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PubMed1993

Protein unfolding pathways explored through molecular dynamics simulations.

Herein we describe the results of molecular dynamics simulations of the bovine pancreatic trypsin inhibitor (BPTI) in solution at a variety of temperatures both with and without disulfide bonds. The reduced form of the protein unfolded at high temperature to an ensemble of conformations with all the properties of the molten globule state. In this account we outline the structural details of the actual unfolding process between the native and molten globule states. The first steps of unfolding involved expansion of the protein, which disrupted packing interactions. The solvent-accessible surface area also quickly increased. The unfolding was localized mostly to the turn and loop regions of the molecule, while leaving the secondary structure intact. Then, there was more gradual unfolding of the secondary structure and non-native turns became prevalent. This same trajectory was continued and more drastic unfolding occurred that resulted in a relatively compact state devoid of stable secondary structure.

Aprotinin↗

PubMed1993

Crystal structure of canine and bovine granulocyte-colony stimulating factor (G-CSF).

The crystal structures of recombinant canine and bovine granulocyte colony stimulating factor (G-CSF) have been determined by X-ray crystallography, using molecular replacement with recombinant human G-CSF as a model. G-CSF is a member of the cytokine family of glycoproteins that stimulate the differentiation and proliferation of blood cells. Human, bovine and canine G-CSF all have a molecular mass of about 19 kDa and share an amino acid sequence identity of about 80%. Two crystal forms of canine G-CSF have been solved. Form I recombinant canine G-CSF (rcG-CSFI; space group C2) contains one molecule in the asymmetric unit while form II canine G-CSF (rcG-CSFII; space group P2(1)) has two molecules in the asymmetric unit and bovine G-CSF (rbG-CSF; space group P2(1)2(1)2(1)) contains one molecule in the asymmetric unit. rcG-CSFI has been refined to an R factor of 20.7% with data to 2.3 A resolution and rcG-CSFII has been refined to an R factor of 19.3% with data to 2.2 A resolution. rbG-CSF has been refined to an R factor of 21.3% with data to 1.7 A resolution. The structure of human, canine and bovine G-CSF is an antiparallel 4-alpha-helical bundle with up-up-down-down connectivity. With the exception of one highly exposed loop (residues 66 to 74), the human, canine and bovine structures are very similar to each other. Using our series of G-CSF crystal structures we developed a function that describes the probability that a particular residue position (i) contributes to a G-CSF receptor binding site based on two principles, (1) high sequence conservation in the primary sequence of human, bovine, canine and murine G-CSF and (2) conservation of high solvent accessibility in the human, bovine and canine crystal structures. On the basis of this probability function as well as a comparison of G-CSF to the crystal structure of human growth hormone (hGH) complexed with the extracellular domain of the human growth hormone receptor (hGHbp), residues that contribute to potential G-CSF receptor binding sites are identified.

Amino Acid Sequence↗

PubMed1994

Locations of anti-AIDS drug binding sites and resistance mutations in the three-dimensional structure of HIV-1 reverse transcriptase. Implications for mechanisms of drug inhibition and resistance.

