bluuebunny/crossref_metadata_2025 huggingface dataset:
Gaussian neighborhood: A new measure of accessibility for residues of protein molecules
Authors: Nauchitel Vladimir V. , Somorjai Rajmund L.
Published: May 2005

We introduce a new method for assessing the extent of residue exposure in proteins. For each atom of every residue a Gaussian‐weighted atomic surroundings value (the G‐neighborhood) is calculated. A normalized sum of G‐neighborhood values over all the atoms of a residue is complementary to conventional surface accessibility characteristics. The G‐0neighborhood value of a residue is a sensitive indicator of its location, strongly dependent on the 3D structure of a the protein. Correlations between secondary structures and patterns of G‐neighborhood values for six different protein molecules are discussed. Comparison of the distribution of hydrophobic and charged residues in the 3D structure for the alcohol‐soluble protein crambin and that of five water‐soluble proteins (cytochrome c, flavodoxin, myoglobin, rhodanese, and Bence–Jones protein) shows striking differences in their G‐neighborhood patterns. Contacts between the prosthetic group and the peptide portion of a protein as well as protein interdomain contacts and monomer–monomer contacts are characterized. © 1993 Wiley‐Liss, Inc.
A novel parameterization scheme for energy equations and its use to calculate the structure of protein molecules
Authors: Snow Mark E.
Published: May 2005

A novel scheme for the parameterization of a type of “potential energy” function for protein molecules is introduced. The function is parameterized based on the known conformations of previously determined protein structures and their sequence similarity to a molecule whose conformation is to be calculated. Once parameterized, minima of the potential energy function can be located using a version of simulated annealing which has been previously shown to locate global and near‐global minima with the given functional form. As a test problem, the potential was parameterized based on the known structures of the rubredoxins from Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Clostridium pasteurianum, which vary from 45 to 54 amino acids in length, and the sequence alignments of these molecules with the rubredoxin sequence from Desulfovibrio gigas. Since the Desulfovibrio gigas rubredeoxin conformation has also been determined, it is possible to check the accuracy of the results. Ten simulated‐annealing runs from random starting conformations were performed. Seven of the 10 resultant conformations have an all‐Cα rms deviation from the crystallographically determined conformation of less than 1.7 Å. For five of the structures, the rms deviation is less than 0.8 Å. Four of the structures have conformations which are virtually identical to each other except for the position of the carboxy‐terminal residue. This is also the conformation which is achieved if the determined crystal structure is minimized with the same potential. The all‐Cα rms difference between the crystal and minimized crystal structures is 0.6 Å. It is further observed that the “energies” of the structures according to the potential function exhibit a strong correlation with rms deviation from the native structure. The conformations of the individual model structures and the computational aspects of the modeling procedure are discussed. © 1993 Wiley‐Liss, Inc.
Crystallization, preliminary X‐ray diffraction study, and crystal packing of a complex between anti‐Hen lysozyme antibody F9.13.7 and Guinea‐fowl lysozyme
Authors: Lescar Julien , Riottot Marie‐Madeleine , Souchon Hélène , Chitarra Véronique , Bentley Graham A. , Navaza Jorge , Alzari Pedro M. , Poljak Roberto J.
Published: May 2005

The complex formed between the Fab fragment of a murine monoclonal anti‐hen egg lysozyme antibody F9.13.7 and the het‐erologous antigen Guinea‐fowl egg lysozyme has been crystallized by the hanging drop technique. The crystals, which diffract X‐rays to 3 Å resolution, belong to the monoclinic space groupP21, witha= 83.7 Å,b= 195.5 Å,c= 50.2 Å, β = 108.5° and have two molecules of the complex in the asymmetric unit The three‐dimensional structure has been determined from a preliminary data set to 4 Å using molecular replacement techniques. The lysozyme–Fab complexes are arranged with their long molecular axes approximately parallel to the crystallo‐graphic unique axis. Fab F9.13.7 binds an anti‐genie determinant that partially overlaps the epitope recognized by antilysozyme antibody HyHEL10. © 1993 Wiley‐Liss, Inc.
Site‐specific proteolytic cleavage of Ku protein bound to DNA
Authors: Paillard Sophie , Strauss François
Published: May 2005

Ku protein, a relatively abundant nuclear protein associated with DNA of mammalian cells, is known to be a heterodimer with subunits of 85 and 72 kDa which binds in vitro to DNA ends and subsequently translocates along the molecule. The functional role played by this protein in the cell, however, remains to be elucidated. We have observed here that Ku protein, purified from cultured monkey cells, is the target of specific endoproteolysis in vitro, by which the 85 kDa subunit is cleaved at a precise site while the 72 kDa subunit remains intact. This cleavage releases an 18 kDa polypeptide and converts Ku protein into a heterodimer composed of the 72 kDa subunit associated with a 69 kDa fragment from the 85 kDa subunit. The proteolyzed form of Ku protein, denoted Ku′, has DNA binding properties similar to those of Ku protein. The proteolytic mechanism, which is inhibited by leupeptin and chymostatin, is extremely sensitive to ionic conditions, in particular to pH, being very active at pH 7.0 and completely inhibited at pH 8.0. In addition, cleavage occurs only when Ku protein is bound to DNA, not free in solution. We suggest that in vivo, such proteolysis might be necessary for Ku protein function at some stage of the cell cycle. © 1993 Wiley‐Liss, Inc.
Length distribution of CDRH3 in antibodies
Authors: Wu Tai Te , Johnson George , Kabat Elvin A.
Published: May 2005

