bluuebunny/crossref_metadata_2025 huggingface dataset:
A multiple‐start Monte Carlo docking method
Authors: Hart Trevor N. , Read Randy J.
Published: May 2005

We present a method to search for possible binding modes of molecular fragments at a specific site of a potential drug target of known structure. Our method is based on a Monte Carlo (MC) algorithm applied tothe translational and rotational degrees of freedom of the probe fragment. Starting from a randomly generated initial configuration, favorable bindingmodes are generated using a two‐step process. An MC run is first prformed in which the energy in the Metropolis algorithm is substituted by a score function that measures the average distance of the probe to the targetsurface. This has the effect of making buried probes move toward the targetsurface and also allows enhanced sampling of deep pockets. In a second MC run, a pairwise atom potential function is used, and the temperature parameter is slowly lowered during the run (Simulated Annealing). We repeat this procedure starting from a large number of different randomly generated initial configurations in order to find all energetically favourable docking modes in a specified region around the target. We test this method using two inhibitor‐receptor systems: Streptomyces griseus Proteinase B in complex with the third domain of the ovomucoid inhibitor from turkey, and dihydrofolate reductase from E. Coli in complex with methotrexate. The method could consistently reproduce the complex found in thecrystal structure searching from random initial positions in cubes ranging from 25 Å to 50 Å about the binding site. In the case of SGPB, we were also successful in docking to the native structure. In addition, we were successful in docking small probes in a search that included the entire protein surface. © 1992 Wiley‐Liss, Inc.
Common features of the conformations of antigen‐binding loops in immunoglobulins and application to modeling loop conformations
Authors: Tramontano Anna , Lesk Arthur M.
Published: May 2005

Using database screening techniques we have examined the relationship between antigen‐binding loops in immunoglobulins, and regions of similar conformation in other protein families. The conformations of most antigen‐binding loops are not unique to immunoglobulins. But in many cases, the geometrical relationship between the loop and the peptides flanking it differs between the immunoglobulins and other structures with the same loop. We assess model building by data base screening, compared with thatbased on canonical structures. © 1992 Wiley‐Liss, Inc.
Detection of native‐like models for amino acid sequences of unknown three‐dimensional structure in a data base of known protein conformations
Authors: Sippl Manfred J. , Weitckus Sabine
Published: May 2005

We present an approach which can be used to identify native‐like folds in a data base of protein conformations in the absence of any sequence homology to proteins in the data base. The method is based on a knowledge‐based force field derived from a set of known protein conformations. A given sequence is mounted on all conformations in the data base andthe associated energies are calculated. Using several conformations and sequences from the globin family we show that the native conformation is identified correctly. In fact the resolution of the force field is high enough to discriminate between a native fold and several closely related conformations. We then apply the procedure to several globins of known sequence but unknown three dimensional structure. The homology of these sequences to globins of known structures in the data base ranges from 49 to 17%. Withone exception we find that for all globin sequences one of the known globinfolds is identified as the most favorable conformation. These results are obtained using a force field derived from a data base devoid of globins of known structure. We briefly discuss useful applications in protein structurlresearch and future development of our approach. © 1992 Wiley‐Liss, Inc.
Pancreatic spasmolytic polypeptide: Crystallization, circular dichroism analysis, and preliminary X‐ray diffraction studies
Authors: Gajhede Michael , Thim Lars , Jørgensen Klavs H. , Melberg Steen G.
Published: May 2005

Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapor diffusion method. Crystals suitable for X‐ray diffraction analysis were grown at pH 4.7 from a solution of 6% saturated ammonium sulfate. The space group is orthorhombic I222 or I212121 with unit cell parameters a = 54.38 Å, b = 72.29 Å, and c = 180.85 Å. There are three molecules of PSP per asymmetric unit and a water content of 46.9%. The crystals diffracts to an estimated resolution of 2.7 Å.

The far‐UV CD spectrum of PSP shows some exceptional features which cannot be accounted for thoroughly in terms of standard secondary structures commonly seen in protein CD spectroscopy. With this limitation, the secondary structure analysis predicts 15% α‐helix, between 10 and 20% antiparallel β‐strand, 10% parallel β‐strand, 15% turn, and 25 to 40% of other structures. © 1992 Wiley‐Liss, Inc.
Modeling of substrate and inhibitor binding to phospholipase A2
Authors: Sessions Richard B. , Dauber‐Osguuthorpe Pnina , Campbell Malcolm M. , Osguthorpe David J.
Published: May 2005

Molecular graphics and molecular mechanics techniques have been used to study the mode of ligand binding and mechanism of action of the enzyme phospholipase A2. A substrate–enzyme complex was constructed based on the crystal structure of the apoenzyme. The complex was minimized to relieve initial strain, and the structural and energetic features of the resultant complex analyzed in detail, at the molecular and residue level. The minimized complex was then used as a basis for examining the action of the enzyme on modified substrates, binding of inhibitors to the enzyme, and possible reaction intermedite complexes. The model is compatible with the suggested mechanism of hydrolysis and with experimental data about stereoselectivity, efficiency of hydrolysis of modified substrates, and inhibitor potency. In conclusion, the model can be used as a tool in evaluating new ligands as possible substrates and in the rational design of inhibitors, for the therapeutic treatment of diseases such as rheumatoid arthritis, atherosclerosis, and asthma. © 1992 Wiley‐Liss, Inc.
Variability of conformations at crystal contacts in BPTI represent true low‐energy structures: Correspondence among lattice packing and molecular dynamics structures
Authors: Kossiakoff Anthony A. , Randal Michael , Guenot Jeanmarie , Eignebrot Charles
Published: May 2005

