The GCN4 leucine zipper is a peptide homodimer that has been the subject of a number of experimental and theoretical investigations into the determinants of affinity and specificity. Here, we utilize this model system to investigate electrostatic effects in protein binding using continuum calculations. A particularly novel feature of the computations made here is that they provide an interaction-by-interaction breakdown of the electrostatic contributions to the free energy of docking that includes changes in the interaction of each functional group with solvent and changes in interactions between all pairs of functional groups on binding. The results show that (1) electrostatic effects disfavor binding by roughly 15 kcal/mol due to desolvation effects that are incompletely compensated in the bound state, (2) while no groups strongly stabilize binding, the groups that are most destabilizing are charged and polar side chains at the interface that have been implicated in determining binding specificity, and (3) attractive intramolecular interactions (e.g., backbone hydrogen bonds) that are enhanced on binding due to reduced solvent screening in the bound state contribute significantly to affinity and are likely to be a general effect in other complexes. A comparison is made between the results obtained in an electrostatic analysis carried out calculationally and simulated results corresponding to idealized data from a scanning mutagenesis experiment. It is shown that scanning experiments provide incomplete information on interactions and, if overinterpreted, tend to overestimate the energetic effect of individual side chains that make attractive interactions. Finally, a comparison is made between the results available from a continuum electrostatic model and from a simpler surface-area dependent solvation model. In this case, although the simpler model neglects certain interactions, on average it performs rather well.

B*2701 differs from all other HLA-B27 subtypes of known peptide specificity in that, among its natural peptide ligands, arginine is not the only allowed residue at peptide position 2. Indeed, B*2701 is unique in binding many peptides with Gln2 in vivo. However, the mutation (Asp74Tyr) responsible for altered selectivity is far away from the B pocket of the peptide binding site to which Gln/Arg2 binds. Here, we present a model that explains this effect. It is proposed that a new rotameric state of the conserved Lys70 is responsible for the unique B*2701 binding motif. This side chain should be either kept away from pocket B through its interaction with Asp74 in most HLA-B27 subtypes, or switched to this pocket if residue 74 is Tyr as in B*2701. Involvement of Lys70 in pocket B would thus allow binding of peptides with Gln2. Binding of Arg2-containing peptides to B*2701 is also possible because Lys70 could adopt another conformation, H-bonded to Asn97, which preserves the same binding mode of Arg2 as in B*2705. This model was experimentally validated by mutating Lys70 into Ala in B*2701. Edman sequencing of the B*2701(K70A) peptide pool showed only Arg2, characteristic of HLA-B27-bound peptides, and no evidence for Gln2. This supports the computational model and demonstrates that allowance of B*2701 for peptides with Gln2 is due to the long-range effect of the polymorphic residue 74 of HLA-B27, by inducing a conformational switch of the conserved Lys70.

A 12-residue peptide designed to form an α-helix and self-associate into an antiparallel 4-α-helical bundle yields a 0.9 Å crystal structure revealing unanticipated features. The structure was determined by direct phasing with the “Shake-and-Bake” program, and contains four crystallographically distinct 12-mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal α-helices hydrogen bonded head-to-tail into columns, which in turn pack side-by-side into sheets spanning the width of the crystal. Within each sheet, the α-helices run antiparallel and are closely spaced (9–10 Å center-to-center). The sheets are more loosely packed against each other (13–14 Å between helix centers). Each sheet is amphiphilic: apolar leucine side chains project from one face, charged lysine and glutamate side chains from the other face. The sheets are stacked with two polar faces opposing and two apolar faces opposing. The result is a periodic biomaterial composed of packed protein bilayers, with alternating polar and apolar interfaces. All of the 30 water molecules in the unit cell lie in the polar interface or between the stacked termini of helices. A section through the sheet reveals that the helices packed at the apolar interface resemble the four-α-helical bundle of the design, but the helices overhang parts of the adjacent bundles, and the helix crossing angles are less steep than intended (7–11° rather than 18°).

