X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase

The metabolic pathway by which 4-chlorobenzoate is degraded to 4-hydroxybenzoate in the soil-dwelling microbe Pseudomonas sp. strain CBS-3 consists of three enzymes including 4-hydroxybenzoyl-CoA thioesterase. The structure of the unbound form of this thioesterase has been shown to contain the so-called "hot dog" fold with a large helix packed against a five-stranded anti-parallel beta-sheet. To address the manner in which the enzyme accommodates the substrate within the active site, two inhibitors have been synthesized, namely 4-hydroxyphenacyl-CoA and 4-hydroxybenzyl-CoA. Here we describe the structural analyses of the enzyme complexed with these two inhibitors determined and refined to 1.5 and 1.8 A resolution, respectively. These studies indicate that only one protein side chain, Ser(91), participates directly in ligand binding. All of the other interactions between the protein and the inhibitors are mediated through backbone peptidic NH groups, carbonyl oxygens, and/or solvents. The structures of the enzyme-inhibitor complexes suggest that both a hydrogen bond and the positive end of a helix dipole moment serve to polarize the electrons away from the carbonyl carbon of the acyl group, thereby making it more susceptible to nucleophilic attack. Additionally, these studies demonstrate that the carboxylate group of Asp(17) is approximately 3.2 A from the carbonyl carbon of the acyl group. To address the role of Asp(17), the structure of the site-directed mutant protein D17N with bound substrate has also been determined. Taken together, these investigations suggest that the reaction mechanism may proceed through an acyl enzyme intermediate.     

Proline residues in transmembrane helices of channel and transport proteins: a molecular modelling study.

Proline residues are commonly found in putative transbilayer helices of many integral membrane proteins which act as transporters, channels and receptors. Intramembranous prolines are often conserved between homologous proteins. It has been suggested that such intrahelical prolines provide liganding sites for cations via exposure of the backbone carbonyl oxygen atoms of residues i-3 and i-4 (relative to the proline). Molecular modelling studies have been carried out to evaluate this proposal. Bundles of parallel proline-kinked helices are considered as simplified models of ion channels. The energetics of K+ ion-helix bundle interactions are explored. It is shown that carbonyl oxygens exposed by the proline-induced kink and at the C-terminus of the helices may provide cation-liganding sites. 'Hybrid' bundles of antiparallel helices, only some of which contain proline residues, are considered as models of transport proteins. Again, proline-exposed carbonyl oxygens are shown to be capable of liganding cations. The roles of alpha-helix dipoles and of the geometry of helix packing are considered in relation to cation-bundle interactions. Implications with respect to modelling of ion channel and transport proteins are discussed.     

Interaction of poly(X) with poly(U): alkali metal ion specificity suggests a four-stranded helix

Interaction of poly(X) with poly(U) to form a regular, ordered structure has been investigated by uv, CD, and ir spectroscopy. The XU complex exhibits a marked dependence of T_m on the nature of the alkali metal ion present in solution. Stability of the complex is surprisingly high and increases with ionization of X residues at N3. The ir evidence shows the U residues are present as the diketo form and X residues as the 6-keto-2-enolate anion and indicates that the X and U carbonyl groups are vibrationally coupled. A mixing curve exhibits a single minimum, at 1:1, but we conclude that the actual combining ratio is twice this value and that the formula of the complex is X₂U₂. We propose a model based on a four-standard helix with size-specific alkali metal complexing to X and U carbonyl oxygens in the axial channel of the helix. This model and previously suggested two-stranded structures are evaluated in terms of the physical and chemical properties of the complex.     

Conduction of Na+ and K+ through the NaK channel: molecular and Brownian dynamics studies

Conduction of ions through the NaK channel, with M0 helix removed, was studied using both Brownian dynamics and molecular dynamics. Brownian dynamics simulations predict that the truncated NaK has approximately a third of the conductance of the related KcsA K⁺ channel, is outwardly rectifying, and has a Michaelis-Menten current-concentration relationship. Current magnitude increases when the glutamine residue located near the intracellular gate is replaced with a glutamate residue. The channel is blocked by extracellular Ca²⁺. Molecular dynamics simulations show that, under the influence of a strong applied potential, both Na⁺ and K⁺ move across the selectivity filter, although conduction rates for Na⁺ ions are somewhat lower. The mechanism of conduction of Na⁺ differs significantly from that of K⁺ in that Na⁺ is preferentially coordinated by single planes of pore-lining carbonyl oxygens, instead of two planes as in the usual K⁺ binding sites. The water-containing filter pocket resulting from a single change in the selectivity filter sequence (compared to potassium channels) disrupts several of the planes of carbonyl oxygens, and thus reduces the filter's ability to discriminate against sodium.     