The structure of five basic pancreatic trypsin inhibitor (BPTI) molecules are compared to establish the extent and nature of the conformational variability resulting from crystal packing effects. BPTI is an ideal system to evaluate such factors because of the availability of high resolution X‐ray models of five different BPTI structures, each in a different crystal packing environment. Differences observed among the structures are found to be distributed throughout the olecules, although the regions that display most variability are associated with the loop structures (residues 14–17 and 24–29). The regions of structure that show the largest rms deviations from the mean of the five packing motifs correlate well with the presence of intermolecular contacts in the crystal lattice. For most of the molecules there is also a correspondence between a larger number of intermolecular contacts and systematically higher B‐factors, although it is not appearent whether this is induced by the crystal contact or results from the fact that the contacts are made predominantly through surface loops. The conformational differences seen among the X‐ray models constitute more than local shifts at the lattice contact surfaces, and in fact involve in some cases the making and breaking of intramolecular H‐bonds. The magnitudes of the differences among packing models are significantly larger than those usually associated with changes induced by mutagenesis; for instance, the structural differences at the site of mutation observed on removing an internal disulfide from the molecule are significantly less than those associated with lattice contact effects. The crystal packing conformations are compared to representative structures of BPTI generated during a 96‐psec molecular dynamics (MD) simulation. This comparison shows a high level of correspondence between the protein flexibility indicated by the X‐ray and MD analyses, and specifically between those regions that are most variable. This suggests that the regions that show most variability among the crystal packing models are not artifacts of crystallization, but rather represent true low‐energy conformers that have been preferentially selected by crystallization factors. © 1992 Wiley‐Liss, Inc.
Multiple protein sequence alignment from tertiary structure comparison: Assignment of global and residue confidence levels
Authors: Russell Robert B. , Barton Geoffrey J.
Published: May 2005

An algorithm is presented for the accurate and rapid generation of multiple protein sequence alignments from tertiary structure comparisons. A preliminary multiple sequence alignment is performed using sequence information, which then determines an initial superposition of the structures. A structure comparison algorithm is applied to all pairs of proteins in the superimposed set and a similarity tree calculated. Multiple sequence alignments are then generated by following the tree from the branches to the root. At each branchpoint of the tree, a structure‐based sequence alignment and coordinate transformations are output, with the multiple alignment of all structures output at the root The algorithm encoded in STAMP (Structural Alignment of Multiple Proteins) is shown to give alignments in good agreement with published structural accounts within the dehydrogenase fold domains, globins, and serine proteinases.

In order to reduce the need for visual verification, two similarity indices are introduced to determine the quality of each generated structural alignment. Sc quantifies the global structural similarity between pairs or groups of proteins, whereas Pij′ provides a normalized measure of the confidence in the alignment of each residue. STAMP alignments have the quality of each alignment characterized by Sc and Pij′ values and thus provide a reproducible resource for studies of residue conservation within structural motifs. © 1992 Wiley‐Liss, Inc.
Purification and crystallization of insecticidal δ‐endotoxin CryIIIB2 from Bacillus thuringiensis
Authors: Cody Vivian , Luft Joseph R. , Jensen Erik , Pangborn Walter , English Leigh
Published: May 2005

CryIIIB2, an insecticidal protein from Bacillus thuringiensis has been crystallized from 0.6 M NaBr and HEPES buffer at pH 7.0 and X‐ray diffraction data collected on a native crystal to 2.4 Å. The insecticidal protein was obtained from a Bacillus thuringiensis (Bt) strain EG7231. Crystals of the endotoxin are orthorhombic, space group C2221, with unit cell dimensions of a = 122.44, b = and c = Å. A unit cell contains one molecule of the 67,000 Da endotoxin per asymmetric unit. © 1992 Wiley‐Liss, Inc.
Structural studies of the retroviral proteinase from avian myeloblastosis associated virus
Authors: Ohiendorf Douglas H. , Foundling Steven I. , Wendoloski John J. , Sedlacek Juraj , Strop Peter , Salemme F. Raymond
Published: May 2005

The structure of the retroviral proteinase from avian myeloblastosisassociated virus (MAV) has been determined and refined at 2.2 Å resolution. This structure is compared with those of homologous proteinases from Rous sarcoma virus (RSV) and human immunodeficiency type 1 virus (HIV). Through comparison with the structure of a proteinase‐inhibitor complex from HIV, a model of a complex between MAV proteinase and a peptide substrate has been generated. Examination of this model suggests structural basis for the diverse specifications of viral proteinases. © 1992 Wiley‐Liss, Inc.
What is the pitch of the α‐helical coiled coil?
Authors: Phillips George N.
Published: May 2005

The α‐helical, coiled‐coil protein motif is increasingly recognized in a variety of functional classes of proteins. The pitch of a coiled coil, or rate of winding of the α‐helices around each other, is a key determinant of both intra‐ and intermolecular interactions. Experimental measurements of the pitch of parallel two‐stranded coiled coils of muscle proteins, and examination of the recently determined structure of another two‐stranded coiled coil, the GCN4 transcription factor protein, suggest that the pitch has an average value of about 140 Å. This value is consistent with the observed number of residues per turn in α‐helices of globular proteins, the determinant of the inter‐helical packing within the coiled‐coil motif. An understanding of the structural determinants of this value for the pitch and possible variations will be important in defining the interactions of coiled‐coil proteins with other macromolecules. © 1992 Wiley‐Liss, Inc.
Crystal structures of two mutants of adenylate kinase from Escherichia coli that modify the Gly‐loop
Authors: Müller Christoph W. , Schulz Georg E.
Published: May 2005