The locations of HIV-1 RT nucleoside and non-nucleoside inhibitor-binding sites and inhibitor-resistance mutations are analyzed in the context of the three-dimensional structure of the enzyme and implications for mechanisms of drug inhibition and resistance are discussed. In order to help identify residues that may play a role in inhibitor binding, solvent accessibilities of amino acids that comprise the inhibitor-binding sites in the structure of HIV-1 RT complexed with a dsDNA template-primer are analyzed. While some mutations that cause resistance to nucleoside analogs, such as AZT, ddI, and ddC, are located near enough to the dNTP-binding site to directly interfere with binding of nucleoside analogs, many are located away from the dNTP-binding site and more likely confer resistance by other mechanisms. Many of the latter mutations are located on the surface of the DNA-binding cleft and may lead to altered template-primer positioning or conformation, causing a distortion of the geometry of the polymerase active site and consequent discrimination between normal and altered dNTP substrates. Other nucleoside analog-resistance mutations located on the periphery of the dNTP-binding site may exert their effects via altered interactions with dNTP-binding site residues. The structure of the hydrophobic region in HIV-1 RT that binds non-nucleoside inhibitors, for example, nevirapine and TIBO, has been analyzed in the absence of bound ligand. The pocket that is present when non-nucleoside inhibitors are bound is not observed in the inhibitor-free structure of HIV-1 RT with dsDNA. In particular it is filled by Tyr181 and Tyr188, suggesting that the pocket is formed primarily by rotation of these large aromatic side-chains. Existing biochemical data, taken together with the three-dimensional structure of HIV-1 RT, makes it possible to propose potential mechanisms of inhibition by non-nucleoside inhibitors. One such mechanism is local distortion of HIV-1 RT structural elements thought to participate in catalysis: the beta 9-beta 10 hairpin (which contains polymerase active site residues) and the beta 12-beta 13 hairpin ("primer grip"). An alternative possibility is restricted mobility of the p66 thumb subdomain, which is supported by the observation that structural elements of the non-nucleoside inhibitor-binding pocket may act as a "hinge" for the thumb.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1995

A continuum model for protein-protein interactions: application to the docking problem.

The prediction of protein-protein interactions in solution is a major goal of theoretical structural biology. Here, we implement a continuum description of the thermodynamic processes involved. The model differs considerably from previous models in its use of "molecular surface" area to describe the hydrophobic component to the free energy of conformational change in solution. We have applied this model to a data set of alternative docked conformations of protein-protein complexes which were generated independently of this work. It was found previously that commonly used energy evaluation techniques fail to distinguish between near-native and certain non-native complexes in this data set. Here, we found that an energy function that takes into account (1) total electrostatic free energy, (2) hydrophobic free energy and (3) loss in side-chain conformational energy was able to reliably discriminate between near-native and non-native configurations but only when molecular surface is used as a descriptor of the hydrophobic effect. It is shown that the molecular surface and the more conventional surface descriptor "solvent accessible surface" give very different quantitative measures of hydrophobicity. In terms of the contribution of different energy components to the free energy of complex formation it was found that loss in side-chain conformational entropy is a second order effect. Electrostatic interaction energy (which is commonly used to score docked conformations) was a poor indicator of complementarity when starting from unbound conformations. It was found that electrostatic desolvation energy and the hydrophobic contribution (based on a molecular surface area descriptor) are much less sensitive to local fluctuations in atomic structure than point-to-point interaction energies and thus may be more suited for use as a scoring function when docking unbound conformations, where atomic complementarity is much less apparent. Whilst a combined energy function was able to distinguish near-native from non-native conformations in the six systems studied here, it remains to be determined to what extent more sizeable conformational changes would influence the results.

Algorithms↗

PubMed1995

Folding pathway mediated by an intramolecular chaperone: characterization of the structural changes in pro-subtilisin E coincident with autoprocessing.

Mechanisms by which many N-terminal propeptides facilitate folding of proteins are unknown. The maturation of such proteins from their precursors involve three steps, namely: (1) folding of the precursor, (2) autoprocessing of the propeptide from the N terminus and (3) degradation of the cleaved propeptide. Using subtilisin E we have analyzed the mechanism of propeptide-mediated protein folding. Two active site mutations allow us to trap intermediates at stages of autoprocessing and degradation. An analysis of these intermediates has shown the existence of a molten-globule-like intermediate on the folding pathway. After autoprocessing of the propeptide, this intermediate undergoes a structural reorganization which reduces solvent-accessible hydrophobic surface area and increases the amount of its tertiary structure. Removal of the propeptide from the mature enzyme in this intermediate state occurs only by proteolytic degradation and contributes to the stability of the active enzyme.