A conformational analysis of leucine enkephalin as a function of pH

The conformations of Leu enkephalin in aqueous solution have been investigated as a function of pH using molecular dynamics simulations. The simulations suggest the peptide backbone exists as a mixture of folded and unfolded forms (approximately 50% each) at neutral pH, but is always unfolded at low or high pH. The folded form at neutral pH possesses a 2 --> 5 hydrogen bond and a close head to tail separation. No significant intramolecular hydrogen bonding of the carbonyl oxygens was observed in either the folded or unfolded forms of the peptide. Analysis of the Gly carbonyl oxygens and terminal groups indicated that, while the conformational population distribution of Leu enkephalin did vary noticeably as a function of pH, their hydration was essentially independent of pH and in agreement with the available NMR data. Further study indicated that the unfolded state of the peptide was not random in nature and consisted of one major unfolded backbone arrangement stabilized by a persistent hydrophobic interaction between the side chains of Tyr and Leu.     

NMR studies on turn mimetic analogs derived from melanocyte-stimulating hormones

Oligomers with alpha-aminooxy acids are reported to form very stable turn and helix structures, and they are supposed to be useful peptidomimetics for drug design. A recent report suggested that homochiral oxa-peptides form a strong eight-member-ring structure by a hydrogen bond between adjacent aminooxy-acid residues in a CDCl3 solution. In order to design an alpha-MSH analog with a stable turn conformation, we synthesized four tetramers and one pentamer, based on alpha-MSH sequence, and determined the solution structures of the molecules by two-dimensional NMR spectroscopy and simulated annealing calculations. The solution conformations of the three peptidomimetic molecules (TLV, TDV, and TLL) in DMSO-d6 contain a stable 7-membered-ring structure that is similar to a gamma-turn in normal peptides. Newly-designed tetramer TDF and pentamer PDF have a ball-type rigid structure that is induced by strong hydrogen bonds between adjacent amide protons and carbonyl oxygens. In conclusion, the aminooxy acids, easily prepared from natural or unnatural amino acids, can be employed to prepare peptidomimetic analogues with well-defined turn structures for pharmaceutical interest.     

Systematics in the interaction of metal ions with the main-chain carbonyl group in protein structures

An analysis of the geometry of metal binding by peptide carbonyl groups in proteins is presented. Such metal ions are predominantly calcium in known protein structures. Cations tend to be located in the peptide plane, near the C = O bond direction. This distribution differs from that observed for water molecules bound to carbonyl oxygens. Most metal ions are bound to carbonyl oxygens of peptides in turns or in regions with no regular secondary structure. More infrequent binding interactions occur at the C-terminal end of alpha-helices or at the edges and sides of beta-sheets, where the geometrical preferences of the metal-carbonyl interaction may be satisfied. In many proteins carbonyl groups that are one, two, or three residues apart along the polypeptide chain bind to the same cation; these structures show a limited number of main-chain conformations around the metal center.     

Influence of the g− conformation of Ser and Thr on the structure of transmembrane helices

In order to study the influence of Ser and Thr on the structure of transmembrane helices we have analyzed a database of helix stretches extracted from crystal structures of membrane proteins and an ensemble of model helices generated by molecular dynamics simulations. Both complementary analyses show that Ser and Thr in the g? conformation induce and/or stabilize a structural distortion in the helix backbone. Using quantum mechanical calculations, we have attributed this effect to the electrostatic repulsion between the side chain Oγ atom of Ser and Thr and the backbone carbonyl oxygen at position i ? 3. In order to minimize the repulsive force between these negatively charged oxygens, there is a modest increase of the helix bend angle as well as a local opening of the helix turn preceding Ser/Thr. This small distortion can be amplified through the helix, resulting in a significant displacement of the residues located at the other side of the helix. The crystal structures of aquaporin Z and the β₂-adrenergic receptor are used to illustrate these effects. Ser/Thr-induced structural distortions can be implicated in processes as diverse as ligand recognition, protein function and protein folding.     