Two mutants of adenylate kinase from Escherichia coli have been crystallized and analyzed by X‐ray diffraction at resolutions of 3.4 and 2.4 Å, respectively. These mutants are Pro‐9→Leu and Gly‐10→Val. They were selected for their positions in the highly conserved Gly‐loop forming a giant anion hole for the β‐phosphate of ATP (GTP) in adenylate kinases, H‐ras‐p21, and other nucleotide‐binding proteins. Mutants at these positions of H‐ras‐p21 cause cancer. In adenylate kinase these mutations cause smallish changes at the active site. Relating the structural changes to the known changes in catalysis indicates that these mutants hinder the induced‐fit movements. As a side result we find that mutant Pro‐9→Leu and wild‐type form one very similar crystal packing contact that is crystallographic in one case and noncrystallographic in the other, while all other packing contacts and the space groups are quite at variance. © 1993 Wiley‐Liss, Inc.
The role of local tight packing of hydrophobic groups in β‐structure
Authors: Vtyurin Nickolie
Published: May 2005

An analysis of the tendency of hydrophobic groups to tight packing on the surface of β‐sheets based on well‐known parameters of β‐sheets and hydrophobic groups was conducted. This analysis shows the existence of very limited numbers and clearly outlined architecture families of regular parts for the majority of β‐structure‐containing domains. Each family of architecture strongly depends on the number of β‐strands in the pure β‐domains and on the existence and number of additional α‐helixes and on the mutual arrangements β‐strands and α‐helixes along the chain in mixed α/β‐domains. This paper demonstrates that the tendency of hydrophobic groups to the local tight packing on the surface of β‐sheets is probably the main reason for the twist of β‐sheets. © 1993 Wiley‐Liss, Inc.
Crystallographic analysis of Thr‐200 → His human carbonic anhydrase II and its complex with the substrate, HCO
Authors: Xue Yafeng , Vidgren Jukka , Svensson L. Anders , Liljas Anders , Jonsson Bengt‐Harald , Lindskog Sven
Published: May 2005

A complex of carbonic anhydrase (CA) with one of its substrates, bicarbonate, has been studied crystallographically. Human isoenzyme II was mutated at position 200 from threonine to histidine, which results in higher affinity for bicarbonate. The HCO ion binds in the active site to the zinc ion as a pseudo‐bidentate ligand which gives the metal a coordination geometry between tetrahedral and trigonal bipyramide. The water/hydroxide normally bound with tetrahedral coordination to the zinc is probably replaced by the OH group of the bicarbonate ion. The importance of residues Thr‐199 and Glu‐106 in controlling the binding orientation of HCO is discussed as well as the catalytic mechanism. Both the complex as well as the uncomplexed mutant were studied at 1.9 Å resolution. © 1993 Wiley‐Liss, Inc.
Preparation and initial characterization of crystals of the photoprotein aequorin from Aequorea victoria
Authors: Hannick Linda I. , Prasher Douglas C. , Schultz Leonard W. , Deschamps Jeffrey R. , Ward Keith B.
Published: May 2005

Crystals of recombinant aequorin, the photoprotein from the jellyfish Aequorea victoria, have been grown from solutions containing sodium phosphate. The crystals grow as thin plates which diffract to beyond 2.2 Å resolution. The crystals are orthorhombic, space group P21212 1; the axes are a = 89.1(1), b = 88.4(1), and c = 52.7(1) Å. The asymmetric unit contains two molecules. Crystals exposed to calcium ion solutions emit a steady glow and slowly deteriorate, confirming that the crystals consist of a charged, competent photoprotein. This represents the first successful preparation of single crystals of a photoprotein suitable for diffraction analysis. © 1993 Wiley‐Liss, Inc.
Crystallization and preliminary crystallographic analysis of ribonuclease H from Thermus thermophilus HB8
Authors: Okumura Mika , Ishikawa Kohki , Kanaya Shigenori , Itaya Mitsuhiro , Morikawa Kosuke
Published: May 2005

Ribonuclease H from an extreme thermophile, Thermus thermophilus HB8, has been crystallized from solutions at low ionic strength. The crystals belong to the hexagonal space group P6 122 (or P6 522), with unit cell parameters a = b = 44.7 Å, c = 314.7 Å. They contain one 18,000 Mr molecule per asymmetric unit and diffract to 2.8 Å resolution. © 1993 Wiley‐Liss, Inc.
Identification of critical contact residues in the NC41 epitope of a subtype N9 influenza virus neuraminidase
Authors: Nuss Jacqueline M. , Whitaker Patricia Bossart , Air Gillian M.
Published: May 2005

