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Robert L Baldwin

Publications and source records attributed to Robert L Baldwin.

16 recordsLinked to original sources
PubMed2006

Limited validity of group additivity for the folding energetics of the peptide group.

The principle of group additivity is a standard feature of analyses of the energetics of protein folding, but it is known that it may not always be valid for the polar peptide group. The neighboring residue effect shows that group additivity is not strictly valid for a heteropeptide. We show here that group additivity fails seriously for peptide groups close to either peptide end, even for a homopeptide that has blocked end groups with no formal charges involved. The failure of group additivity is caused by the electrostatic character of the solvation of peptide polar groups and is illustrated with values of the electrostatic solvation free energy (ESF) calculated by DelPhi. Solvation free energies and enthalpies are known experimentally for monoamides and are often used to model the solvation of peptide groups, but ESF results show that monoamide values are very different from those of peptide groups. A main cause of the difference is that peptide solvation depends on the dipole-dipole interactions made between adjacent peptide groups, which vary with peptide conformation. Ligands that interact with the peptide backbone by an electrostatic mechanism could show a similar peptide end effect, and hydrogen exchange results from the literature confirm that exchange rates are position-dependent close to peptide ends.

Hydrogen Bonding↗
PubMed2006

Intrinsic backbone preferences are fully present in blocked amino acids.

The preferences of amino acid residues for ,psi backbone angles vary strikingly among the amino acids, as shown by the backbone angle found from the (3)J(H(alpha),H(N)) coupling constant for short peptides in water. New data for the (3)J(H(alpha),H(N)) values of blocked amino acids (dipeptides) are given here. Dipeptides exhibit the full range of coupling constants shown by longer peptides such as GGXGG and dipeptides present the simplest system for analyzing backbone preferences. The dipeptide coupling constants are surprisingly close to values computed from the coil library (conformations of residues not in helices and not in sheets). Published coupling constants for GGXGG peptides agree closely with dipeptide values for all nonpolar residues and for some polar residues but not for X = D, N, T, and Y, which are probably affected by polar side chain-backbone interactions in GGXGG peptides. Thus, intrinsic backbone preferences are already determined at the dipeptide level and remain almost unchanged in GGXGG peptides and are strikingly similar in the coil library of conformations from protein structures. The simplest explanation for the backbone preferences is that backbone conformations are strongly affected by electrostatic dipole-dipole interactions in the peptide backbone and by screening of these interactions with water, which depends on nearby side chains. Strong backbone electrostatic interactions occur in dipeptides. This is shown by calculations both of backbone electrostatic energy for different conformers of the alanine dipeptide in the gas phase and by electrostatic solvation free energies of amino acid dipeptides.

Alanine↗
PubMed2004

Protein chemical shifts arising from alpha-helices and beta-sheets depend on solvent exposure.

The NMR chemical shifts of certain atomic nuclei in proteins ((1)H(alpha),(13)C(alpha), and (13)C(beta)) depend sensitively on whether or not the amino acid residue is part of a secondary structure (alpha-helix, beta-sheet), and if so, whether it is helix or sheet. The physical origin of the different chemical shifts of atomic nuclei in alpha-helices versus beta-sheets is a problem of long standing. We report that the chemical shift contributions arising from secondary structure (secondary structure shifts) depend strongly on the extent of exposure to solvent. This behavior is observed for (1)H(alpha), (13)C(alpha), and (13)C(beta) (sheet), but not for(13)C(beta) (helix), whose secondary structure shifts are small. When random coil values are subtracted from the chemical shifts of all(1)H(alpha) nuclei (Pro residues excluded) and the residual chemical shifts are summed to plot the mean values against solvent exposure, the results give a funnel-shaped curve that approaches a small value at full-solvent exposure. When chemical shifts are plotted instead against E(local), the electrostatic contribution to conformational energy produced by local dipole-dipole interactions, a well characterized dependence of (1)H(alpha) chemical shifts on E(local) is found. The slope of this plot varies with both the type of amino acid and the extent of solvent exposure. These results indicate that secondary structure shifts are produced chiefly by the electric field of the protein, which is screened by water dipoles at residues in contact with solvent.