Side-Chain to Main-Chain Hydrogen Bonding Controls the Intrinsic Backbone Dynamics of the Amyloid Precursor Protein Transmembrane Helix

Many transmembrane helices contain serine and/or threonine residues whose side chains form intrahelical H-bonds with upstream carbonyl oxygens. Here, we investigated the impact of threonine side-chain/main-chain backbonding on the backbone dynamics of the amyloid precursor protein transmembrane helix. This helix consists of a N-terminal dimerization region and a C-terminal cleavage region, which is processed by γ-secretase to a series of products. Threonine mutations within this transmembrane helix are known to alter the cleavage pattern, which can lead to early-onset Alzheimer’s disease. Circular dichroism spectroscopy and amide exchange experiments of synthetic transmembrane domain peptides reveal that mutating threonine enhances the flexibility of this helix. Molecular dynamics simulations show that the mutations reduce intrahelical amide H-bonding and H-bond lifetimes. In addition, the removal of side-chain/main-chain backbonding distorts the helix, which alters bending and rotation at a diglycine hinge connecting the dimerization and cleavage regions. We propose that the backbone dynamics of the substrate profoundly affects the way by which the substrate is presented to the catalytic site within the enzyme. Changing this conformational flexibility may thus change the pattern of proteolytic processing.     

Conformational changes of an exchangeable apolipoprotein, apolipophorin III from Locusta migratoria, at low pH: correlation with lipid binding.

Locusta migratoria apolipophorin III (apoLp-III) is a helix bundle exchangeable apolipoprotein that reversibly binds to lipoprotein surfaces. Structural reorganization of its five amphipathic alpha-helices enables the transition from the lipid-free to lipid-bound state. ApoLp-III-induced transformation of dimyristoylphosphatidylcholine (DMPC) bilayer vesicles into smaller discoidal complexes is enhanced as a function of decreasing pH, with maximal transformation occurring at pH 3.5. Over the entire pH range studied, apoLp-III retains nearly all of its secondary structure content. Whereas no changes in fluorescence emission maximum of the two Trp residues in apoLp-III were observed in the pH range from 7.0 to 4.0, a further decrease in pH resulted in a strong red shift. Near-UV circular dichroism spectra of apoLp-III showed well-defined extrema (at 286 and 292 nm) between pH 7.0 and pH 4.0, which were attributed to Trp115. Below pH 4.0, these extrema collapsed, indicating a less rigid environment for Trp115. Similarly, the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate in the presence of apoLp-III increased 4-fold below pH 4.0, indicating exposure of hydrophobic sites in the protein in this pH range. Taken together, the data suggest two conformational states of the protein. In the first state between pH 7.0 and pH 4.0, apoLp-III retains a nativelike helix bundle structure. The second state, found between pH 3.0 and pH 4.0, is reminiscent of a molten globule, wherein tertiary structure contacts are disrupted without a significant loss of secondary structure content. In both states DMPC vesicle transformation is enhanced by lowering the solution pH, reaching an optimum in the second state. The correlation between tertiary structure and lipid binding activity suggests that helix bundle organization is a determinant of apoLp-III lipid binding activity.     

Three-dimensional structure and dynamics of wine tannin-saliva protein complexes. A multitechnique approach

The interactions between the B3 (catechin-4alpha,8-catechin) red wine tannin and the human salivary protein fragment IB7(14) (SPPGKPQGPPPQGG) were monitored by (1)H magic angle spinning NMR, circular dichroism, electrospray ionization mass spectrometry, and molecular modeling. It is found that the secondary structure of IB7(14) is made of a type II helix (collagen helix) and random coil. The central glycine 8 appears to act as a flexible rotula separating two helix II regions. Three tannin molecules tightly complex the peptide, without modifying its secondary structure, but seem to reduce its conformational dynamics. The binding dissociation constant is in the millimolar range. B3 tannins with a "tweezers" conformation bind to the hydrophilic side of the saliva peptide, suggesting that the principal driving forces toward association are governed by hydrogen bonding between the carbonyl functions of proline residues and both the phenol and catechol OH groups. These findings are further discussed in the frame of an astringency phenomenon.     