We have examined amino acids on influenza virus neuraminidase (NA) subtype N9 (A/tern/Australia/G70c/75) which are in contact with monoclonal antibody NC41 to analyze individual interactions important for antibody recognition. The crystal structure of NA complexed with NC41 Fab1 shows antibody contacts at 19 amino acid residues on the NA surface which are localized on five polypeptide loops surrounding the enzyme active site. Fifteen mutant NA genes were constructed to encode a protein which contained a single amino acid substitution and these were tested for effects of the replacement on NC41 binding. Our data revealed that NAs with changes at 368, 400, and 434 completely lost NC41 recognition. NAs with side chains replaced at residues 346 and 373 exhibited binding reduced to less than 50% of wild‐type binding. Changes in seven other contacting residues, including substituted side chains which differed considerably from wild‐type NA in size and charge, had no significant effect on NC41 binding. These results indicate that only a few of the many residues which make up an epitope are crucial for interaction and provide the critical contacts required for antibody recognition. This implies that antibody escape mutants are selected only if they contain changes at these crucial sites, or changes which introduce bulky side chains that sterically prevent antibody attachment. © 1993 Wiley‐Liss, Inc.
Metal poison inhibition of carbonic anhydrase
Authors: Lindahl Martin , Svensson L. Anders , Liljas Anders
Published: May 2005

Carbonic anhydrase is inhibited by the “metal poison” cyanide. Several spectroscopic investigations of carbonic anhydrase where the natural zinc ion has been replaced by cobalt have further strengthened the view that cyanide and cyanate bind directly to the metal. We have determined the structure of human carbonic anhydrase II inhibited by cyanide and cyanate, respectively, by X‐ray crystallography. It is shown that the inhibitors replace a molecule of water, which forms a hydrogen bond to the peptide nitrogen of Thr‐199 in the native structure. The coordination of the zinc ion is hereby left unaltered compared to the native crystal structure, so that the zinc coordinates three histidines and one molecule of water or hydroxyl ion in a tetrahedral fashion. The binding site of the two inhibitors is identical to what earlier has been suggested to be the position of the substrate (CO2) when attacked by the zinc bound hydroxyl ion. The peptide chain undergoes no significant alterations upon binding of either inhibitor. © 1993 Wiley‐Liss, Inc.
A geometrical constraint approach for reproducing the native backbone conformation of a protein
Authors: Saitoh Shin , Nakai Takahisa , Nishikawa Ken
Published: May 2005

It is known that the backbone conformation of a protein can be reproduced with precision once a correct contact map (two‐dimensional representation showing residue pairs in contact) is given as geometrical constraints. There is, however, no way to infer the correct contact map for a protein of unknown structure. We started with one‐dimensional constraints using the quantity N14 (the number of neighboring residues within the radius of 14 Å). Since the plot of N14 along a chain shows a good correlation with the corresponding amino acid sequence, the N14 profile obtained from the X‐ray structure is predictable from the sequence. Construction of backbone conformations under a given N14 profile was carried out in the following two steps: (1) a contact map from the N14 profile was produced by taking the product of N14 values of every two residues; (2) backbone conformations were generated by applying the distance geometry technique to distance constraints given by the contact map. If present, disulfide bonds in a protein, as well as the secondary structure, were treated as additional constraints, and both cases with or without the additional information were examined. The method was tested for 11 proteins of known structure, and the results indicated that the reproduced conformation was fairly good, using an X‐ray structure for comparison, for small proteins of less than 80 residues long. The basic assumption and effectiveness of the present method were compared with those of previous studies employing the geometrical constraint approach. It has become clear that the specific, one‐dimensional information (e.g., N14 profile) is more effective than nonspecific, two‐dimensional constraints, such as average interresidue distances between particular types of amino acids. © 1993 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray crystallographic analysis of arylesterase from Pseudomonas fluorescens
Authors: Kim Kyeong Kyu , Hwang Kwang Yeon , Choi Kang Duk , Kang Joo Hyun , Yoo Ook Joon , Suh Se Won
Published: May 2005

Large crystals of arylesterase from Pseudomonas fluorescens have been grown at room temperature using ammonium sulfate as a precipitant. They grow to dimensions of 0.7 × 0.7 × 0.6 mm3 within a month. The crystals belong to the trigonal space group P31 (or P32), with unit cell dimensions of a= 147.12 Å and c= 131.08 Å. The asymmetric unit seems to contain six molecules of dimeric aryles‐terase, with corresponding crystal volume per protein mass (VM) of 2.53 Å3/Da and solvent fraction of 51.5% by volume. The crystals diffract to at least 2.2 Å Bragg spacing when exposed to X‐rays from a rotating‐anode source. X‐ray data have been collected to 2.9 Å Bragg spacing from native crystals. © 1993 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray diffraction studies of peroxidase from a fungus Arthromyces ramosus
Authors: Kunishima Naoki , Fukuyama Keiichi , Wakabayashi Sadao , Sumida Motoo , Takaya Masamitsu , Shibano Yuji , Amachi Teruo , Matsubara Hiroshi
Published: May 2005

Peroxidase (donor: H2O2 oxi‐doreductase [EC 1.11.1.7]) was purified from the culture broth of the hyphomycete Arthromyces ramosus in the early log phase to show a single band on SDS‐PAGE. The crystals of A. ramosus peroxidase (ARP) were formed by salting out with ammonium sulfate at room temperature and pH 7.5. The repeated seeding technique was employed to grow the crystals to the size large enough for X‐ray diffraction study. The crystals were characterized as tetragonal, space group P42212, with unit cell dimensions of a = b = 74.5 Å, c = 117.6 Å. The asymmetric unit contains one molecule of peroxidase. They diffract X‐rays to at least 2.0 Å resolution and are stable to X‐rays. © 1993 Wiley‐Liss, Inc.
“Ensemble” iterative relaxation matrix approach: A new NMR refinement protocol applied to the solution structure of crambin
Authors: Bonvin Alexandre M. J. J. , Rullmann J. Antoon C. , Lamerichs Rolf M. J. N. , Boelens Rolf , Kaptein Robert
Published: May 2005

