The emerging science of very early detection of disease outbreaks.

A surge of development of new public health surveillance systems designed to provide more timely detection of outbreaks suggests that public health has a new requirement: extreme timeliness of detection. The authors review previous work relevant to measuring timeliness and to defining timeliness requirements. Using signal detection theory and decision theory, the authors identify strategies to improve timeliness of detection and position ongoing system development within that framework.

Strategies for searching sequence databases.

We provide a detailed overview of the choices inherent in performing a sequence database search, including the choice of algorithm, substitution matrix and gap model. Each of these choices has implications that can be described as restrictions on the underlying model of sequence evolution, the expected degree of divergence between the query sequence and the database sequences (if one uses an evolutionary based matrix), as well as the sensitivity and selectivity of the search. We conclude with a series of recommendations for researchers performing these searches based on our experience and literature studies.

Classification of the environment of protein residues.

We have studied the classification of the environment of residues within protein structures. Eisenberg's original idea created environmental categories to discriminate between similar residues [Bowie et al., Science (1991), 253, 164-170]. These environments grouped residues based upon their buried surface area, polarity of the surrounding environment, and secondary structure element in which the residue is found. However, Eisenberg's original categories led to incomplete discrimination between residues that only partially substitute for each other. We have expanded on Eisenberg's original idea of environmental categories, by both considering additional contacts in the calculation of the solvent-accessible molecular surface area and by subdividing the environmental plot into regions based upon its theoretical features. Our alternative surface area calculations were used in conjunction with the polarity of the environment of the residue to define a new set of environmental categories. These new categories were able to discriminate between residues such as threonine, valine, and aspartic acid while reflecting the propensity of these residues to substitute for each other.

The first solvation shell of magnesium and calcium ions in a model nucleic acid environment: an ab initio study.

The interaction of organophosphate anions with divalent metal ions is central to many biological catalytic events. While experimental structural studies can give insight into the likely geometries that can be adopted, quantum mechanics allows for a more complete exploration of the competing forms. Ab initio quantum mechanical calculations have been performed on a series of complexes comprised of dimethyl phosphate, a divalent metal ion (either Mg(II) or Ca(II)) and water of hydration. An additional series of complexes were studied that included a Cl(I) ion to provide for charge neutrality. The most stable orientation of the hydrated metal ion complexed with the phosphate anion occurs when the metal ion is in a unidentate, rather than bidentate, orientation. The question of whether the divalent metal ion is located in the phosphinyl (-PO2(-)-) plane depends on the identity of the divalent metal ion and on the charge state of the complex.

Is Ca(II) ion binding to prothrombin fragment 1 intrinsically cooperative, or is the cooperative binding accounted for by self-association?

The recent suggestion that the apparent cooperativity seen in the binding of Ca(II) ions to prothrombin fragment 1 is due to protein aggregation is evaluated. Since (1) we find that the Ca(II) ion binding is not dependent upon protein concentration, (2) the analytical expression for the equilibrium constant of the aggregation model is unrealistically large when evaluated at realistic Ca(II) ion concentrations, and (3) a very simple allosteric cooperative binding model (Monod) can be shown to fit the experimental data, we conclude that the aggregation explanation for the apparent cooperativity in the Ca(II) ion binding by prothrombin fragment 1 is not correct.

The first solvation shell of magnesium ion in a model protein environment with formate, water, and X-NH3, H2S, imidazole, formaldehyde, and chloride as ligands: an Ab initio study.

The first coordination shell of an Mg(II) ion in a model protein environment is studied. Complexes containing a model carboxylate, an Mg(II) ion, various ligands (NH3, H2S, imidazole, and formaldehyde) and water of hydration about the divalent metal ion were geometry optimized. We find that for complexes with the same coordination number, the unidentate carboxylate-Mg(II) ion is greater than 10 kcal mol-1 more stable than the bidentate orientation. Imidazole was found to be the most stable ligand, followed in order by NH3, formaldehyde, H2O, and H2S.

A simple approach for the distribution of computationally intense tasks in an heterogeneous environment: distribution of the MDPP image-processing package.

A method has been developed to link the display ability of a high-resolution graphics workstation with the computational power of a local mainframe or a remote supercomputer via an electronic data network. The method allows this link to be established in a manner largely transparent to the user. The application of the method is illustrated by our successful distribution of the computationally intensive portions of an imaging program (MDPP) from a small VAX workstation to a VAX mainframe and Cray Y-MP8/832 using a simple message-passing technique. This technique can be applied to almost any configuration of networked machines.

Calcium ion binding to human and bovine factor X.