Circular Dichroism↗

PubMed1995

Acid and thermal denaturation of barnase investigated by molecular dynamics simulations.

The transition in barnase from the native state to a partially unfolded conformation has been studied by molecular dynamics simulations with explicit water molecules at 360 K and low pH(450 ps), and at 600 K and neutral pH (three simulations of 120, 250 and 200 ps each). The use of several simulations provides evidence that the results are not sensitive to initial conditions. To mimic low pH conditions, the acidic sidechains in barnase were neutralized and the two histidine residues were doubly protonated. Runs at 300 K showed that the solvated structures at low pH (300 ps) and neutral pH (310 ps) are very similar. The main structural differences involved the acidic residues, histidine residues, and the beta-turn connecting strands 4 and 5. When the temperature is raised to 360 K at low pH and to 600 K at neutral pH the barnase molecule begins to unfold. The molecule rapidly expands (Rg changes from 13.9 A to 15.3 A in 450 ps at 360 K and from 13.7 A to between 15.1 and 15.5 A in 120 ps at 600 K). However, the expansion is not uniform. In all the simulations, the chain termini, loops and the N-terminal parts of the main alpha-helix (helix 1) show a continuous and progressive unfolding. An essential step in the denaturation process is that the major alpha-helix (helix 1) separates from the beta-sheet; this is coupled to the exposure of the principal hydrophobic core, many of whose non-polar side chains become solvated by hydrogen-bonded water molecules. The barnase-water interaction energy improves during unfolding at the expense of the barnase self-energy. The deterioration of the intramolecular van der Waals energy suggests that the rupture of the tight packing during the initial unfolding phase contributes to the energy barrier of the denaturation process. The mutationally well-analyzed Asp8-Arg110-Asp12 double salt-bridge on the barnase surface is found to be marginally stable in the folded form in the simulations. A Poisson-Boltzmann calculation indicates that the salt-bridge is unstable; this is probably due to an overestimate of the solvation energy. A detailed analysis of the main hydrophobic core reveals that increase in solvent-accessible surface area and penetration of water molecules are simultaneous in the high-temperature simulation; at lower temperatures there is significant cavity formation and the entrance of the water molecules is somewhat delayed. The cavities occur in the neighborhood of the hydrophobic sidechains; the region formed by the sidechains of Val10, Leu14, Leu20, Tyr24, Ala74, Ile76 and Tyr90 is involved. The loosening of the core packing is coupled to an increase in the number of dihedral transitions.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacterial Proteins↗

PubMed1996

Structure, interactions and dynamics of PRD1 virus I. Coupling of subunit folding and capsid assembly.

Bacteriophage PRD1, which infects Escherichia coli and Salmonella typhimurium, consists of an icosahedral capsid enclosing a membrane-packaged double-stranded DNA genome. The viral shell has been investigated using time and temperature resolved Raman and ultraviolet-resonance Raman spectroscopy to reveal novel features of the capsid structure and its pathway of assembly from P3 subunits. Raman spectra show that the shell is thermostable to 50 degrees C, and disassembles between 50 and 70 C degrees with only a small change in P3 conformation. However, the products of thermal disassembly depend sensitively upon total protein concentration. Characterization by analytical ultracentrifugation indicates that below 8 mg/ml, the purified shell disassembles primarily into P3 trimers; at higher concentrations, larger multimers of P3 are formed. Guanidine hydrochloride (GuHCl) dissociation of the P3 shell yields similar results. Purified P3 trimers, isolated either by heat or GuHCl treatment, exhibit structure sensitivity between 30 and 50 degrees C. Thus, shell disassembly diminishes P3 thermostability. Both the lower temperature transition (30 degrees C to 50 degrees C) of the trimer and the higher temperature transition (50 degrees C to 70 degrees C) of the shell involve a conversion of approximately 5% of the P3 peptide backbone from alpha-helix to beta-strand. Deuterium exchange of the P3 peptide backbone reveals more rapid exchange in the shell than in the trimer, consistent with the observed non-specific polymerization of trimers at high concentration. Conversely, the exchange of indole 1NH groups shows that approximately 65% of tryptophan residues are protected against exchange in the assembled shell. The results suggest a mechanism for shell assembly in which the specific association of trimers into the correct shell architecture involves stabilization of a subunit alpha-helical domain and sequestering of selected side-chains from solvent access. We propose a capsid assembly model which couples P3 shell formation with the final step in folding of the P3 subunit.