We report the 0.75 Å crystal structure of a racemic mixture of the 12-residue designed peptide “Alpha-1” (Acetyl-ELLKKLLEELKG), the L-enantiomer of which is described in the accompanying paper. Equivalent solutions of the centrosymmetric bilayers were determined by two direct phasing programs in space groups P1 and P1. The unit cell contains two l-alpha-helices and two d-alpha-helices. The columnar-sheet bilayer motif seen in l-Alpha-1 is maintained in the d,l-Alpha-1 structure except that each sheet of head-to-tail helices is composed of one enantiomer and is related to its neighboring sheets by inversion symmetry. Comparison to the l-Alpha-1 structure provides further insight into peptide design. The high resolution and small asymmetric unit allowed building an intricate model (R = 13.1%, Rfree = 14.5%) that incorporates much of the discrete disorder of peptide and solvent. Ethanolamine and 2-methyl-2,4-pentanediol (MPD) molecules bind near helix termini. Rigid body analysis identifies sites of restricted displacements and torsions. Side-chain discrete disorder propagates into the backbone of one helix but not the other. Although no side chain in Alpha-1 is rigid, the environments in the crystal restrict some of them to no or only one active torsion.

Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (VH), in addition to the well-known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv-binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between protein A or fragments of protein A and variants of the hu4D5 antibody Fab fragment. Analysis of binding isotherms obtained for titration of hu4D5 Fab with intact protein A suggests that 3–4 of the five immunoglobulin binding domains of full length protein A can bind simultaneously to Fab with a Ka of 5.5 ± 0.5 × 105 M−1. A synthetic single immunoglobulin binding domain, Z-domain, does not bind appreciably to hu4D5 Fab, but both the E and D domains are functional for hu4D5 Fab binding. Thermodynamic parameters for titration of the E-domain with hu4D5 Fab are n = 1.0 ± 0.1, Ka = 2.0 ± 0.3 × 105 M−1, and ΔH = −7.1 ± 0.4 kcal mol−1. Similar binding thermodynamics are obtained for titration of the isolated VH domain with E-domain indicating that the E-domain binding site on Fab resides within VH. E-domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2 ± 0.1, Ka > 1.0 × 107 M−1, and ΔH = −24.6 ± 0.6 kcal mol−1. Fc does not compete with Fab for binding to E-domain indicating that the two antibody fragments bind to different sites. Amide 1H and 15N resonances that undergo large changes in NMR chemical shift upon Fv binding map to a surface defined by helix-2 and helix-3 of E-domain, distinct from the Fc-binding site observed in the crystal structure of the B-domain/Fc complex. The Fv-binding region contains negatively charged residues and a small hydrophobic patch which complements the basic surface of the region of the VH domain implicated previously in protein A binding.

The oligopeptide-binding protein OppA provides a useful model system for studying the physical chemistry underlying noncovalent interactions since it binds a variety of readily synthesized ligands. We have studied the binding of eight closely related tripeptides of the type Lysine-X-Lysine, where X is an abnormal amino acid, by isothermal titration calorimetry (ITC) and X-ray crystallography. The tripeptides fall into three series of ligands, which have been designed to examine the effects of small changes to the central side chain. Three ligands have a primary amine as the second side chain, two have a straight alkane chain, and three have ring systems. The results have revealed a definite preference for the binding of hydrophobic residues over the positively charged side chains, the latter binding only weakly due to unfavorable enthalpic effects. Within the series of positively charged groups, a point of lowest affinity has been identified and this is proposed to arise from unfavorable electrostatic interactions in the pocket, including the disruption of a key salt bridge. Marked entropy-enthalpy compensation is found across the series, and some of the difficulties in designing tightly binding ligands have been highlighted.

Conformational changes occurring within the NS3 protease domain from the hepatitis C virus Bk strain (NS31–180) under different physico-chemical conditions either in the absence or in the presence of its cofactor Pep4A were investigated by limited proteolysis experiments. Because the surface accessibility of the protein is affected by conformational changes, when comparative experiments were carried out on NS31–180 either at different glycerol concentrations or in the presence of Pep4A, differential peptide maps were obtained from which protein regions involved in the structural changes could be inferred. The surface topology of isolated NS31–180 in solution was essentially consistent with the crystal structure of the protein with the N-terminal segment showing a high conformational flexibility. At higher glycerol concentration, the protease assumed a more compact structure showing a decrease in the accessibility of the N-terminal segment that either was forced to interact with the protein or originate intermolecular interactions with neighboring molecules. Binding of the cofactor Pep4A caused the displacement of the N-terminal arm from the protein moiety, leading this segment to again adopt an open and flexible conformation, thus suggesting that the N-terminus of the protease contributes only marginally to the stability of the complex. The observed conformational changes might be directly correlated with the activation mechanism of the protease by either the cosolvent or the cofactor peptide because they lead to tighter packing of the substrate binding site.