Human and bovine factor X contain 11 and 12 glutamyl residues, respectively, within the first 40 amino terminal residues that are post-translationally modified to gamma-carboxyglutamyl (Gla) residues. We have measured calcium ion binding to human factor X by equilibrium dialysis. This is the first examination of calcium ion binding to human factor X. We have also re-examined the equilibrium dialysis binding of calcium ions to bovine facor X in order to compare the two species. The data was analysed using a variety of models that allow for more than one class of binding site and for co-operativity among binding sites. Calcium ion binding to human factor X fits a model that had two classes of sites: one class with a single site that had an affinity of 0.1 mM and a second class with 19 equivalent, non-interacting sites with an average affinity of 3.5 mM. There was no evidence for co-operativity in calcium ion binding. Calcium ion binding to bovine factor X was best stimulated by a model that assumed one tight site, four co-operative sites, and 18 equivalent, non-interacting sites. To examine the co-operativity seen in calcium ion binding to bovine factor X, calcium ion binding to isolated Gla region (residues 1-44) and Gla-domainless factor X was measured by equilibrium dialysis. Calcium ion binding to Gla-domainless factor X was simulated by a model that had two classes of sites: one class with a single site that had an affinity of 0.25 mM, and a second class that had 15 sites with very low affinity sites (greater than 15 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Divalent metal ion mediated interaction of proteins with negatively charged membranes. A model study employing molecular mechanics.

Previous molecular mechanics calculations on the effect of Ca(II) and Mg(II) ions on the conformation of the 18-23 loop of bovine prothrombin [Maynard et al. 1988, Int. J. Peptide Protein Res. 31, 137-149] are extended to include the effect of a model phospholipid head group methyl[L-seryl] phosphate. Whereas the conformation of the Gla-21 Pro-22 amide bond remains decidedly trans in the absence of the model head group, in its presence, the cis Ca(II) ion induced (but not Mg(II] form is significantly lowered in relative energy. The low energy Ca(II) structures establish a coordination sphere with more ligands than do the low energy Mg(II) ion structures.

Generation of potential structures for the G-domain of chloroplast EF-Tu using comparative molecular modeling.

Comparative molecular modeling has been used to generate several possible structures for the G-domain of chloroplast elongation factor Tu (EF-Tu(chl)) based on the crystallographic data of the homologous E. coli protein. EF-Tu(chl) contains a 10 amino acid insertion not present in the E. coli protein and this region has been modeled based on its predicted secondary structure. The insertion appears to lie on the surface of the protein. Its orientation could not be determined unequivocally but several likely structures for the nucleotide binding domain of EF-Tu(chl) have been developed. The effects of the presence of water in the Mg2+ coordination sphere and of the protonation state of the GDP ligand on the conformation of the guanine nucleotide binding site have been examined. Relative binding constants of several guanine nucleotide analogs for EF-Tu(chl) have been obtained. The interactions between EF-Tu(chl) and GDP predicted to be important by the models that have been developed are discussed in relation to the nucleotide binding properties of this factor and to the interactions proposed to be important in the binding of guanine nucleotides to related proteins.

The role of hydrated divalent metal ions in the bridging of two anionic groups. An ab initio quantum chemical and molecular mechanics study of dimethyl phosphate and formate bridged by calcium and magnesium ions.

Ab initio quantum chemical (Gaussian82) and molecular mechanics (AMBER2.0) computational techniques are employed to investigate the interaction of two anions (formate an dimethylphosphate) and a central divalent metal cation (magnesium or calcium). These systems are models for the essential GDP binding unit of the G-proteins (e.g., EF-Tu or the ras oncogene proteins) and for protein/phospholipid interactions, both of which are mediated by divalent metal cations. Various levels of hydration are utilized to examine coordination of differences between magnesium and calcium ions. Two different orientations of formate and dimethyl phosphate in direct ion contact with a magnesium ion and two waters of hydration were energy minimized with both quantum and molecular mechanics techniques. The structures and energy differences between the two orientations determined by either of the computational techniques are similar. Magnesium ion has a strong propensity to assume six coordination whereas calcium ion preferentially assumes a coordination greater than six. Likewise, water molecules attached to magnesium ion are held more rigidly than those of calcium ion, thus calcium ion is more accommodating in the exchange of water for negative ligands.

Salt or ion bridges in biological systems: a study employing quantum and molecular mechanics.