Amino Acids↗
PubMed2004

Origin of the neighboring residue effect on peptide backbone conformation.

Unfolded peptides in water have some residual structure that may be important in the folding process, and the nature of the residual structure is currently of much interest. There is a neighboring residue effect on backbone conformation, discovered in 1997 from measurements of (3)J(HN alpha) coupling constants. The neighboring residue effect appears also in the "coil library" of Protein Data Bank structures of residues not in alpha-helix and not in beta-structure. When a neighboring residue (i - 1 or i + 1) belongs to class L (aromatic and beta-branched amino acids, FHITVWY) rather than class S (all others, G and P excluded), then the backbone angle of residue i is more negative for essentially all amino acids. Calculated values of peptide solvation (electrostatic solvation free energy, ESF) predict basic properties of the neighboring residue effect. We show that L amino acids reduce the solvation of neighboring peptide groups more than S amino acids. When tripeptides from the coil library are excised to allow solvation, the central residues have more negative values of but less negative values of with L than with S neighbors. The coil library values of <(3)J(HN alpha)>, which vary strikingly among the amino acids, are correlated with the neighboring residue effect seen by ESF. Moreover, values for the "blocking effect" of side chains on the hydrogen exchange rates of peptide NH protons are correlated with ESF values.

Amino Acid Sequence↗
PubMed2003

Role of backbone solvation and electrostatics in generating preferred peptide backbone conformations: distributions of phi.

The "coil library," consisting of the phi, psi values of residues outside secondary structure in high-resolution protein structures, has chiefly the beta, alpha(R), alpha(L), and polyproline II backbone conformations. In denatured proteins, the 20 aa have different average values of the (3)J(HN)alpha coupling constant, related to the backbone angle phi by the Karplus relation. Average (3)J(HN)alpha values obtained from the distributions of phi, g(phi), of the coil library agree with NMR results, and so the coil library can be and is being used to model denatured proteins. Here, Monte Carlo simulations of backbone conformations in denatured proteins are used to test two physics-based models: the random coil model of Brant and Flory [(1965) J. Am. Chem. Soc. 87, 2788-2791 and 2791-2800] and an electrostatic screening model (ESM) that includes electrostatic solvation. The random coil model represents hindered rotation about phi and psi backbone angles, nonbonded interactions, and dipole-dipole interactions. In the ESM, the nonbonded interactions term is replaced by the use of hard sphere repulsion and allowed regions in the Ramachadran maps. These models were tested by using the amino acid sequences of three small proteins. There are two main conclusions: (i) The g(phi) distributions of the coil library contain detailed, specific information, so that prediction of the g(phi) distributions of the different amino acids is a demanding test of the energy function. (ii) The ESM is partly successful in predicting the g(phi) distributions. Electrostatic solvation is primarily responsible, and steric clash between pairs of atoms connected by torsion angles is not responsible.

Amino Acids↗
PubMed2002

Relation between peptide backbone solvation and the energetics of peptide hydrogen bonds.

The H-bond inventory approach is used commonly to interpret data involving changes in the number or types of protein hydrogen bonds. I point out here that this approach gives an incorrect answer either for the standard free energy or enthalpy of the reaction between simple amides and water. On the other hand, an electrostatic solvation approach fits almost within error the polar solvation free energies of small molecules, including amides. The electrostatic solvation approach is used here to discuss the relation between peptide backbone solvation and the enthalpy change for forming an alanine helix.

Hydrogen Bonding↗
PubMed2002

Circular dichroism spectra of short, fixed-nucleus alanine helices.