Effective inhibitors of matrix metalloproteinases (MMPs), a family of connective tissue-degrading enzymes, could be useful for the treatment of diseases such as cancer, multiple sclerosis, and arthritis. Many of the known MMP inhibitors are derived from peptide substrates, with high potency in vitro but little selectivity among MMPs and poor bioavailability. We have discovered nonpeptidic MMP inhibitors with improved properties, and report here the crystal structures of human stromelysin-1 catalytic domain (SCD) complexed with four of these inhibitors. The structures were determined and refined at resolutions ranging from 1.64 to 2.0 Å. Each inhibitor binds in the active site of SCD such that a bulky diphenyl piperidine moiety penetrates a deep, predominantly hydrophobic S′1 pocket. The active site structure of the SCD is similar in all four inhibitor complexes, but differs substantially from the peptide hydroxamate complex, which has a smaller side chain bound in the S′1 pocket. The largest differences occur in the loop forming the “top” of this pocket. The occupation of these nonpeptidic inhibitors in the S′1 pocket provides a structural basis to explain their selectivity among MMPs. An analysis of the unique binding mode predicts structural modifications to design improved MMP inhibitors.

Insulin-like growth factor (IGF-1) contains three disulfide bonds. In the presence of denaturant and thiol catalyst, IGF-1 shuffles its native disulfide bonds and denatures to form a mixture of scrambled isomers. The composition of scrambled IGF varies under different denaturing conditions. Among the 14 possible scrambled IGF isomers, the yield of the beads-form isomer is shown to be directly proportional to the strength of the denaturing condition. This paper demonstrates a new approach to quantify the extent of unfolding of the denatured protein.

The pressure-induced unfolding of lysozyme was investigated in an aqueous guanidinium chloride solution by means of ultraviolet spectroscopy. Assuming a two-state transition model, volume changes were calculated from the slope of free energy vs. pressure plots over a temperature range of 10 to 60 °C. Between 25 and 60 °C, almost constant volume changes were observed in the transition region, which was reflected in almost identical slopes of the free energy change vs. pressure plots. On the other hand, the different slopes were observed in the pressure dependence of free energy change at temperatures lower than 25 °C. These data were interpreted as suggesting that a two-state model is not appropriate at low temperature, but instead one or more intermediates are present under these conditions. The volume changes for unfolding became less negative at temperatures higher than 25 °C.

Production of folded and biologically active protein from Escherichia coli derived inclusion bodies can only be accomplished if a scheme exists for in vitro naturation. Motivated by the need for a rapid and statistically meaningful method of determining and evaluating protein folding conditions, we have designed a new fractional factorial protein folding screen. The screen includes 12 factors shown by previous experiments to enhance protein folding and it incorporates the 12 factors into 16 different folding conditions. By examining a 1/256th fraction of the full factorial, multiple folding conditions were determined for the ligand binding domains from glutamate and kainate receptors, and for lysozyme and carbonic anhydrase B. The impact of each factor on the formation of biologically active material was estimated by calculating factor main effects. Factors and corresponding levels such as pH (8.5) and l-arginine (0.5 M) consistently had a positive effect on protein folding, whereas detergent (0.3 mM lauryl maltoside) and nonpolar additive (0.4 M sucrose) were detrimental to the folding of these four proteins. One of the 16 conditions yielded the most folded material for three out of the four proteins. Our results suggest that this protein folding screen will be generally useful in determining whether other proteins will fold in vitro and, if so, what factors are important. Furthermore, fractional factorial folding screens are well suited to the evaluation of previously untested factors on protein folding.

Apomyoglobin from sperm whale is often used for studies of ligand binding, protein folding, and protein stability. In an effort to describe its conformational properties in solution, homonuclear and heteronuclear (13C and 15N) NMR methods were applied to the protein in its native state. Assignments were confirmed for nuclear Overhauser effects (NOEs) involving side chain and backbone protons in the folded regions of the structure. These NOEs were used to derive distance restraints. The shifts induced by the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS) were inspected in the regions remote from its binding site and served as an indicator of conformational flexibility. 3JαH-NH values were obtained to assess dihedral angle averaging and to provide additional restraints. A family of structures was calculated with X-PLOR and an ab initio simulated annealing protocol using holomyoglobin as a template. Where the structure appeared well defined by chemical shift, line width, ANS perturbation, and density of NOEs, the low resolution model of apomyoglobin provides a valid approximation for the structure. The new model offers an improved representation of the folded regions of the protein, which encompass the A, B, E, helices as well as parts of the G and H helices. Regions that are less well defined at this stage of calculations include the CD corner and the end of the H-helix. The EF-F-FG segment remains uncharacterized.