Equilibrium geometries and binding energies of model "salt" or "ion" bridge systems have been computed by ab initio quantum chemistry techniques (GAUSSIAN82) and by empirical force field techniques (AMBER2.0). Formate and dimethyl phosphate served as anions in the model compounds while interacting with several organic cations, including methyl ammonium, methyl guanidinium, and divalent metal ion (either Mg2+ or Ca2+) without and with an additional chloride; and a divalent metal ion (either Mg2+ or Ca2+), chloride, and four water molecules of hydration about the metal ion. The majority of the quantum chemical computations were performed using a split-valence basis set. For the model compounds studied we find that the ab initio optimized geometries are in remarkably good agreement with the molecular mechanics geometries. Several calculations were also performed using diffuse fractions. The formate anion binds these model cations more strongly than does dimethyl phosphate, while the organic cation methyl ammonium binds model anions more strongly than does methyl guanidinium. Finally, in model compounds including organic anions, Mg2+ or Ca2+ and four molecules of water, and a chloride anion, we find that the equilibrium structure of the magnesium complex involves a solvent separated ion pair (the magnesium ion is six coordinate), whereas the calcium ion complex remains seven coordinate. Molecular mechanics overestimates binding energies, but the estimates may be close enough to actual binding energies to give useful insight into the details of salt bridges in biological systems.

A theoretical study of the effect of methylation or ethylation at O6-guanine in the structure and energy of DNA double strands.

Quantum and molecular mechanical calculations were employed to examine the effect on binding energies and structure of methylation and ethylation at O6-guanine in double-stranded DNA. Ab initio quantum chemical calculations (STO-3G, 3-21G) were initially used to pseudo-optimize the structure of the 9-methyl derivative of O6-methylguanine. The distal orientation for the O6-methyl group was found to be lower in energy than the proximal orientation. The geometry determined for the distal O6-methyl group was in agreement with recent X-ray work. These results were used in supplementary parameterization of the AMBER molecular mechanics force field necessary for the minimization of DNA double strands containing O6-methylguanosine. Resulting calculations with AMBER on two 5-mer DNA sequences containing the promutagenic G(GM)A subsequence showed that the proximal orientation, while higher in energy in the isolated molecule, is both less disruptive to the DNA double helix and more stable than the distal orientation. Binding energies and degree of destabilization upon methylation were found to be functions of the adjacent bases around a GGA subsequence. Sequence-dependent destabilization could play a role in the repair of alkylated bases. Quantum and molecular mechanics calculations indicate that the O6-methyl and O6-ethylguanines behave energetically in a very similar manner. These calculations suggest that the necessity for the different repair mechanisms for methylation and ethylation lesions cannot be simply explained by energy differences or observed structural differences.

Chemical modification of bovine prothrombin fragment 1 in the presence of Tb3+ ions. Sequence studies on 3-gamma-MGlu-fragment.

Chemical modification of the gamma-carboxyglutamyl (Gla) residues of bovine prothrombin fragment 1 using the formaldehyde-morpholine method in the presence of 100 Kappm Tb3+ ions at pH 5.0 provided a modified protein containing 3 gamma-methyleneglutamyl residues (gamma-MGlu) and 7 Gla residues (bovine 3-gamma-MGlu-fragment 1). The modified protein bound the same number of Ca2+ ions as the native protein (six to seven), exhibited 28Mg2+-binding properties identical to native fragment 1 (five Mg2+ ions bound), exhibited the metal ion-promoted quenching of the intrinsic fluorescence in a manner similar to the native protein, but did not bind to phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles in the presence of Ca2+ ions. Modification of the bovine protein using [14C]formaldehyde-morpholine provided a 14C-labeled 3-gamma-MGlu-fragment 1 suitable for sequence analysis. Edman sequencing of the peptides released by a tryptic digest of the reduced and carboxymethylated bovine [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7, 8, and 33 had been converted to [14C]gamma-methyleneglutamyl residues. In addition Lys97 was found to contain a 14C label. Similar analysis of the human [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7 and 32 were major modification sites and that Gla residues at positions 6 and 14 were partially modified. Lysine 96 was also modified in the human protein. The incorporation of a 14C label at Lys97 in bovine 3-gamma-MGlu-fragment 1 protein is not responsible for the loss of Ca2+-promoted binding to PS/PC vesicles. We suggest that Gla residues 7, 8, and 33 are elements of the first Ca2+-binding site; occupancy of this site establishes the Ca2+-specific conformation which is essential for the Ca2+-promoted interaction of the bovine protein with PS/PC vesicles. These studies also suggest that the loss of Gla residues at positions 7 and 32 prevents the formation of the initial Ca2+-binding site in the human protein.

Effect of calcium (II) and magnesium (II) ions on the 18-23 gamma-carboxyglutamic acid containing cyclic peptide loop of bovine prothrombin. An AMBER molecular mechanics study.