Bacteriophages↗

PubMed1996

Implications of N and C-terminal proximity for protein folding.

We have investigated the proximity of the N and C termini in protein structures, and developed a model to test the theoretical possibility that proteins fold with their termini closely associated. On average, the distance between the termini is not significantly different from what would be expected based on chance. However, the theoretical model indicated that it is possible to greatly decrease the N-to-C terminal distance by allowing small (approximately six amino acid residues) solvent-accessible terminal fragments to move. Subsequent to this distance minimization method, more than 90% of the proteins studied had smaller-than-expected N-to-C distances, but only minor structural modification.

Computer Simulation↗

PubMed1996

The solution structure of bovine ferricytochrome b5 determined using heteronuclear NMR methods.

The solution structure of a recombinant form of cytochrome b5 containing 104 amino acid residues has been determined using three-dimensional NMR spectroscopy. Using protein enriched in 15N the majority of the polypeptide backbone resonances have been assigned to reveal numerous chemical shift differences to those reported previously for smaller fragments of cytochrome b5. By using 3D NMR methods the extensive spectral overlap of resonance cross-peaks in 2D NMR spectra could be satisfactorily resolved. The large number of sequence-specific assignments made for this form of the protein allowed the identification of over 1130 NOEs, giving an average of 14 NOEs per assigned residue, and 52 dihedral angles (phi). This data was used in an ab initio simulated annealing protocol to determine the solution structure for bovine microsomal cytochrome b5. A series of 50 structures was generated using distance restraints derived from the magnitude of the NOE and torsional angles based on the measured JHN-HA coupling constants. From an initial round of simulated annealing a family of 36 structures was selected on the basis of good covalent geometry and minimal restraint violations. A single cycle of simulated annealing refinement produced 36 converged structures that exhibited an average r.m.s.d. of 0.73 A for the backbone atoms. The determination of the solution structure of cytochrome b5 is the first using NMR methods for any form of this protein. It is also the only cytochrome whose structure has been determined in the oxidised or paramagnetic state. The results show that despite significant line broadening and pseudocontact shifts for resonances lying close to the paramagnetic haem centre, and despite extensive spectral overlap that prevents complete resonance assignment, the topology of the polypeptide backbone can be derived. The conformation for cytochrome b5 determined in this study reveals several small, but significant, differences in structure to that determined previously by crystallography for a smaller fragment of this protein. For example, NMR data do not support a short beta strand as the first element of secondary structure at the N terminus nor is it likely that a beta-bulge structure forms between residues 75 to 79. The data obtained in this study are more consistent with a turn in this region of the protein linking helices 5 and 6 and leads to cytochrome b5 containing only three clearly defined beta strands. Four of the six helices together with the antiparallel beta strands make up a haem binding pocket in which the solvent-accessible area of the protoporphyrin IX centre remains very similar to that found in the crystal structure. The remaining helices and the beta strands form a second structural domain on which the four helix bundle that surrounds the haem is based. THe derivation of the solution structure of cytochrome b5 will allow a greater understanding of the functional properties of cytochrome b5 including its role in biological electron transfer and molecular recognition together with insight into haem protein folding and stability.

Animals↗

PubMed1996

Synthesis and activity of piperazine-containing antirhinoviral agents and crystal structure of SDZ 880-061 bound to human rhinovirus 14.