Conformational free energy calculations have been carried out for proline-containing alanine-based pentadecapeptides with the sequence Ac-(Ala)n-Pro-(Ala)m-NHMe, where n + m = 14, to figure out the positional preference of proline in α-helices. The relative free energy of each peptide was calculated by subtracting the free energy of the extended conformation from that of the α-helical one, which is used here as a measure of preference. The highest propensity is found for the peptide with proline at the N-terminus (i.e., Ncap + 1 position), and the next propensities are found at Ncap, N′ (Ncap − 1), and C′ (Ccap + 1) positions. These computed results are reasonably consistent with the positional propensities estimated from X-ray structures of proteins. The breaking in hydrogen bonds around proline is found to play a role in destabilizing α-helical conformations, which, however, provides the favored hydration of the corresponding N–H and C=O groups. The highest preference of proline at the beginning of α-helix appears to be due to the favored electrostatic and nonbonded energies between two residues preceding proline and the intrinsic stability of α-helical conformation of the proline residue itself as well as no disturbance in hydrogen bonds of α-helix by proline. The average free energy change for the substitution of Ala by Pro in a α-helix is computed to be 4.6 kcal/mol, which is in good agreement with the experimental value of ∼4 kcal/mol estimated for an oligopeptide dimer and proteins of barnase and T4 lysozyme.

The change in heat capacity ΔCp for the folding of ribonuclease A was determined using differential scanning calorimetry and thermal denaturation curves. The methods gave equivalent results, ΔCp = 1.15 ± 0.08 kcal mol−1 K−1. Estimates of the conformational stability of ribonuclease A based on these results from thermal unfolding are in good agreement with estimates from urea unfolding analyzed using the linear extrapolation method.

Refolding of b*C40A/C82A/P27A is comprised of several kinetically detectable folding phases. The slowest phase in refolding originates from trans → cis isomerization of the Tyr47–Pro48 peptide bond being in cis conformation in the native state. This refolding phase can be accelerated by the peptidyl-prolyl cis/trans isomerase human cytosolic cyclophilin (Cyp18) with a kcat/KM of 254,000 M−1 s−1. The fast refolding phase is not influenced by the enzyme.

The mechanism by which the type Iα regulatory subunit (RIα) of cAMP-dependent protein kinase is localized to cell membranes is unknown. To determine if structural modification of RIα is important for membrane association, both beef skeletal muscle cytosolic RI and beef heart membrane-associated RI were characterized by electrospray ionization mass spectrometry. Total sequence coverage was 98% for both the membrane-associated and cytosolic forms of RI after digestion with AspN protease or trypsin. Sequence data indicated that membrane-associated and cytosolic forms of RI were the same RIα gene product. A single RIα phosphorylation site was identified at Ser81 located near the autoinhibitory domain of both membrane-associated and cytosolic RIα. Because both R subunit preparations were 30–40% phosphorylated, this post-translational modification could not be responsible for the membrane compartmentation of the majority of RIα. Mass spectrometry also indicated that membrane-associated RIα had a higher extent of disulfide bond formation in the amino-terminal dimerization domain. No other structural differences between cytosolic and membrane-associated RIα were detected. Consistent with these data, masses of the intact proteins were identical by LCQ mass spectrometry. Lack of detectable structural differences between membrane-associated and cytosolic RIα strongly suggests an interaction between RIα and anchoring proteins or membrane lipids as more likely mechanisms for explaining RIα membrane association in the heart.

To understand general aspects of stability and folding of c-type cytochromes, we have studied the folding characteristics of cytochrome c553 from Desulfovibrio vulgaris (Hildenborough). This cytochrome is structurally similar but lacks sequence homology to other heme proteins; moreover, it has an abnormally low reduction potential. Unfolding of oxidized and reduced cytochrome c553 by guanidine hydrochloride (GuHCl) was monitored by circular dichroism (CD) and Soret absorption; the same unfolding curves were obtained with both methods supporting that cytochrome c553 unfolds by an apparent two-state process. Reduced cytochrome c553 is 7(3) kJ/mol more stable than the oxidized form; accordingly, the reduction potential of unfolded cytochrome c553 is 100(20) mV more negative than that of the folded protein. In contrast to many other unfolded cytochrome c proteins, upon unfolding at pH 7.0 both oxidized and reduced heme in cytochrome c553 become high-spin. The lack of heme misligation in unfolded cytochrome c553 implies that its unfolded structure is less constrained than those of cytochromes c with low-spin, misligated hemes.

