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PubMed1992

A model of the platelet factor 4 complex with heparin.

A model of heparin bound to bovine platelet factor 4 (BPF4) was completed using a graphically designed heparin molecule and the crystallographic coordinates of the native bovine platelet factor 4 tetramer. The oligosaccharides had a chain length of at least eight disaccharide units with the major repeating disaccharide unit consisting of (1----4)-O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1----4)-(2-deoxy-2-sulfamino-2-D-glucopyranosyl 6-sulfate). Each disaccharide unit carried a -4.0 charge. The structure of BPF4 was solved to 2.6 A resolution with R = 0.237. Each monomer of BPF4 contains an alpha-helix lying across 3 strands of antiparallel beta-sheet. Each helix has four lysines, which have been implicated in heparin binding. These lysine residues are predominantly on one side of the helix and are solvent accessible. Electrostatic calculations performed on the BPF4 tetramer show a ring of strong, positive charge which runs perpendicularly across the helices. Included in this ring of density is His-38, which has been shown by NMR to have a large pKa shift when heparin binds to BPF4. Our model of heparin bound to PF4 has the anionic polysaccharide perpendicular to the alpha-helices, wrapped about the tetramer along the ring of positive charge, and salt linked to all four lysines on the helix of each monomer.

Amino Acid Sequence↗

PubMed1993

Characterization of the backbone dynamics of an anti-digoxin antibody VL domain by inverse detected 1H-15N NMR: comparisons with X-ray data for the Fab.

The dynamic behavior of the polypeptide backbone of a recombinant antidigoxin antibody VL domain has been characterized by measurements of 15NT1 and T2 relaxation times, 1H-15N NOE values, and 1H-2H exchange rates. These data were acquired with 2D inverse detected heteronuclear 1H-15N NMR methods. The relaxation data are interpreted in terms of model free spectral density functions and exchange contributions to transverse relaxation rates R2 (= 1/T2). All characterized residues display low-amplitude picosecond time-scale librational motions. Fifteen residues undergo conformational changes on the nanosecond timescale, and 24 residues have significant R2 exchange contributions, which reflect motions on the microsecond to millisecond time-scale. For several residues, microsecond to millisecond motions of nearby aromatic rings are postulated to account for some or all of their observed R2 exchange contributions. The measured 1H-2H exchange rates are correlated with hydrogen bonding patterns and distances from the solvent accessible surface. The degree of local flexibility indicated by the NMR measurements is compared to crystallographic B-factors derived from X-ray analyses of the native Fab and the Fab/digoxin complex. In general, both the NMR and X-ray data indicate enhanced flexibility in the turns, hypervariable loops, and portions of beta-strands A, B, and G. However, on a residue-specific level, correlations among the various NMR data, and between the NMR and X-ray data, are often absent. This is attributed to the different dynamic processes and environments that influence the various observables. The combined data indicate that certain regions of the VL domain, including the three hypervariable loops, undergo dynamic changes upon VL:VH association and/or complexation with digoxin. Overall, the 26-10 VL domain exhibits relatively low flexibility on the ps-ns timescale. The possible functional consequences of this result are considered.

Digoxin↗

PubMed1995

Progress of 1D protein structure prediction at last.

Accuracy of predicting protein secondary structure and solvent accessibility from sequence information has been improved significantly by using information contained in multiple sequence alignments as input to a neural network system. For the Asilomar meeting, predictions for 13 proteins were generated automatically using the publicly available prediction method PHD. The results confirm the estimate of 72% three-state prediction accuracy. The fairly accurate predictions of secondary structure segments made the tool useful as a starting point for modeling of higher dimensional aspects of protein structure.

Amino Acid Sequence↗

PubMed1994

The effect of substituted laminin A chain-derived peptides on the conformation and activation kinetics of plasminogen.

Conversion of the zymogen plasminogen (Pg) to the active enzyme plasmin is catalyzed by proteinases such as tissue-type plasminogen activator (t-PA). Interaction of Pg with small ligands such as lysine or macromolecular ligands such as fibrin induces a dramatic conformational change in the zymogen which enhances its efficacy as a t-PA substrate, thereby increasing catalytic efficiency of the activation reaction. We have previously demonstrated that a synthetic peptide derived from amino acids 2091-2108 of the laminin A chain (designated LamA2091-2108) can significantly enhance t-PA-catalyzed Pg activation. To probe the mechanism of this stimulatory reaction, we have determined the effect of substituted LamA2091-2108 derivatives on Pg activation by t-PA. Substitution of charged residues in LamA2091-2108 with neutral amino acids decreases the kcat/Km observed in the presence of native LamA2091-2108. Furthermore, fluorescence-quenching experiments demonstrate that whereas LamA2091-2108 alters the solvent accessibility of Pg Trp residues, charge-substituted peptides have little effect on Pg conformation. These data suggest that LamA2091-2108 stimulates Pg activation by inducing a conformational change in the zymogen similar to that observed upon binding of other ligands such as lysine and fibrin.