Very short alanine peptide helices can be studied in a fixed-nucleus, helix-forming system [Siedlicka, M., Goch, G., Ejchart, A., Sticht, H. & Bierzynski, A. (1999) Proc. Natl. Acad. Sci. USA 96, 903-908]. In a 12-residue sequence taken from an EF-hand protein, the four C-terminal peptide units become helical when the peptide binds La(3+), and somewhat longer helices may be made by adding alanine residues at the C terminus. The helices studied here contain 4, 8, or 11 peptide units. Surprisingly, these short fixed-nucleus helices remain almost fully helical from 4 to 65 degrees C, according to circular dichroism results reported here, and in agreement with titration calorimetry results reported recently. These peptides are used here to define the circular dichroism properties of short helices, which are needed for accurate measurement of helix propensities. Two striking properties are: (i) the temperature coefficient of mean peptide ellipticity depends strongly on helix length; and (ii) the intensity of the signal decreases much less rapidly with helix length, for very short helices, than supposed in the past. The circular dichroism spectra of the short helices are compared with new theoretical calculations, based on the experimentally determined direction of the NV(1) transition moment.

Alanine↗
PubMed2002

Polyproline II structure in a sequence of seven alanine residues.

A sequence of seven alanine residues-too short to form an alpha-helix and whose side chains do not interact with each other-is a particularly simple model for testing the common description of denatured proteins as structureless random coils. The (3)J(HN alpha) coupling constants of individual alanine residues have been measured from 2 to 56 degrees C by using isotopically labeled samples. The results display a thermal transition between different backbone conformations, which is confirmed by CD spectra. The NMR results suggest that polyproline II is the dominant conformation at 2 degrees C and the content of beta strand is increased by approximately 10% at 55 degrees C relative to that at 2 degrees C. The polyproline II conformation is consistent with recent studies of short alanine peptides, including structure prediction by ab initio quantum mechanics and solution structures for both a blocked alanine dipeptide and an alanine tripeptide. CD and other optical spectroscopies have found structure in longer "random coil" peptides and have implicated polyproline II, which is a major backbone conformation in residues within loop regions of protein structures. Our result suggests that the backbone conformational entropy in alanine peptides is considerably smaller than estimated by the random coil model. New thermodynamic data confirm this suggestion: the entropy loss on alanine helix formation is only 2.2 entropy units per residue.

Alanine↗
PubMed2002

Origin of the different strengths of the (i,i+4) and (i,i+3) leucine pair interactions in helices.

Pairs of leucine side chains, spaced either (i,i+3) or (i,i+4), are known to stabilize alanine-based peptide helices, Experiments with new peptide sequences confirm that the (i,i+4) pair interaction is markedly stronger than the (i,i+3) pair interaction. This result is not expected from reported Monte Carlo simulations, which predict that the (i,i+3) interaction is slightly stronger. The interaction strength can be predicted from recently reported measurements of buried non-polar surface area, obtained from structures in the Protein Data Bank: the agreement is reasonable for the (i,i+3) LL interaction but underestimates the (i,i+4) LL interaction. Solvation of peptide groups in the helix backbone may contribute to the different strengths of the two LL pair interactions because different chi(1) leucine rotamers are used and the (i,i+3) pair shields two peptide groups whereas the (i,i+4) pair shields only one. A rough estimate of the backbone solvation effect, based on the difference between the helix propensities of leucine and alanine, agrees with the size of the difference between the (i,i+3) and (i,i+4) leucine pair interactions.

Circular Dichroism↗
PubMed2002

Making a network of hydrophobic clusters.

The early events in protein folding are often difficult to track. A hydrophobic collapse, during which nonpolar residues are buried away from the polar solvent, has been proposed, but little is known about the role of this collapse in protein folding. In his Perspective, Baldwin discusses the report by Klein-Seetharaman et al., who shed some light on this issue. A network of hydrophobic clusters stabilizes at least one nonnative interaction in unfolded hen lysozyme. In conjunction with earlier studies, the results suggest that this network has a beneficial effect on the folding of the protein.

Animals↗
PubMed2002

The enthalpy of the alanine peptide helix measured by isothermal titration calorimetry using metal-binding to induce helix formation.