Comparison of protein and deoxyribonucleic acid backbone structures in fd and Pf1 bacteriophages

The conformations of the protein and nucleic acid backbones in the filamentous viruses fd and Pf1 are characterized by one- and two-dimensional solid-state NMR experiments on oriented virus solutions. Striking differences are observed between fd and Pf1 in both their protein and DNA structures. The coat proteins of fd and Pf1 are almost entirely alpha helical and in both viruses most of the helix is oriented parallel to the filament axis. fd coat protein is one stretch of alpha helix that is slightly slued about the filament axis. In Pf1 coat protein two distinct sections of alpha helix are present, the smaller of which is tilted with respect to the filament axis by about 20 degrees. The DNA backbone structure of fd is completely disordered. By contrast, the DNA backbone of Pf1 is uniformly oriented such that all of the phosphodiester groups have the O-P-O plane of the nonesterified oxygens approximately perpendicular to the filament axis.     

Side-Chain to Main-Chain Hydrogen Bonding Controls the Intrinsic Backbone Dynamics of the Amyloid Precursor Protein Transmembrane Helix

Many transmembrane helices contain serine and/or threonine residues whose side chains form intrahelical H-bonds with upstream carbonyl oxygens. Here, we investigated the impact of threonine side-chain/main-chain backbonding on the backbone dynamics of the amyloid precursor protein transmembrane helix. This helix consists of a N-terminal dimerization region and a C-terminal cleavage region, which is processed by γ-secretase to a series of products. Threonine mutations within this transmembrane helix are known to alter the cleavage pattern, which can lead to early-onset Alzheimer’s disease. Circular dichroism spectroscopy and amide exchange experiments of synthetic transmembrane domain peptides reveal that mutating threonine enhances the flexibility of this helix. Molecular dynamics simulations show that the mutations reduce intrahelical amide H-bonding and H-bond lifetimes. In addition, the removal of side-chain/main-chain backbonding distorts the helix, which alters bending and rotation at a diglycine hinge connecting the dimerization and cleavage regions. We propose that the backbone dynamics of the substrate profoundly affects the way by which the substrate is presented to the catalytic site within the enzyme. Changing this conformational flexibility may thus change the pattern of proteolytic processing.     

Energy-minimized conformation of gramicidin-like channels. I. Infinitely long poly-(L,D)-alanine beta 6.3-helix.

The energy-minimized conformation of an infinitely long poly-(L,D)-alanine in single-stranded beta 6.3-helix was calculated by the molecular mechanics method. When energy minimization was started from a wide range of initial geometries, six optimized conformations were obtained and identified as the right- and left-handed counterparts of the beta 4.5-, beta 6.3-, and beta 8.2-helices. It was found that their conformation energies increase in this order, the beta 4.5-helix having the lowest energy. The backbone dihedral angles of the energy-minimized beta 6.3-helix were: phi L = -116 degrees (or -131 degrees), psi L = 122 degrees (or 111 degrees), phi D = 131 degrees (or 116 degrees), psi D = -111 degrees (or -122 degrees), omega L = 173 degrees (or 173 degrees), and omega D = -173 degrees (or -173 degrees) for the right-handed (or left-handed) helix. This helix was composed of 6.30 residues/turn with a pitch of 4.97 A. All the alpha-carbons of L- and D-configurations appeared on one common circular helix. Interestingly, small deviations (approximately 7 degrees) of the peptide bonds from the planar structure caused a considerable lowering of the conformation energy, and, at the same time, they produced more favorable fitting of the hydrogen bonds; the carbonyl oxygens and the nearest-neighbor alpha-hydrogens also took more favorable relative positions.     