Amino Acid Sequence↗

PubMed1995

Intrinsic fluorescence of the chloroplast H(+)-ATPase.

We have examined the intrinsic fluorescence properties of a highly purified chloroplast H(+)-ATPase (CF0F1) preparation [R. D. Kirch and P. Graber (1992) Acta Physiol. Scand. 746, 9-12). Unlike the catalytic CF1 portion alone, CF0F1 fluorescence was dominated by tryptophan fluorescence both at 277-nm excitation, favoring tyrosine excitation, and at 295-nm excitation, favoring tryptophan excitation. A broad tryptophan fluorescence peak was observed with a maximum at around 335 nm and a broad shoulder around 350 nm. Denaturation of the enzyme complex with guanidine-HCl resulted in a significant increase (approximately 40%) in tyrosine fluorescence. The fluorescence spectrum (lambda ex = 295 nm) of the inhibitory epsilon subunit isolated from CF1 resembled that of CF1, indicating the presence of two tryptophan species located in different environments. Fluorescence quenching by potassium iodide indicated a substantial increase in the solvent accessibility of one of the two tryptophans following isolation of epsilon from CF1. Thus, when epsilon binds to CF1, a tryptophan residue becomes partially buried, probably at an interface between epsilon and another (possibly gamma) CF1 subunit. Removal of the epsilon subunit from CF1 leads to an increase in tyrosine fluorescence of a magnitude similar to that obtained upon denaturation of the CF0F1 complex. The results suggest that the reversible association of the epsilon subunit with CF0F1 or with isolated CF1 may be monitored by following changes in the intrinsic fluorescence of the enzyme complex.

Chloroplasts↗

PubMed1995

Interactions of peroxyquinols with cytochromes P450 2B1, 3A1, and 3A5: influence of the apoprotein on heterolytic versus homolytic O-O bond cleavage.

In studies of the mechanisms involved in oxygen activation by cytochromes P450 (P450s), organic hydroperoxides are frequently used to model the putative peroxyferrous complex formed immediately prior to oxygen-oxygen bond cleavage during the reaction cycle of this monooxygenase. Heterolysis of the O-O bond by ferric P450 generates the catalytically competent oxo-ferryl intermediate analogous to peroxidase Compound I, and homolysis produces the Compound II analog capable only of one-electron oxidations. As P450s have been shown to catalyze both modes of O-O bond scission, the present investigation was focused on the influence of the apoprotein on the relative contributions of these competing processes. Liver microsomes from rats treated with the P450-inducing agents phenobarbital, beta-naphthoflavone, and dexamethasone, as well as purified P450s 2B1, 3A1, and 3A5 were incubated with 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2, 5-cyclohexadienone (BHTOOH). Ratios of heterolysis to homolysis were determined by analyzing the products derived from this hydroperoxide. The data demonstrate that BHTOOH is cleaved with a ratio of approximately 1.0 with all of the liver microsomal or purified P450s investigated, except with liver microsomes from dexamethasone-treated rats or with P450s 3A1 and 3A5. In these cases, heterolysis predominated over homolysis by factors of 2.5 to 4.0. On the other hand, microsomes rich in P450 2B1 catalyzed predominantly heterolysis with analogs of BHTOOH containing smaller alkyl substituents. The data are consistent with a requirement for solvent access to the peroxyferrous complex and general acid catalysis of heterolytic O-O bond cleavage by water.

Animals↗

PubMed1998

Evaluation of methods for the quantitation of cysteines in proteins.

Several methods for the quantitation of cysteines in proteins have been evaluated and compared. Titration of protein sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) under carefully controlled conditions has extended the detection limits of this method with high accuracy and reproducibility. Results are reported for a variety of enzymes containing a range of total cysteines with different degrees of solvent accessibility and reactivity. A papain amplification assay has also been examined, in which reactivation of the disulfide-blocked active site cysteine of papain can be achieved by a coupled reaction with protein sulfhydryl groups. Detection of sulfhydryls by this amplification assay can be extended, by increasing the enzyme assay times, to achieve over a 40-fold increase in sensitivity over the improved DTNB titration method. Alternatively, titration of enzyme cysteinyl residues with either bromobimane or a maleimide derivative of naphthopyranones has the advantage that a fluorescent product results upon modification of the sulfhydryl group. Reaction of bromobimane with several different enzymes results in nonspecific background fluorescence that limits the detection range of this method unless the products are separated. In contrast, low background fluorescence and high quantum yields with maleimide naphthopyranoses has allowed detection of protein cysteinyl residues with very high sensitivities.