The structure in solution of crambin, a small protein of 46 residues, has been determined from 2D NMR data using an iterative relaxation matrix approach (IRMA) together with distance geometry, distance bound driven dynamics, molecular dynamics, and energy minimization. A new protocol based on an “ensemble” approach is proposed and compared to the more standard initial rate analysis approach and a “single structure” relaxation matrix approach. The effects of fast local motions are included and R‐factor calculations are performed on NOE build‐ups to describe the quality of agreement between theory and experiment. A new method for stereospecific assignment of prochiral groups, based on a comparison of theoretical and experimental NOE intensities, has been applied. The solution structure of crambin could be determined with a precision (rmsd from the average structure) of 0.7 Å on backbone atoms and 1.1 Å on all heavy atoms and is largely similar to the crystal structure with a small difference observed in the position of the side chain of Tyr‐29 which is determined in solution by both J‐coupling and NOE data. Regions of higher structural variability (suggesting higher mobility) are found hi the solution structure, in particular for the loop between the two helices (Gly‐20 to Pro‐22). © 1993 Wiley‐Liss, Inc.
Perturbation of Trp 138 in T4 lysozyme by mutations at Gln 105 used to correlate changes in structure, stability, solvation, and spectroscopic properties
Authors: Pjura Philip , McIntosh Lawrence P. , Wozniak Joan A. , Matthews Brian W.
Published: May 2005

In order to correlate between spectroscopic and structural changes in a protein, the environment of Trp 135 in T4 lysozyme was deliberately perturbed by the replacement of Gln 105 with alanine (Q105A), glycine (Q105G), and glutamic acid (Q105E). In wild‐type lysozyme, Trp 135 is buried, but the indole nitrogen is hydrogen‐bonded to the side‐chain of Gln 105. In the Q105G and Q105A mutant structures, the indole nitrogen becomes accessible to solvent. Crystallographic analysis shows that the structures of all of the mutants are similar to wild‐type. There are, however, distinct rearrangements of the local solvent structure in response to the new side‐chains. There are also small but significant changes in the relative orientations of the two domains of the protein that appear to result from a series of small, concerted movements of side‐chains adjacent to residue 105. Evaluation of the fluorescence and phosphorescence of the mutant proteins in terms of their observed three‐dimensional structures shows that large spectral changes do not necessarily imply large changes in structure or in static solvent accessibility. Increases in polar relaxation about the excited state of tryptophan may be the result of only small increases in local dynamics or solvent exposure. 1H‐NMR was also used to monitor the effects of the substitutions on Trp 138. In Q105E, but not in Q105G, Q105A and WT, the Hε1 chemical shift of Trp 138 is very pH‐dependent, apparently reflecting the titration of Glu 105 which has a spectroscopically determined pKa of 6.0. The elevation of the pKa of Glu 105 in Q105E is also reflected in the pH dependence of the stability of this mutant. © 1993 Wiley‐Liss, Inc.
Novel mechanism‐based substrates of dihydrofolate reductase and the thermodynamics of ligand binding: A comparison of theory and experiment for 8‐methylpterin and 6,8‐dimethylpterin
Authors: Cummins Peter L. , Gready Jill E.
Published: May 2005

Molecular dynamics simulation and free energy perturbation techniques have been used to study the relative binding free energies of the designed mechanism‐based pterins, 8‐methylpterin and 6,8‐dimethylpterin, to dihydrofolate reductase (DHFR), with co‐factor nicotinamide adenine dinucleotide phosphate (NADPH). The calculated free energy differences suggest that DHFR.NADPH.6,8‐dimethylpterin is thermodynamically more stable than DHFR.NADPH.8‐methylpterin by 2.4 kcal/mol when the substrates are protonated and by 1.3 kcal/mol when neutral. The greater binding strength of 6,8‐dimethylpterin may be attributed largely to hydration effects. In terms of an appropriate model for the pH‐dependent kinetic mechanism, these differences can be interpreted consistently with experimental data obtained from previous kinetic studies, i.e., 6,8‐dimethylpterin is a more efficient substrate of vertebrate DHFRs than 8‐methylpterin. The kinetic data suggest a value of 6.6 ± 0.2 for the pKa of the active site Glu‐30 in DHFR.NADPH. We have also used experimental data to estimate absolute values for thermodynamic dissociation constants of the active (i.e., protonated) forms of the substrates: these are of the same order as for the binding of folate (0.1–10 μM). The relative binding free energy calculated from the empirically derived dissociation constants for the protonated forms of 8‐methylpterin and 6,8‐dimethylpterin is 1.4 kcal/mol, a value which compares reasonably well with the theoretical value of 2.4 kcal/mol. © 1993 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray diffraction data of two heparin‐binding fragments of human fibronectin
Authors: Pechik Lgor , Nachman Joseph , Ingham Kenneth , Gilliland Gary L.
Published: May 2005