The effect of calcium (II) and magnesium (II) ions on the conformation of the 18-23 cyclic peptide loop of bovine prothrombin are investigated by the molecular mechanics program AMBER (Assisted Model Building with Energy Refinement). The work is an extension of an earlier paper (Eastman et al., Int. J. Peptide Protein Res. 27, 1986, 530-553) that employed the program ECEPP (Empirical Conformational Energy Program for Peptides). In the absence of either metal ion, or in the presence of either one Ca(II) or one Mg(II) ion, the lowest-energy forms found by AMBER have the Gla21-Pro22 peptide bond in a trans conformation. In the presence of two Ca(II) or Mg(II) ions, the loop form of lowest energy is decidedly cis. The coordination about the Ca(II) and Mg(II) ions is different in both the single and double metal cases. In addition, the peptide chains that emerge from the loop are oriented parallel to each other in the lowest-energy complex with two Ca(II) ions, but are not parallel in the lowest-energy complex with two Mg(II) ions.

Studies on Ca(II) binding to gamma-carboxyglutamic acid. Use of thermal decarboxylation to probe metal ion/gamma-carboxyglutamic acid interactions.

The thermal decarboxylation of N-benzyloxycarbonyl-L-gamma-carboxyglutamic acid alpha-methyl ester [Z)-L-Gla-OMe) has been studied. In the presence of increasing amounts of calcium or magnesium ions, lyophilized powders of (Z)-L-Gla-OMe exhibit a corresponding increase in thermal stability. Both magnesium and calcium form relatively tight, thermally stable complexes with (Z)-L-Gla-OMe at high metal ion concentrations. Differences between Ca(II) and Mg(II) binding are noted at low metal ion concentrations, where (Z)-L-Gla-OMe is in excess. Under these conditions, complex formation with Mg(II) apparently favors a 2:1 Gla-magnesium ion complex in which both Gla residues are unstable to thermal decarboxylation. Calcium ion complexes, however, are found to favor a 3:1 Gla-calcium ion complex in which 1 of the 3 Gla residues is thermally stable.

Relative affinity of Ca(II) and Mg(II) ions for human and bovine prothrombin and fragment 1.

Equilibrium dialysis results are presented for Ca(II) and Mg(II) ion binding to human and bovine prothrombin and fragment 1. Ca(II) ions bind cooperatively, Mg(II) does not.

Mg(II) binding by bovine prothrombin fragment 1 via equilibrium dialysis and the relative roles of Mg(II) and Ca(II) in blood coagulation.

The first direct equilibrium dialysis titration of the blood coagulation protein bovine prothrombin fragment 1 with Mg(II) is presented. Fragment 1 has fewer thermodynamic binding sites for Mg(II) than Ca(II), less overall binding affinity, and significantly less cooperativity. Several nonlinear curve fitting models were tested for describing the binding of fragment 1 with Mg(II), Ca(II), and mixed metal binding data. The Mg(II) data is represented by essentially five equivalent, noninteracting sites; for Ca(II), a model with three tight, cooperative sites and four "loose", equal affinity, noninteracting sites provides the best model. Based on the reported equilibrium dialysis data and in conjunction with other experimental data, a model for the binding of Ca(II) and Mg(II) to bovine prothrombin fragment 1 is proposed. The key difference between the binding of these divalent ions is that Ca(II) apparently causes a specific conformational change reflected by the cooperativity observed in the Ca(II) titration. The binding of Ca(II) ions to the three tight, cooperative sites establishes a conformation that is essential for phospholipid X Ca(II) X protein binding. The filling of the loose sites with Ca(II) ions leads to charge reduction and subsequent phospholipid X Ca(II) X protein complex interaction. Binding of Mg(II) to bovine prothrombin fragment 1 does not yield a complex with the necessary phospholipid-binding conformation. However, Mg(II) is apparently capable of stabilizing the Ca(II) conformation as is observed in the mixed metal ion binding data and the synergism in thrombin formation.

Determination of magnesium binding to macromolecules.

An equilibrium dialysis technique for examining magnesium binding to macromolecules is described. The technique is used to determine the binding constants of magnesium to human prothrombin. This procedure should be of great utility for many biochemical systems which exhibit magnesium affinity.

Synthesis of a gamma-carboxyglutamic acid containing heptapeptide corresponding to bovine prothrombin residues 17-23.

The vitamin K-dependent proteins involved in blood coagulation processes each contain a small cystine loop with the sequence -Gla-Cys-X-Gla-Gla-X-Cys-. A method for the synthesis of the heptapeptide derivative representing the 17-23 sequence of bovine prothrombin, Z-Gla-Cys-Leu-Gla-Gla-Pro-Cys-OBzl, has been developed. The 25Mg2+ n.m.r. spectrum of the heptapeptide derivative has been obtained and is compared to the n.m.r. spectra obtained from the interaction of 25Mg2+ with Z-Gla-OMe and Z-Gla-Gla-OMe.