A series of antipicornaviral agents containing piperazinyl moieties was synthesized with the objective of obtaining a compound with a broad spectrum of antirhinovirus activity, high potency (< or = 0.003 microgram/ml), and low cytotoxicity (> or = 30 micrograms/ml). Five compounds of this series were evaluated in detail for efficacy against various HRV serotypes. The agent SDZ 880-061, containing the benzothiazine moiety SDZ 108-075, which is particularly active against HRV14, and the thiazolyl acetic acid ester group of SDZ 89-124, which is potent against HRV1B, indeed has a relatively broad antiviral spectrum. SDZ 880-061 inhibited 85% of 89 HRV serotypes tested at a concentration of < or = 3 micrograms/ml. The 3.0 A resolution X-ray structure of SDZ 880-061 bound to HRV14 has revealed the binding characteristics of this potent compound. It binds in the same pocket as other capsid-binding antiviral agents characterized to date, leaving the innermost portion of the pocket vacant. The binding causes similar, although less extensive, alterations of the HRV14 VP1 backbone conformation (residues 100 to 110, 151 to 159, and 213 to 224) compared to other antiviral agents analyzed structurally. Although the contacts between SDZ 880-061 and HRV14 are mostly of hydrophobic character, the inhibitor has three relatively short polar interactions with residues of VP1 that represent potential hydrogen bonds. The amount of solvent-accessible surface area of SDZ 880-061 buried in the complex (613 A2) is within the range of that observed in protein-protein interfaces. The observed influence of time of addition or removal of SDZ 880-061 on virus yield and on the infectious-center formation indicates that the compound primarily interferes with HRV14 cellular attachment. Since it is assumed that uncoating requires virion instability and/or flexibility, the finding that SDZ 880-061 has only a marginal effect on uncoating may be due to the fact that it does not completely fill the hydrophobic pocket.

Antiviral Agents↗

PubMed1996

Crystal structure of the pyridoxal-5'-phosphate dependent cystathionine beta-lyase from Escherichia coli at 1.83 A.

Cystathionine beta-lyase (CBL) is a member of the gamma-family of PLP-dependent enzymes, that cleaves C beta-S bonds of a broad variety of substrates. The crystal structure of CBL from E. coli has been solved using MIR phases in combination with density modification. The structure has been refined to an R-factor of 15.2% at 1.83 A resolution using synchroton radiation diffraction data. The asymmetric unit of the crystal cell (space group C222(1)) contains two monomers related by 2-fold symmetry. A homotetramer with 222 symmetry is built up by crystallographic and non-crystallographic symmetry. Each monomer of CBL can be described in terms of three spatially and functionally different domains. The N-terminal domain (residues 1 to 60) consists of three alpha-helices and one beta-strand. It contributes to tetramer formation and is part of the active site of the adjacent subunit. The second domain (residues 61 to 256) harbors PLP and has an alpha/beta-structure with a seven-stranded beta-sheet as the central part. The remaining C-terminal domain (residues 257 to 395), connected by a long alpha-helix to the PLP-binding domain, consists of four helices packed on the solvent-accessible side of an antiparallel four-stranded beta-sheet. The fold of the C-terminal and the PLP-binding domain and the location of the active site are similar to aminotransferases. Most of the residues in the active site are strongly conserved among the enzymes of the transsulfuration pathway. Additionally, CBL is homologous to the mal gamma gene product indicating an evolutionary relationship between alpha and gamma-family of PLP-dependent enzymes. The structure of the beta, beta, beta-trifluoroalanine inactivated CBL has been refined at 2.3 A resolution to an R-factor of 16.2%. It suggests that Lys210, the PLP-binding residue, mediates the proton transfer between C alpha and S gamma.

Alanine↗

PubMed1996

Use of a conformationally restricted secondary structural element to display peptide libraries: a two-stranded alpha-helical coiled-coil stabilized by lactam bridges.