The goal of this study is to use the model system described earlier to make direct measurements of the enthalpy of helix formation at different temperatures. For this we studied model alanine peptides in which helix formation can be triggered by metal (La(3+)) binding. The heat of La(3+) interaction with the peptides at different temperatures is measured by isothermal titration calorimetry. Circular dichroism spectroscopy is used to follow helix formation. Peptides of increasing length (12-, 16-, and 19-aa residues) that contain a La(3+)-binding loop followed by helices of increasing length, are used to separate the heat of metal binding from the enthalpy of helix formation. We demonstrate that (i) the enthalpy of helix formation is -0.9 +/- 0.1 kcal/mol; (ii) the enthalpy of helix formation is independent of the peptide length; (iii) the enthalpy of helix formation does not depend significantly on temperature in the range from 5 to 45 degrees C, suggesting that the heat capacity change on helix formation is very small. Thus, the use of metal binding to induce helix formation has an enormous potential for measuring various thermodynamic properties of alpha-helices.

Alanine↗
PubMed2002

Role of backbone solvation in determining thermodynamic beta propensities of the amino acids.

There is a paradox concerning the beta propensities of the amino acids: the amino acids with the highest beta propensities such as valine and isoleucine have the highest tendency to desolvate the peptide backbone, which should result in a loss of stability. Nevertheless, backbone solvation, calculated as electrostatic solvation free energy (ESF), is highly correlated with mutant stability in the zinc-finger system studied by Kim and Berg [Kim, C. A. & Berg, J. M. (1993) Nature (London) 362, 267-270], and valine and isoleucine are among the most stabilizing amino acids. This inverse correlation between stability and ESF can be explained, because the mutant ESF differences in the unfolded protein are larger than in the native protein. Consequently, mutations such as Ala to Val destabilize the unfolded form more than the native protein. By comparing mutant Delta ESF values in isolated beta-strands versus beta-sheets, we conclude that amino acids with high beta propensities should exert their stabilizing effects at early stages in folding. This deduction agrees with the studies by Clarke and coworkers [Lorch, M., Mason, J. M., Clarke, A. R. & Parker, M. J. (1999) Biochemistry 38, 1377-1385, and Lorch, M., Mason, J. M., Sessions, R. B. & Clarke, A. R. (2000) Biochemistry 39, 3480-3485] of the thermodynamics of folding of the beta-sheet protein CD2.d1.

Amino Acid Sequence↗
PubMed2002

Sulfate anion stabilization of native ribonuclease A both by anion binding and by the Hofmeister effect.

Data are reported for T(m), the temperature midpoint of the thermal unfolding curve, of ribonuclease A, versus pH (range 2-9) and salt concentration (range 0-1 M) for two salts, Na(2)SO(4) and NaCl. The results show stabilization by sulfate via anion-specific binding in the concentration range 0-0.1 M and via the Hofmeister effect in the concentration range 0.1-1.0 M. The increase in T(m) caused by anion binding at 0.1 M sulfate is 20 degrees at pH 2 but only 1 degree at pH 9, where the net proton charge on the protein is near 0. The 10 degrees increase in T(m) between 0.1 and 1.0 M Na(2)SO(4), caused by the Hofmeister effect, is independent of pH. A striking property of the NaCl results is the absence of any significant stabilization by 0.1 M NaCl, which indicates that any Debye screening is small. pH-dependent stabilization is produced by 1 M NaCl: the increase in T(m) between 0 and 1.0 M is 14 degrees at pH 2 but only 1 degree at pH 9. The 14 degree increase at pH 2 may result from anion binding or from both binding and Debye screening. Taken together, the results for Na(2)SO(4) and NaCl show that native ribonuclease A is stabilized at low pH in the same manner as molten globule forms of cytochrome c and apomyoglobin, which are stabilized at low pH by low concentrations of sulfate but only by high concentrations of chloride.

Anions↗