Cysteine↗

PubMed1999

Rate and equilibrium constants for protein unfolding and refolding determined by hydrogen exchange-mass spectrometry.

Studies of protein unfolding and refolding may help us understand the more general problem of protein folding. Recent studies from this laboratory demonstrated that the unfolding and refolding of a large protein, rabbit muscle aldolase (M(r) 157 kDa), can be studied by combining amide hydrogen exchange and mass spectrometry. Results of these studies indicated that aldolase has three unfolding domains which likely unfold sequentially. Urea was used to increase the populations of partially unfolded states which were labeled with deuterium following a brief exposure to D(2)O. Electrospray ionization mass spectra of both the intact protein and its peptic fragments had multiple envelopes of isotope peaks from which the populations of unfolded forms were determined. The present study extends the previous investigations to include different urea concentrations and kinetic modeling of data taken as the system approaches equilibrium. Analysis of these results gives rate and equilibrium constants describing the unfolding and refolding processes characteristic of aldolase destabilized in urea. The change in solvent-accessible surface, which has been used as a reaction coordinate for protein folding, is estimated from the dependence of the equilibrium and rate constants on the concentration of urea.

Animals↗

PubMed1994

Proline-protein interactions: protection of structural and functional integrity of M4 lactate dehydrogenase.

A well defined labile isozyme, rabbit muscle M4-lactate dehydrogenase was denatured under freeze-thaw, heat and GuHCl treatment in the presence and absence of proline, and the corresponding structural changes of the enzyme were monitored through fluorescence and CD spectral studies. The data reveal that proline confers protection to the structural integrity of the enzyme, thereby protecting its activity. This was attributed to its property of forming hydrophilic colloids in aqueous media with a hydrophobic backbone interacting with protein. Unlike other osmolytes, proline is proposed to act on the enzyme stability not only by inducing preferential hydration of proteins but also through the interactions of its multimeric hydrophobic backbone with the solvent-accessible hydrophobic regions of the enzyme.

Animals↗

PubMed1997

Three-dimensional cryoelectron microscopic reconstruction of the 2.25-MDa homomultimeric phosphoenolpyruvate synthase from Staphylothermus marinus.

The phosphoenolpyruvate synthase (EC 2.7.9.2) of the hyperthermophilic archaeon Staphylothermus marinus forms a 2.25 MDa homomultimeric complex of 24 subunits. Here, computational analysis of low-dose cryoelectron micrographs was used to ascertain that the major rotational symmetry axes were 2-fold, 3-fold, and 4-fold. These symmetry considerations were used to perform a three-dimensional reconstruction to a spatial resolution of 4 nm. This assembly has an octahedral architecture with a solvent accessible interior and with ill-defined yet seemingly flexible appendages on the periphery. This macromolecular assembly is unusually large in mass compared with most other known globular proteins--especially other identified phosphoenolpyruvate synthases which are usually dimeric--and its elaborate quaternary arrangement might represent an adaptation to its extreme environment.

Archaeal Proteins↗

PubMed1998

Characterization of a cytochrome P450 from the acidothermophilic archaea Sulfolobus solfataricus.

We report the cloning, expression, purification, and molecular characterization of a cytochrome P450 (CYP119) from the thermophilic archaea Sulfolobus solfataricus. This protein displays an absorption spectra in the reduced, oxidized, and carbonyl adduct analogous to those of other P450 enzymes. We demonstrate that P450 (CYP119) exhibits remarkable thermo- and pressure stability, with a melting temperature 40 degrees higher than that of the extensively studied cytochrome P450cam (CYP101) and an optical spectra completely resistant to the formation of the inactive P420 by hydrostatic pressure up to 2 kbar. CO flash photolysis experiments, as well as construction of a CYP119 homology model, suggest an open active site with greater solvent access than P450 (CYP101) and similar to that of P450 (CYP102). This communication represents the first molecular characterization of an extremophilic cytochrome P450.

Amino Acid Sequence↗

PubMed2000

Highly reactive cysteine residues in rodent hemoglobins.