The hydrophilic subunit of the mannose transporter (IIABMan) of Escherichia coli is a homodimer that contains four tryptophans per monomer, three in the N-terminal domain (Trp12, Trp33, and Trp69) and one in the C-terminal domain (Trp182). Single and double Trp-Phe mutants of IIABMan and of the IIA domain were produced. Fluorescence emission studies revealed that Trp33 and Trp12 are the major fluorescence emitters, Trp69 is strongly quenched in the native protein and Trp182 strongly blue shifted, indicative of a hydrophobic environment. Stabilities of the Trp mutants of dimeric IIAMan and IIABMan were estimated from midpoints of the GdmHCl-induced unfolding transitions and from the amount of dimers that resisted dissociation by SDS (sodium dodecyl sulfate), respectively. W12F exhibited increased stability, but only 6% of the wild-type phosphotransferase activity, whereas W33F was marginally and W69F significantly destabilized, but fully active. Second site mutations W33F and W69F in the background of the W12F mutation reduced protein stability and suppressed the functional defect of W12F. These results suggest that flexibility is required for the adjustments of protein–protein contacts necessary for the phosphoryltransfer between the phosphorylcarrier protein HPr, IIAMan, IIBMan, and the incoming mannose bound to the transmembrane IICMan–IIDMan complex.

Desulforedoxin (Dx), isolated from the sulfate reducing bacterium Desulfovibrio gigas, is a small homodimeric (2 × 36 amino acids) protein. Each subunit contains a high-spin iron atom tetrahedrally bound to four cysteinyl sulfur atoms, a metal center similar to that found in rubredoxin (Rd) type proteins. The simplicity of the active center in Dx and the possibility of replacing the iron by other metals make this protein an attractive case for the crystallographic analysis of metal-substituted derivatives. This study extends the relevance of Dx to the bioinorganic chemistry field and is important to obtain model compounds that can mimic the four sulfur coordination of metals in biology. Metal replacement experiments were carried out by reconstituting the apoprotein with In3+, Ga3+, Cd2+, Hg2+, and Ni2+ salts. The In3+ and Ga3+ derivatives are isomorphous with the iron native protein; whereas Cd2+, Hg2+, and Ni2+ substituted Dx crystallized under different experimental conditions, yielding two additional crystal morphologies; their structures were determined by the molecular replacement method. A comparison of the three-dimensional structures for all metal derivatives shows that the overall secondary and tertiary structures are maintained, while some differences in metal coordination geometry occur, namely, bond lengths and angles of the metal with the sulfur ligands. These data are discussed in terms of the entatic state theory.

In this work with ab initio computations, we describe relevant interactions between protein active sites and ligands, using as a test case arthropod hemocyanins. A computational analysis of models corresponding to the oxygenated and deoxygenated forms of the hemocyanin active site is performed using the Density Functional Theory approach. We characterize the electron density distribution of the binding site with and without bound oxygen in relation to the geometry, which stems out of the crystals of three hemocyanin proteins, namely the oxygenated form from the horseshoe crab Limulus polyphemus, and the deoxygenated forms, respectively, from the same source and from another arthropod, the spiny lobster Panulirus interruptus. Comparison of the three available crystals indicate structural differences at the oxygen binding site, which cannot be explained only by the presence and absence of the oxygen ligand, since the geometry of the ligand site of the deoxygenated Panulirus hemocyanin is rather similar to that of the oxygenated Limulus protein. This finding was interpreted in the frame of a mechanism of allosteric regulation for oxygen binding. However, the cooperative mechanism, which is experimentally well documented, is only partially supported by crystallographic data, since no oxygenated crystal of Panulirus hemocyanin is presently available. We address the following question: is the local ligand geometry responsible for the difference of the dicopper distance observed in the two deoxygenated forms of hemocyanin or is it necessary to advocate the allosteric regulation of the active site conformations in order to reconcile the different crystal forms? We find that the difference of the dicopper distance between the two deoxygenated hemocyanins is not due to the small differences of ligand geometry found in the crystals and conclude that it must be therefore stabilized by the whole protein tertiary structure.