A model for an alpha-helical peptide library based on a lactam bridged stabilized two-stranded alpha-helical coiled-coil is described. Sites for library display were incorporated in the middle of the peptide sequence of the most solvent accessible sites of a coiled-coil. A comparison was made between this coiled coil and a native coiled-coil based on the same sequence but lacking the lactam bridges. A lactam bridged peptide where the hydrophobic repeat consisted of all alanine residues, such that the tendency to dimerize would be diminished, was also prepared. This enabled us to determine the role tertiary interactions play in maintaining library positions in a helical conformation. The consensus sequence derived from a Zn finger library screened against an IgA reactive against the lipopolysaccharide of Shigella flexneri was transplanted into the peptides. CD spectroscopy revealed that although both coiled-coils are highly helical at 100 microM, the lactam bridge enhanced dimerization and allow the peptide to maintain its coiled-coil conformation at lower peptide concentrations. The helical content of the alanine based peptide was 69% and was independent of concentration over a range of 1.4 to 1410 microM. Urea denaturation studies indicated that the coiled-coils were considerably more stable than the alanine based peptide and that the lactam bridge coiled-coil was more stable than the native coiled coil by 1.6 kcal/mol. The lactam bridged coiled-coil was found to inhibit binding of the Zn finger peptide to the IgA in a concentration dependent manner with an IC50 of 5.0 microM whereas the peptide lacking the lactam bridges was much less effective in inhibiting binding. The alanine peptide was less active than the lactam bridged coiled-coil but more effective than the native coiled-coil with an IC50 of 16 microM. The versatility of the lactam stabilized coiled-coil template was demonstrated by incorporating five Gly residues into the library display sites. While the native coiled-coil adopted a random conformation the lactam bridged coiled-coil was 59% helical. Incorporation of a Cys-Gly-Gly linker to the N terminus and formation of a disulfide bond stabilized the peptide to the extent that it adopted a highly helical coiled-coil (94%) with a [urea]1/2 value of 5.9 M. Since the five glycine residues represent one of the most destabilizing combinations of amino acids that would be encountered at the five library display sites (with the exception of Pro), this stabilized coiled-coil should maintain its folded conformation regardless of the amino acids occupying the library positions.

Amino Acid Sequence↗

PubMed1996

Microsecond protein folding through a compact transition state.

Dynamic NMR methods have been employed to measure the folding and unfolding rate constants of two extremely fast-folding proteins. lambda 6-85, a truncated, monomeric form of the N-terminal domain of lambda repressor, refolds with a lifetime of approximately 250 microseconds. These methods have also been applied to a thermostable lambda 6-85 variant with alanine substituted for glycine residues 46 and 48 in the third helix (G46A/G48A). Both proteins exhibit linear ln (kf,u) versus [urea] plots, consistent with two-state folding for both proteins. When extrapolated to 0M urea, the data indicate that G46A/G48A folds with a lifetime of less than 20 microseconds. The slopes of the ln (kf,u) versus [urea] curves (mu and mf) indicate that the modest Gly-->Ala double mutation dramatically changes the transition state solvent accessibility. The transition state for lambda 6-85 has a fractional accessibility (mu/(mu-mf)) of 0.61, whereas the transition state for G46A/G48A is much more native-like, with a fractional accessibility of 0.16. The extraordinary change in the folding pathway that these mutations induce suggests that the intrinsic stability of helix 3 is an important determinant of the folding mechanism.

DNA-Binding Proteins↗

PubMed1996

Hyperthermophile protein folding thermodynamics: differential scanning calorimetry and chemical denaturation of Sac7d.