Two different heparin‐binding fragments of human fibronectin have been crystallized in forms which are suitable for crystal structure analyses. The 30 kDa hep‐2A fragment, consisting of type III domains 12–14, was crystallized from solutions containing ammonium sulfate or polyethylene glycol 6000. The crystals grown in ammonium sulfate solutions were orthorhombic with space group I222 or I212121 with a = 68.1 Å, b = 88.6 Å, and c = 144.9 Å. The crystals grown in polyethylene glycol solutions are hexagonal with space group P6122 or P6522 witha a = b = 66.7 Å and c = 245.7 Å. The 40 kDa hep‐2B fragment, consisting of type III domains 12–15, was also crystallized from solutions containing ammonium sulfate with the addition of glycerol. Glycerol proved an effective agent for reducing the number of crystals in the crystallization experiments, and thus, increasing the size of the crystals in these experiments. This crystal form is nearly isomorphous to the orthorhombic form of the hep‐2A fragment with space group I222 or I212121 and a = 67.5 Å, b = 87.0 Å, and c = 144.3 Å. All crystal forms diffract to at least 3.5 Å resolution and contain a single molecule in the asymmetric unit. © 1993 Wiley‐Liss, Inc.
Picosecond timescale rigid‐helix and side‐chain motions in deoxymyoglobin
Authors: Furois‐Corbin Sylvie , Smith Jeremy C. , Kneller Gerald R.
Published: May 2005

The contribution of rigidbody motions to the atomic trajectories in a 100 ps molecular dynamics simulation of deoxymyoglobin is examined. Two typesof rigid‐body motions are considered: one in which the helices are rigid units and one in which the side‐chains are rigid units. Using a quaternionbased algorithm, fits of the rigid reference structures are made to each time frame of the simulation to derive trajectories of the rigid‐body motions. The fitted trajectories are analysed in terms of atomic position fluctuations, mean‐square displacements as a function of time, velocity autocorrelation functions and densities of states. The results are compared with the corresponding quantities calculated from the full trajectory. The relative contribution of the rigid helix motions to the helix atom dynamics depends on which quantity is examined and on which subset of atoms is chosen: rigid‐helix motions contribute 86% of the rms helix backbone atomic position fluctuations, but 30% of the helix,: atom (backbone and side‐chain) mean square displacements and only 1.1% of total kinetic energy. Only very low‐frequency motions contribute to the rigid‐helix dynamics; the rigid‐body analysis allows characteristic rigid‐helix vibrations to be identified and described. Treating the side‐chains as rigid bodies is foundto be an excellent approximation to both their diffusive and vibrationalmean‐square displacements: 96% of side‐chain atom mean‐square displacements originate from rigid side‐Chain motions. However, the errors in theside‐chain atomic positional fits are not always small. An analysis is madeof factors contributing to the positional error for different types of side‐chain. © Wiley‐Liss, Inc.
Calcium‐independent subtilisin by design
Authors: Gallagher Travis , Bryan Philip , Gilliland Gary L.
Published: May 2005

A version of subtilisin BPN′ lacking the high affinity calcium site (site A) has been produced through genetic engineering methods, and its crystal structure refined at 1.8 Å resolution. This protein and the corresponding version containing the calcium A site are describedand compared. The deletion of residues 75–83 was made in the context of four site‐specific replacements previously shown to stabilize subtilisin. The helix that in wild type is interrupted by the calcium binding loop, is continuous in the deletion mutant, with normal geometry. A few residues adjacent to the loop, principally those that were involved in calcium coordination, are repositioned and/or destabilized by the deletion. Because refolding is greatly facilitated by the absence of the Caloop, this proteinoffers a new vehicle for analysis and dissection of the folding reaction. This is among the largest internal changes to a protein to be described at atomic resolution. © Wiley‐Liss, Inc.
Improved calculations of compactness and a reevaluation of continuous compact units
Authors: Zehfus Micheal H.
Published: May 2005

A new method for calculating compactness (Z) that uses look‐up table‐based algorithms for area and volume computations is introduced. These algorithms can be used in any iterative area orvolume calculation, are more than 1000 times faster than the originalalgorithms, and have equal or better precision. With the faster algorithms it is now possible to calculate the compactness of all continuous units in a protein, and to precisely locate the optimal compact units without the screening functions and limited resolution used previously. These methods have been incorporated into a fully automatic domain finding algorithm, and this method has been applied to the 21 proteins originally analyzed as well as 12 additional proteins. This method is robust, and yields similar units even when applied to coordinates of protein crystals grown under different experimental conditions. © 1993 Wiley‐Liss, Inc.
The structure of a centrosymmetric protein crystal
Authors: Zawadzke Laura E. , Berg Jeremy M.
Published: May 2005

Crystals of racemic rubredoxin, prepared by independent chemical synthesis of the two enantiomers, have been grown and characterized. The unit cell contains two molecules, one of each enantiomer. Examination of the intensity distribution in the diffraction pattern revealed that the crystals are centrosymmetric. This was confirmed by solution of thestructure to 2 Å resolution via molecular replacement methods. The electron density maps are of very high quality due to the fact that the phaseof each reflection must be exactly 0° or exactly 180°. These results demonstrate the feasibility of using synthetic racemic proteins to yield centrosymmetric protein crystals with electron density maps that have very low phase error and model bias. © 1993 Wiley‐Liss, Inc.
Mutational tests of the NMR‐docked structure of the staphylococcal nuclease–metal–3′,5′‐pdTp complex
Authors: Chuang Woei‐Jer , Weber David J. , Gittis Apostolos G. , Mildvan Albert S.
Published: May 2005