Peptaibol zervamicin IIb structure and dynamics refinement from transhydrogen bond J couplings

Zervamicin IIB (Zrv-IIB) is a channel-forming peptaibol antibiotic of fungal origin. The measured transhydrogen bond (3h)J(NC') couplings in methanol solution heaving average value of -0.41 Hz indicate that the stability of the Zrv-IIB helix in this milieu is comparable to the stability of helices in globular proteins. The N-terminus of the peptide forms an alpha-helix, whereas 3(10)-helical hydrogen bonds stabilize the C-terminus. However, two weak transhydrogen bond peaks are observed in a long-range HNCO spectrum for HN Aib(12). Energy calculations using the Empirical Conformation Energy Program for Peptides (ECEPP)/2 force field and the implicit solvent model show that the middle of the peptide helix accommodates a bifurcated hydrogen bond that is simultaneously formed between HN Aib(12) and CO Leu(8) and CO Aib(9). Several lowered (3h)J(NC') on a polar face of the helix correlate with the conformational exchange process observed earlier and imply dynamic distortions of a hydrogen bond pattern with the predominant population of a properly folded helical structure. The refined structure of Zrv-IIB on the basis of the observed hydrogen bond pattern has a small ( approximately 20 degrees ) angle of helix bending that is virtually identical to the angle of bending in dodecylphosphocholine (DPC) micelles, indicating the stability of a hinge region in different environments. NMR parameters ((1)HN chemical shifts and transpeptide bond (1)J(NC') couplings) sensitive to hydrogen bonding along with the solvent accessible surface area of carbonyl oxygens indicate a large polar patch on the convex side of the helix formed by three exposed backbone carbonyls of Aib(7), Aib(9), and Hyp(10) and polar side chains of Hyp(10), Gln(11), and Hyp(13). The unique structural features, high helix stability and the enhanced polar patch, set apart Zrv-IIB from other peptaibols (for example, alamethicin) and possibly underlie its biological and physiological properties.     

Structural consequences of replacement of an alpha-helical Pro residue in Escherichia coli thioredoxin

While it is well known that introduction of Pro residues into the interior of protein alpha-helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein alpha-helix. We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1-1.5 kcal/mol in the temperature range 280-330 K. This paper describes the structure of the P40S mutant at a resolution of 1.8 A. In wild-type thioredoxin, P40 is located in the interior of helix two, a long alpha-helix that extends from residues 32 to 49 with a kink at residue 40. Structural differences between the wild-type and P40S are largely localized to the above helix. In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36. The helix remains kinked. In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39. However, these are absent in P40S. Instead, these main chain atoms are hydrogen bonded to water molecules. The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin.     

Solution structure of the eukaryotic pore-forming cytolysin equinatoxin II: implications for pore formation

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. The structure of the actinoporin equinatoxin II in aqueous solution, determined from NMR data, consists of two short helices packed against opposite faces of a beta-sandwich structure formed by two five-stranded beta-sheets. The protein core has extensive hydrophobic interfaces formed by residues projecting from the internal faces of the two beta-sheets. 15N relaxation data show uniform backbone dynamics, implying that equinatoxin II in solution is relatively rigid, except at the N terminus; its inferred rotational correlation time is consistent with values for monomeric proteins of similar mass. Backbone amide exchange rate data also support the view of a stable structure, even though equinatoxin II lacks disulfide bonds. As monitored by NMR, it unfolds at around 70 degrees C at pH 5.5. At 25 degrees C the structure is stable over the pH range 2.5-7.3 but below pH 2.5 it undergoes a slow transition to an incompletely unfolded structure resembling a molten globule. Equinatoxin II has two significant patches of positive electrostatic potential formed by surface-exposed Lys and Arg residues, which may assist its interaction with charged regions of the lipid head groups. Tyr and Trp residues on the surface may also contribute by interacting with the carbonyl groups of the acyl chains of target membranes. Data from mutational studies and truncated analogues identify two regions of the protein involved in membrane interactions, the N-terminal helix and the Trp-rich region. Once the protein is anchored, the N-terminal helix may penetrate the membrane, with up to four helices lining the pore, although other mechanisms of pore formation cannot be ruled out.     

A new model for the K+-induced macromolecular structure of guanosine 5'-monophosphate in solution.