Recombinant Sac7d protein from the thermoacidophile Sulfolobus acidocaldarius is shown to be stable towards acid, thermal and chemical denaturation. The protein maintains a compact native fold between pH 0 and 10 in 0.3 M KCl and 25 degrees C as indicated by near and far UV circular dichroism spectra. Thermal unfolding followed by differential scanning calorimetry (DSC) occurs as a reversible, two-state transition from pH 0 to 10, with a maximal Tm of 90.7 degrees C between pH 5 and 9. At pH 0 the protein unfolds with a Tm of 63.3 degrees C. Plots of the enthalpy of unfolding as a function of Tm are linear and yield an anomalously low delta Cp of 497 (+/-20) cal deg-1 mol-1 using the Kirchhoff relation. Guanidine hydrochloride and urea-induced chemical denaturation of Sac7d occur reversibly and can be followed by circular dichroism. Global non-linear regression of the chemical denaturation data constrained by DSC determined values for delta Hm and Tm yields a delta Cp of unfolding of 858 (+/-21) cal deg-1 mol-1. The higher delta Cp is in good agreement with that predicted from the buried polar and apolar surface areas using the NMR solution structure. It is similar to values reported for mesophile proteins of comparable size, indicating that the packing and change in solvent-accessible surface area on unfolding are not unusual. Similarly, guanidine hydrochloride and urea m-values are in good agreement with those expected for a protein of 66 residues. Possible explanations for the difference in delta Cp determined by application of the Kirchhoff relation to DSC data and that determined by the global fit are discussed. Protein stability curves defined by either delta Cp values are similar to those observed for small mesophile proteins. Although the protein is thermally stable, it is marginally stable thermodynamically with a free energy of unfolding of 1.6 (+/-0.1) kcal mol-1 at the growth temperature of 80 degrees C. The large number of potential ion pairs on the surface of this hyperthermophile protein do not result in an inordinate increase in stability. Post-translational modification, possibly lysine monomethylation, appears to be the single most important stabilizing factor that distinguishes the native hyperthermophile protein from small mesophile proteins. Additional stabilization in vivo is expected from compatible osmolytes (polyamines) and DNA-binding.

Archaeal Proteins↗

PubMed1997

Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and Bayesian scoring functions.

We explore the ability of a simple simulated annealing procedure to assemble native-like structures from fragments of unrelated protein structures with similar local sequences using Bayesian scoring functions. Environment and residue pair specific contributions to the scoring functions appear as the first two terms in a series expansion for the residue probability distributions in the protein database; the decoupling of the distance and environment dependencies of the distributions resolves the major problems with current database-derived scoring functions noted by Thomas and Dill. The simulated annealing procedure rapidly and frequently generates native-like structures for small helical proteins and better than random structures for small beta sheet containing proteins. Most of the simulated structures have native-like solvent accessibility and secondary structure patterns, and thus ensembles of these structures provide a particularly challenging set of decoys for evaluating scoring functions. We investigate the effects of multiple sequence information and different types of conformational constraints on the overall performance of the method, and the ability of a variety of recently developed scoring functions to recognize the native-like conformations in the ensembles of simulated structures.

Bayes Theorem↗

PubMed1997

Dissecting the energetics of a protein-protein interaction: the binding of ovomucoid third domain to elastase.

An understanding of the structural basis for protein-protein interactions, and molecular recognition in general, requires complete characterization of binding energetics. Not only does this include quantification of the changes that occur in all of the thermodynamic parameters upon binding, including the enthalpy, entropy and heat capacity, but a description of how these changes are modulated by environmental conditions, most notably pH. Here, we have investigated the binding of turkey ovomucoid third domain (OMTKY3), a potent serine protease inhibitor, to the serine protease porcine pancreatic elastase (PPE) using isothermal titration calorimetry and structure-based thermodynamic calculations. We find that near neutral pH the binding energetics are influenced by a shift in the pKa of an ionizable group, most likely histidine 57 in the protease active site. Consequently, the observed binding energetics are strongly dependent upon solution conditions. Through a global analysis, the intrinsic energetics of binding have been determined, as have those associated with the pKa shift. The protonation energetics suggest that the drop in pKa is largely due to desolvation of the histidine residue. The resulting deprotonation is necessary for the enzymatic function of elastase. Intrinsically, at 25 degrees C the binding of OMTKY3 to PPE is characterized by an almost negligible enthalpy change, a large positive entropy change, and a large negative heat capacity change. These parameters are consistent with a model of the OMTKY3-PPE complex, which shows a large and significantly apolar protein-protein interface. Thermodynamic calculations based upon changes that occur in polar and apolar solvent-accessible surface area are in very good agreement with the measured intrinsic binding energetics.