In the X‐ray structure of the staphylococcal nuclease–Ca2+ −3′,5′‐pdTp complex, the conformation of the inhibitor 3′,5′‐pdTp is distroteed Lys‐70* and Lys‐71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5 : 183‐201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme‐bound 3′,5′‐pdTp in the staphylococcal nuclease–metal–pdTp Complex determined by NMR methods was docked into the X‐ray structure of the enzyme [Weber, D. J., Serpersu, E. H., Gittis, A. G., Lattman, E. E., Mildvan, A. S. (preceding paper)]. In the NMR‐docked structure, the 5′‐phophate of 3′,5′‐pdTp overlaps with that in the X‐ray Structure. However the 3′‐phosphate accepts a hydrogen bond from Lys‐49 (2.89Å) rather than from Lys‐84 (8.63 Å), and N3 of thymine donates a hydrogen bond to the OH of Tyr‐115 (3.16 Å) which does not occur in the X‐ray structure (5.28 Å). These interactions have been tested by binding studies of 3′,5′‐pdTp, Ca2+, and Mn2+ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two‐dimensional NMR spectroscopy, but bound Ca2+ 9.1‐ to 9.9‐fold more weakly than the wild‐type enzyme. While thye K84A mutation did not significantly weaken 3′,5′‐pdTp binding to the enzyme (1.5 ± 0.7 fold), the K49A mutation weakened 3′,5′‐pdTp binding to the enzyme by the factor of 4.4 ± 1.8‐fold. Similarly, the Y115A mutation weakened 3′,5′‐pdTp binding to the enzyme 3.6 ± 1.6‐fold. Comparable weakening effects of these mutations were found on the binding of Ca2+‐3′,5′‐pdTp. These results are more readily explained by the NMR‐docked structure of staphylococcal nuclease‐metal‐3′,5′‐pdTp than by the X‐ray structure. © 1993 Wiley‐Liss, Inc.
Structural relationships of homologous proteins as a fundamental principle in homology modeling
Authors: Hilbert Martina , Böhm Gerald , Jaenicke Rainer
Published: May 2005

Protein structure prediction is based mainly on the modeling of proteins by homology to known structures; this knowledgebased approach is the most promising method to date. Although it is used in the whole area of protein research, no general rules concerning the quality and applicability of concepts and procedures used in homology modeling have been put forward yet. Therefore, the main goal of the present work is to provide tools for the assessment of accuracy of modeling at a given level of sequence homology. A large set of known structures from different conformational and functional classes, but various degrees of homology was selected. Pairwise structure superpositions were performed. Starting with the definition of the structurally conserved regions and determination of topologically correct sequence alignments, we correlated geometrical properties with sequence homology (defined by the 250 PAM Dayhoff Matrix) and identity. It is shown that both the topological differences of the protein backbones and the relative positions of corresponding side chains diverge with decreasing sequence identity. Below 50% identity, the deviation in regions that are structurally not conserved continually increases, thus implying that with decreasing sequence identity modeling has to take into account more and more structurally diverging loop regions that are difficult to predict. © 1993 Wiley‐Liss, Inc.
Influence of protein flexibility on the redox potential of rubredoxin: Energy minimization studies
Authors: Shenoy Vaishali S. , Ichiye Toshiko
Published: May 2005

A theoretical investigation of the protein contribution to the redox potential of the iron–sulfur protein rubredoxin is presented. Structures of the oxidized and reduced forms of the protein were obtained by energy minimizing the oxidized crystal structure of Clostridium pasteurianum rubredoxin with appropriate charges and parameters. By including 102 crystal waters, structures close to the original crystal structure were obtained (rms difference of 1.16 Å), even with extensive minimization, thus allowing accurate calculations of comparative energies. Our calculations indicate an energy change of about –60 kcal/mol (2.58 eV) in the protein alone upon reduction. This energy change was due to both the change in charge of the redox site and the subsequent relaxation of the protein. An energy minimization procedure for the relaxation gives rms differences between the oxidized and reduced states of about 0.2 Å. The changes were small and occurred in both the backbone and sidechain mainly near the Fe–S center but contributed about – 16 kcal/mol (0.69 eV) to the total protein contribution. Although the neglect of certain effects such as electronic polarization may make the relaxation energies calculated an upper limit, the results indicate that protein relaxation contributes substantially to the redox potential. © 1993 Wiley‐Liss, Inc.
Molecular skins: A new concept for quantitative shape matching of a protein with its small molecule mimics
Authors: Masek Brian B. , Merchant Arshad , Matthew James B.
Published: May 2005

A novel analytical method for comparing molecular shapes by optimizing the intersection of molecular “SKINS” has been developed. This method provides a quantitative measure of the shape similarity by maximizing the intersection volume of molecular surfaces with a finite thickness; a molecular skin. We report shape matching of a small tripeptide inhibitor (DFKi) of elastase class proteins with the 56 residue turkey ovomucoid inhibitor (TOMI). To match a large elastase inhibitor such as TOMI with a small inhibitor or drug, we found that it is necessary to use a skin match rather than molecular volume. Skin based comparisons of TOMI protein with DFKi successfully found the alignment expected from comparison of their respective crystallographic complexes with elastase (i.e. HLE/TOMI complex and PPE/tripeptide complex). In the skin comparison of the tripeptide with the TOMI protein, blind searching for skin matches involved optimization of the skin intersection from 172 starting positions randomly selected from a set of 500 points on the TOMI van der Waals surface [within 9.5 Å of the Leu‐18 on the TOMI binding loop (1 point/Å2)]. The tripeptide center of mass was placed at these points and its orientation was randomized before optimization was initiated. The best skin intersection, 86.4 Å3, was found thre times and corresponds to the experimental alignment. The next best skin intersection was 78.1 Å3 giving a discrimination factor in this case of 10%. Searches over the entire surface of the TOMI protein did not identify any new matches with skin intersection greater than 78.1 Å3. Matching the DFKi with a TOMI structure relaxed from its crystal conformation by molecular dynamics gives similar results. © 1993 Wiley‐Liss, Inc.
A reduced representation of proteins for use in restraint satisfaction calculations
Authors: Herzyk Pawel , Hubbard Rodrick E.
Published: May 2005

