) has been investigated by the hydrogen-deuterium exchange-2D NMR (HDEx-2D NMR) technique (Udgaonkar & Baldwin, 1988; Rder et al., 1988) to gain insight into the mechanism and pathways of protein folding. The HDEx-2D NMR technique combines rapid mixing and 2D NMR methods to follow the protection of backbone amide deuterons from exchange with solvent protons as a function of folding time. The technique depends on the difference in the exchange rates of hydrogen-bonded and non-hydrogen-bonded amide residues so that as the protein folds, the amide residues involved in hydrogen bonding are protected from exchange with solvent to give structural information about early folding events. The observed time course for deuterium protection was followed for 24 backbone amide residues that form stable hydrogen bonds in RNase T1. The time courses are biphasic with 60-80% of the protein molecules showing rapid hydrogen bond formation (12-119 s-1) in the alpha-helix and the beta-sheet. The remaining 20-40% of the molecules are protected in a slow phase with a rate constant that has a lower limit of 0.01 s-1. If the rate constants in this first phase are arbitrarily subdivided into two classes, fast (> or = 25 s-1) and intermediate (< 25 s-1), then the amide residues that are found in the hydrophobic core are in the fast class while those located on the periphery of the three-dimensional structure are in the intermediate class.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1992

Temperature and guanidine hydrochloride dependence of the structural stability of ribonuclease T1.

The thermal unfolding of ribonuclease T1 has been studied by high-sensitivity differential scanning calorimetry as a function of temperature, [GuHCl], and scanning rate. The destabilizing effect of GuHCl has revealed that the kinetics of the unfolding transition become extremely slow as the transition temperature decreases. At pH 5.3 and zero GuHCl, the unfolding transition is centered at 59.1 degrees C; upon increasing the GuHCl concentration, the transition occurs at lower temperatures and exhibits progressively slower kinetics; so, for example, at 3 M GuHCl, the transition temperature is 40.6 degrees C and is characterized by a time constant close to 10 min. Under all conditions studied (pH 5.3, pH 7.0, [GuHCl] < 3 M), the transition is thermodynamically reversible. The slow kinetics of the transition induce significant distortions in the shape of the transition profiles that can be mistakenly interpreted as deviations from a two-state mechanism. Determination of the thermodynamic parameters from the calorimetric data has required the development of an analytical formalism that explicitly includes the thermodynamics as well as the kinetics of the transition. Using this formalism, it is shown that a two-state slow-kinetics model is capable of accurately describing the structural stability of ribonuclease T1 as a function of temperature, GuHCl concentration, and scanning rate. Multidimensional analysis of the calorimetric data has been used to estimate the intrinsic thermodynamic parameters for protein stability, the interaction parameters with GuHCl, and the time constant for the unfolding transition and its temperature dependence.

Aspergillus↗
PubMed1992

Contribution of the hydrophobic effect to globular protein stability.

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.

Amino Acids↗
PubMed1992

Thermodynamics of ribonuclease T1 denaturation.

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.

Calorimetry, Differential Scanning↗
PubMed1992

Urea denaturation of barnase: pH dependence and characterization of the unfolded state.

To investigate the pH dependence of the conformational stability of barnase, urea denaturation curves were determined over the pH range 2-10. The maximum conformational stability of barnase is 9 kcal mol-1 and occurs between pH 5 and 6. The dependence of delta G on urea concentration increases from 1850 cal mol-1 M-1 at high pH to about 3000 cal mol-1 M-1 near pH 3. This suggests that the unfolded conformations of barnase become more accessible to urea as the net charge on the molecule increases. Previous studies suggested that in 8 M urea barnase unfolds more completely than ribonuclease T1, even with the disulfide bonds broken [Pace, C.N., Laurents, D. V., & Thomson, J.A. (1990) Biochemistry 29, 2564-2572]. In support of this, solvent perturbation difference spectroscopy showed that in 8 M urea the Trp and Tyr residues in barnase are more accessible to perturbation by dimethyl sulfoxide than in ribonuclease T1 with the disulfide bonds broken.

Bacterial Proteins↗