Animals↗

PubMed1997

Imino proton exchange and base-pair kinetics in the AMP-RNA aptamer complex.

We report on the dynamics of base-pair opening in the ATP-binding asymmetric internal loop and flanking base-pairs of the AMP-RNA aptamer complex by monitoring the exchange characteristics of the extremely well resolved imino protons in the NMR spectrum of the complex. The kinetics of imino proton exchange as a function of basic pH or added ammonia catalyst are used to measure the apparent base-pair dissociation constants and lifetimes of Watson-Crick and mismatched base-pairs, as well as the solvent accessibility of the unpaired imino protons in the complex. The exchange characteristics of the imino protons identify the existence of four additional hydrogen bonds stabilizing the conformation of the asymmetric ATP-binding internal loop that were not detected by NOEs and coupling constants alone, but are readily accommodated in the previously reported solution structure of the AMP-RNA aptamer complex published from our laboratory. The hydrogen exchange kinetics of the non-Watson-Crick pairs in the asymmetric internal loop of the AMP-RNA aptamer complex have been characterized and yield apparent dissociation constants (alphaKd) that range from 10(-2) to 10(-7). Surprisingly, three of these alphaKd values are amongst the lowest measured for all base-pairs in the AMP-RNA aptamer complex. Comparative studies of hydrogen exchange of the imino protons in the free RNA aptamer and the AMP-RNA aptamer complex establish that complexation stabilizes not only the bases within the ATP-binding asymmetric internal loop, but also the flanking stem base-pairs (two pairs on either side) of the binding site. We also outline some preliminary results related to the exchange properties of a sugar 2'-hydroxyl proton of a guanosine residue involved in a novel hydrogen bond that has been shown to contribute to the immobilization of the bound AMP by the RNA aptamer, and whose resonance is narrow and downfield shifted in the spectrum.

Adenosine Monophosphate↗

PubMed1997

Crystal structure of a decameric complex of human serum amyloid P component with bound dAMP.

Serum amyloid P component (SAP) is a glycoprotein that binds in a calcium-dependent fashion to a variety of ligands including other proteins, glycosaminoglycans and DNA. SAP is universally associated with the amyloid deposits in all forms of amyloidoses including Alzheimer's disease. Small-molecule ligands that displace SAP from amyloid fibrils and thereby expose the fibrils to proteolytic clearance mechanisms hold potential as drugs for the prevention and treatment of amyloidosis. We have carried out a screen for novel SAP ligands and have identified 2'-deoxyadenosine-5'-monophosphate (dAMP) as a ligand. The crystal structure of the SAP-dAMP complex determined at 2.8 A resolution (R = 0.232, R(free) = 0.252) reveals a decamer in which all interactions between SAP pentamers are mediated by the ligand. The stability of the decamer in solution has been demonstrated by gel filtration chromatography. The two calcium ions of SAP are bridged by the dAMP phosphate group and five hydrogen bonds are formed between the protein and the ligand, including specific interactions made by the adenine base. This mode of dAMP binding is not compatible with the nucleotide being part of double-helical DNA. The SAP-dAMP decamer is stabilized mainly by base-stacking of adjacent ligand molecules and possibly by electrostatic interactions involving the dAMP phosphate groups; decamerization buries 1000 A2 (2.6%) of the pentamer solvent-accessible surface. Ligand-induced decamerization of SAP, which utilizes the high cooperativity of a multiple-site interaction, may be a strategy to overcome the problems for drug design associated with the rather modest affinities of SAP for small-molecule ligands.

Computer Graphics↗