The (31)P NMR spectra of (TMA)(2)(5'-GMP), where TMA is [(CH(3))(4)N](+) and 5'-GMP is guanosine 5'-monophosphate, and K(2)(5'-GMP), containing various amounts of KCl or TMACl, have been obtained at 2 degrees C. Variable-temperature spectra have also been obtained for K(2)(5'-GMP). The TMA(+) ion serves to neutralize the charge on the dianionic 5'-GMP and permits the added K(+) to bond preferentially in structure-forming sites. (1)H NMR spectra (one- and two-dimensional) have been obtained for K(2)(5'-GMP) and used to assign the proton resonances in the self-associated structures and determine that all residues have the anti glycosidic conformation. The (31)P and (1)H NMR spectra are very complex and indicate the presence of a large number of molecular environments and a structural variation dependent upon the mole ratio of 5'-GMP to K(+). A new model for the solution structure is proposed in which the 5'-GMP forms a pseudo-four-stranded helix with guanine-guanine hydrogen bonding forming a continuous helical strand, rather than the usual planar G-tetrad structure. The guanine-guanine hydrogen bonding sites are the same as that found in a G-tetrad. The K(+) ions would be located in the center of the helix and bonding to the carbonyl oxygens. They are interacting with the phosphates as well. Integration data from the largest sized species give an estimate of 14.3 +/- 1.1 residues in a helical structure.     

The effect of H<Subscript>3</Subscript>O<Superscript>+</Superscript> on the membrane morphology and hydrogen bonding of a phospholipid bilayer

At the 2017 meeting of the Australian Society for Biophysics, we presented the combined results from two recent studies showing how hydronium ions (H₃O⁺) modulate the structure and ion permeability of phospholipid bilayers. In the first study, the impact of H₃O⁺ on lipid packing had been identified using tethered bilayer lipid membranes in conjunction with electrical impedance spectroscopy and neutron reflectometry. The increased presence of H₃O⁺ (i.e. lower pH) led to a significant reduction in membrane conductivity and increased membrane thickness. A first-order explanation for the effect was assigned to alterations in the steric packing of the membrane lipids. Changes in packing were described by a critical packing parameter (CPP) related to the interfacial area and volume and shape of the membrane lipids. We proposed that increasing the concentraton of H₃O⁺ resulted in stronger hydrogen bonding between the phosphate oxygens at the water–lipid interface leading to a reduced area per lipid and slightly increased membrane thickness. At the meeting, a molecular model for these pH effects based on the result of our second study was presented. Multiple μs-long, unrestrained molecular dynamic (MD) simulations of a phosphatidylcholine lipid bilayer were carried out and showed a concentration dependent reduction in the area per lipid and an increase in bilayer thickness, in agreement with experimental data. Further, H₃O⁺ preferentially accumulated at the water–lipid interface, suggesting the localised pH at the membrane surface is much lower than the bulk bathing solution. Another significant finding was that the hydrogen bonds formed by H₃O⁺ ions with lipid headgroup oxygens are, on average, shorter in length and longer-lived than the ones formed in bulk water. In addition, the H₃O⁺ ions resided for longer periods in association with the carbonyl oxygens than with either phosphate oxygen in lipids. In summary, the MD simulations support a model where the hydrogen bonding capacity of H₃O⁺ for carbonyl and phosphate oxygens is the origin of the pH-induced changes in lipid packing in phospholipid membranes. These molecular-level studies are an important step towards a better understanding of the effect of pH on biological membranes.     

Thermodynamic effects of replacements of Pro residues in helix interiors of maltose-binding protein

Introduction of Pro residues into helix interiors results in protein destabilization. It is currently unclear if the converse substitution (i.e., replacement of Pro residues that naturally occur in helix interiors would be stabilizing). Maltose-binding protein is a large 370-amino acid protein that contains 21 Pro residues. Of these, three nonconserved residues (P48, P133, and P159) occur at helix interiors. Each of the residues was replaced with Ala and Ser. Stabilities were characterized by differential scanning calorimetry (DSC) as a function of pH and by isothermal urea denaturation studies as a function of temperature. The P48S and P48A mutants were found to be marginally more stable than the wild-type protein. In the pH range of 5-9, there is an average increase in T(m) values of P48A and P48S of 0.4 degrees C and 0.2 degrees C, respectively, relative to the wild-type protein. The other mutants are less stable than the wild type. Analysis of the effects of such Pro substitutions in MBP and in three other proteins studied to date suggests that substitutions are more likely to be stabilizing if the carbonyl group i-3 or i-4 to the mutation site is not hydrogen bonded in the wild-type protein.