A reduced representation of paroteins has been developed for use in restraint satisfaction calculations with dynamic simulated annealing. Each amino acid residue is represented by up to four spherical virtual atoms. The virtual bonds and excluded volume of these atoms has ben parameterized by analysis of 83 protein structures determined at high resolution by X‐ray crystallography. The use of the new representation in NOE distance restraint satisfaction has been compared with the standard all‐atom represntation for the determination of the structures of crambin, eshistatin, and protein G. Using the reduced representation, there is a 30‐fold decrease in the computer time needed for generatin a single structure, and up to a 20‐fold decrease in the time taken to produce an acceptable structure compared to using the all‐atom representation. The root mean square deviation between the mean structure obtain with all‐atom and reduced representation si between 1.5 and 1.7 Å for Cα atoms. The new representation is adequate for describing the “low‐resolution” features of protein structure such as the general fold and the positions of the secondary structure for more detailed refinement with the full all‐atom representation. © 1993 Wiley‐Liss, Inc.
Growth and analysis of crystal forms of toxic shock syndrome toxin 1
Authors: Earhart Cathleen A. , Prasad G. Sridhar , Murray Debra L. , Novick Richard P. , Schlievert Patrick M. , Ohlendorf Douglas H.
Published: May 2005

Native toxic shock syndrom toxin 1 (TSST‐1) purified from Staphylococcus aurius has been crystallized in four different forms. The highest resolution data (2.05 Å) was collected from orthorhombic crystals belonging to the space group C2221. The unit cell dimension are a = 108.7 Å, b = 177.5 Å, c = 97.6 Å. Rotation function analysis of this from indicates that there is trimer of toxin molecules in the asymmetric unit with a local 3‐fold axis parallel to the crystallographic c axis. Crystals of a double mutant of TSST‐1 have been grown which has a single molecule in the asymmetric unit and diffract to 1.9 Å. The space group is P21 with unit cell parameters of a = 44.4 Å, b = 34.0 Å, c = 55.2 Å, β = 93.0°. © 1993 Wiley‐Liss, Inc.
Orientation and rotational dynamics of spin‐labeled phalloidin bound to actin in muscle fibers
Authors: Naber Nariman , Ostap E. Michael , Thomas David D. , Cooke Roger
Published: May 2005

We have used electron paramagnetic resonance spectroscopy (EPR) to investigate the orientational distribution of actin in thin filaments of glycerinated muscle fibers in rigor, relaxation, and contraction. A spin‐labeled derivative of a mushroom toxin, phalloidin (PHSL), was bound to actin in the muscle fibers (PHSL–fibers). The EPR spectrum of unoriented PHSL–labeled myofibrils consisted of three sharp lines with a splitting between the outer extrema (2T) of 42.8 ± 0.1 G, indicating that the spin labels undergo restricted nanosecond rotational motion within an estimated halfcone angle of 76°. When the PHSL–fiber bundle was oriented parallel to the magnetic field, the splitting between the zero‐crossing points (2T′) was 42.7 ± 0.1 G. When the fiber bundle was perpendicular to the magnetic field, 2T′ decreased to 34.5 ± 0.2 G. This anisotropy shows that the motion of the probe is restricted in orientation by its binding site on actin, so that the EPR spectrum of PHSL–fiber bundles would be sensitive to small changes in the mean axial orientation of the PHSL–actin interface. No differences in the EPR spectra were observed in fibers during rigor, relaxation, or contraction, indicating that the mean axial orientation of the PHSL binding site changes by less than 5°, and that the amplitude of nanosecond probe rotational motion, which should be quite sensitive to the local environment of the phalloidin, changes by no more than 1°. These results rule out large changes in the overall geometry of the actin filament and in the local conformation of actin near the phalloidin binding site during the generation of isometric tension in muscle fibers. © 1993 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray analysis of nonglycosylated tissue inhibitor of metalloproteinases‐1, N30QN78Q TIMP‐1
Authors: Tolley Shirley P. , Davies Gideon J. , O'Shea Mark , Cockett Mark I. , Docherty Andrew J. P. , Murphy Gillian
Published: May 2005

A nonglycosylated (N30QN78Q) form of the human tissue inhibitor of metalloproteinases, TIMP‐1, has been prepared and crystallized in a form suitable for X‐ray diffraction analysis. Small single crystals have been grown using sodium tartrate as a precipitant. The crystals are in space group P21, with cell dimensions a = 35.28, b = 53.95, c = 48.56, and β = 96.0°. There is a single molecule of TIMP‐1 in the asymmetric unit. The crystals diffract to at least 2.3 Å resolution. Complete data have been collected to 2.9 Å and a search for heavymetal derivatives is in progress. © 1993 Wiley‐Liss, Inc.