Two hemoglobins with cysteine residues highly reactive toward electrophiles have been identified and characterized. Cys-125beta of guinea pig hemoglobin has a low pK(a) and forms conjugates with electrophiles more quickly than glutathione and several orders of magnitude more quickly than other protein thiols. This cysteine is capable of intercepting benzoquinone, a known carcinogenic metabolite, before other protein nucleophiles can be modified. Cys-13beta of mouse hemoglobin was observed to conjugate with electrophiles as quickly as glutathione. The structural basis of reactivity is different in the two hemoglobins and is analyzed in terms of hydrogen-bonding, solvent accessibility, and helix-dipole contributions. Complementing a previously characterized highly reactive cysteine in rat hemoglobin, identification of these cysteines suggests that the reactivity of these hemoglobins could represent a common function as a detoxification sink against carcinogens.

Amino Acid Sequence↗

PubMed2001

Association of the Glu298Asp polymorphism in the endothelial nitric oxide synthase gene with essential hypertension resistant to conventional therapy.

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO) which, after diffusing into vascular smooth muscle cells, activates guanylate cyclase leading to vasodilatation. A polymorphism (894G to T) in exon 7 of the eNOS gene causes the conversion of Glu to Asp in position 298. The recently described crystal structure of the heme domain of eNOS protein shows that Glu298 is fully solvent accessible and distant from regions integral to enzyme function. Searching for phenotypic expression of eNOS gene variants, we genotyped a group of patients with essential hypertension (H, n = 119) for the Glu298Asp polymorphism and compared them with age- and sex-matched healthy normals (N, n = 85). To specify phenotypic expression further, the hypertensive patients were subdivided into one group that responded well to regular antihypertensive therapy (CH, n = 45) and one group that was resistant to the therapy (RH, n = 74). Patients with BP higher than 140/90 mmHg when on adequate lifestyle modification and triple-combination therapy (including diuretics) were considered resistant. In RH and H groups, a significantly higher frequency of T alleles (P = 0.022 and P = 0.046, respectively) was found compared to normotonics (N). In well-controlled hypertonics, the same tendency was found, but did not reach statistical significance. The Glu298Asp polymorphism may contribute to the complex pathogenesis of essential hypertension and may be a factor in the resistance of these patients to conventional antihypertensive therapy. The presence of this allele may thus be predictive of the patients' therapeutic response.

Adrenergic beta-Antagonists↗

PubMed2001

Unfolding of apomyoglobin examined by synchrotron footprinting.

A new method to examine the structure and stability of proteins using footprinting is applied to examine the unfolding of apomyoglobin. Unlike previous cleavage based footprinting methods, synchrotron X-ray protein footprinting is based on a quantitative determination of the extent and the site of radiolytic modification of amino acid side chains, analyzed using mass spectrometry. The amino acids most susceptible to radiolytic oxidation (cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine, proline, and leucine) serve as convenient probes of protein structure to monitor changes in solvent accessibility. To determine if the technique can measure quantitative properties of proteins relevant to structure and function, we examined the equilibrium unfolding of apomyoglobin in urea and compared the results to data derived from fluorescence studies under the same conditions.

Amino Acid Sequence↗

PubMed2000

Mechanistic Studies on Prolyl-4-Hydroxylase: Demonstration That the Ferryl Intermediate Does Not Exchange with Water.

Prolyl-4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens. In contrast to deacetoxy/deacetylcephalosporin C synthase, p-hydroxyphenylpyruvate hydroxylase, lysyl hydroxylase and alpha-ketoisocaproate oxygenase, no incorporation of (18)O-labeled water into the hydroxylated product was found for the human type I prolyl-4-hydroxylase when N-Cbz-Gly-L-Phe-L-Pro-Gly-OEt was used as a substrate. This suggests that the ferryl intermediate for this enzyme is not solvent accessible. Copyright 2000 Academic Press.

Journal Article↗

PubMed1994

Changes in rheumatoid factor and monoclonal IgG antibody specificity after site-specific mutations in antigenic region of beta 2-microglobulin.

Antigenic epitopes on the SKDWSFY region (positions 57-63) of human beta 2-microglobulin (beta 2m) reacting in ELISA with polyclonal and monoclonal human IgM rheumatoid factors (RF) as well as with monoclonal mouse IgG anti-beta 2m antibodies were studied after site-specific mutation of single or double beta 2m amino acid residues. Single site-specific mutations at lysine 58 and serine 61 produced a major decrease (50-100%) in reactivity of polyclonal IgM RF with beta 2m. Mutations at serine 57, tryptophane 60, and phenylalanine 62 also induced substantial decrements (25-49%) in IgM RF reactivity. beta 2m mutants containing double amino acid mutations including one residue outside the SKDWSFY region along with four different mutations at tryptophane 60 also often showed marked decrements in IgM RF anti-beta 2m reactivity. Site-specific mutations at virtually all residues within the SKDWSFY beta 2m region produced a major decrease in reactivity of two of three monoclonal human IgM RF. Results with nine murine IgG anti-beta 2m mAbs often showed an entirely different profile. Site-specific mutation of aspartic acid at position 59 produced a 50-100% decrease in binding of nine mAbs. Serine at position 61 and tyrosine at 63 also appeared to represent important residues for antigenic determinants reacting with anti-beta 2m mAbs. Results of mapping discrete epitopes using glycine substitution within overlapping 7-mers of beta 2m linear sequence compared with site-specific mutation of single beta 2m residues were similar in many instances, but also often showed marked differences. A marked change in antigenic contribution of single amino acid residues to beta 2m antigenic epitopes frequently depended on what residues were mutated to. Our findings emphasize that single residues within a small seven amino acid, solvent-accessible antigenic stretch of primary beta 2m sequence vary considerably in their contribution to the reactive antigenic structures for IgM RF as well as monoclonal IgG antibodies.

Amino Acid Sequence↗

PubMed1993

Hydration of proteins. A comparison of experimental residence times of water molecules solvating the bovine pancreatic trypsin inhibitor with theoretical model calculations.

A 1 ns trajectory from a molecular dynamics study of 1.4 ns total length was used for a detailed analysis of the residence times of water molecules located near 227 selected bovine pancreatic trypsin inhibitor (BPTI) atoms. The simulation was performed using the GROMOS force field, with apolar hydrogen atoms treated as united atoms, and the SPC/E water model. The system consisted of 568 BPTI atoms and 2371 water molecules. The theoretical results are in good agreement with experimental data available from nuclear magnetic resonance spectroscopy. The residence times of individual water molecules coming near a given BPTI atom, as obtained from the simulation, vary greatly and range between 10 and 500 ps. The effective residence time, calculated using a correlation function technique from the presence of all individual water molecules visiting the hydration shell of a given BPTI atom, never exceeds 200 ps. The average residence time near backbone and side-chain atoms is approximately 39 ps and 24 ps, respectively. The shortest residence times, on average, are found near charged atoms (19 ps), whereas near non-polar and polar side-chain atoms the residence times are 25 ps and 36 ps, respectively. There is no apparent correlation between the residence times of the hydration water molecules of solvent-accessible residues and their location in different regular or non-regular secondary structures.

Amino Acid Sequence↗

PubMed1993

Bacteriophage T7 RNA polymerase. 19F-nuclear magnetic resonance observations at 5-fluorouracil-substituted promoter DNA and RNA transcript.

We have substituted 5-fluorodeoxyuridine (5-FdU) in place of thymidine in defined positions along synthetic bacteriophage T7 promoter DNA sequences. None of the fluoro-substitutions in the promoter DNA sequence reduced transcription yields with T7 RNA polymerase significantly. Substitutions on the coding template strand reduced transcription yields when placed at +3, but not at +4. 19F-n.m.r. spectra from transcription reactions and gel analysis of transcription products show that T7 RNA polymerase correctly and efficiently utilizes 5-FUTP as a RNA substrate analog. The fluorine atom provides a sensitive probe for monitoring the local environment, base sequence and solvent exposure at the DNA major groove through its 19F-n.m.r. resonance. Buffer dependencies of the fluorine chemical shift and digestion patterns with DNase I suggest that the T7 promoter base-pairs near the transcription start site are distorted with a more open minor groove and less solvent accessible major groove. Previous chemical footprinting data of promoter-polymerase complexes yield a picture that T7 RNA polymerase recognizes major groove features in the region from positions -7 to -11 and minor groove features on the same side of DNA flanking both sides of this region. Consistent with this, 19F-n.m.r. observations identify two additional positions, -8 and -17, involved in promoter recognition on this side of the DNA helix. On the other hand, our observations also implicate the opposite side of the DNA helix, primarily at positions -14 and -15, as major groove recognition sites for T7 RNA polymerase. In addition, n.m.r. spectra from 5-FdU-substituted base-pairs -2 and -3, suggest either additional interactions on the same side of the DNA helix as -14 and -15, or distortions in the DNA structure.

Bacteriophage T7↗