Sequences of the third complementarity determining region of antibody heavy chains (CDRH3s) are listed according to their length. Human sequences vary from 2 to 26 amino acids residues, but less extensively in other species. When combined with the other five complementarity determining regions, this enormous length variation of CDRH3, together with amino acid substitutions in their sequences, can provide a very large number of antibody specificities and can influence the shape of antibody combining sites.©1993 Wiley‐Liss,Inc.
Computer modeling and folding of four‐helix bundles
Authors: Rey Antonio , Skolnick Jeffrey
Published: May 2005

In the context of simplified models of globular proteins, the requirements for the unique folding to a four‐helix bundle have been addressed through a new Monte Carlo procedure. In particular, the relative importance of secondary versus tertiary interactions in determining the nature of the folded structure is examined. Various cases spanning the extremes where tertiary interactions completely dominate to that where tertiary interactions are negligible have been explored. Not surprisingly, the folding to unique four‐helix bundles is found to depend on an adequate balance of the secondary and tertiary interactions. Moreover, because the simplified model is composed of spheres representing α‐carbons and side chains, the geometry of the latter being based on small real amino acids, the role played by the side chains, and the problems associated with packing and hard‐core repulsions, are considered. Also, possible folding intermediates and their relationship with the experimentally observed molten globule state are explored. From these studies, a general set of rules is extracted which should aid in the further design of more detailed protein models adequate to more fully investigate the protein folding problem. Finally, the relationship between our conclusions and experimental work with specifically designed sequences is briefly discussed. © 1993 Wiley‐Liss, Inc.
A vector projection approach to predicting HIV protease cleavage sites in proteins
Authors: Chou Kuo‐Chen , Zhang Chun‐Ting , Kézdy Ferenc J
Published: May 2005

A vector projection method is proposed to predict the cleavability of oligopeptides by extended‐specificity site proteases. For an enzyme with eight specificity subsites the substrate octapeptide can be uniquely expressed as a vector in an 8‐dimensional space, whose eight bases correspond to the amino acids at the eight subsites, P1, P1′, P2′, P3′ and P4′, respectively. The component of such a characteristic vector on each of the eight bases is defined as the frequency of an amino acid occurring at a given site. These frequencies were derived from a set of octapeptides known to be cleaved by HIV protease. The cleavability of an octapeptide can then be estimated from the projection of its characteristic vector on an idealized, optimally cleavable vector. The high ratio of correct prediction vs. total prediction for the data in both the training and the testing sets indicates that the new method is self‐consistent and efficient. It provides a rapid and accurate algorithm for analyzing the specificity of any multisubsite enzyme for which there is no coupling between subsites. In particular, it is useful for predicting the cleavability of an oligopeptide by either HIV‐1 or HIV‐2 protease, and hence offers a supplementary means for finding effective inhibitors of HIV protease as potential drugs against AIDS. © Wiley‐Liss, Inc.
A computer model of the interleukin‐4/receptor complex
Authors: Bamborough Paul , Grant Guy H. , Hedgecock Charles J. R. , West Susan P. , Richards W. Graham
Published: May 2005

Interleukin‐4 is a member of the cytokine family, a group of related messenger proteins which collectively help to moderate and control the immune response. It is believed that the folding topology of the β‐sheets of the interleukin‐4 receptor (IL4R) is the same as that seen in the crystal structure of CD4. Although the sequence identity is low, homology modeling techniques have been used to model the IL4R structure from CD4. Refinement by molecular dynamics leads to a suggested structure which has been docked to interleukin‐4 (IL4). Several residues of apparent importance for binding are identified. © 1993 Wiley‐Liss, Inc.
A homology model of human interferon α‐2
Authors: Murgolo Nicholas J. , Windsor William T. , Hruza Alan , Reichert Paul , Tsarbopoulos Anthony , Baldwin Samuel , Huang Eric , Pramanik Birendra , Ealick Steven , Trotta Paul P.
Published: May 2005

An atomic coordinate five α‐helix three‐dimensional model is presented for human interferon α‐2 (HuIFNα2). The HuIFNα2 structure was constructed from murine interferon β (MuIFNβ) by homology modeling using the STEREO and IMPACT programs. The HuIFNα2 model is consistent with its known biochemical and biophysical properties including epitope mapping. Lysine residues predicted to be buried in the model were primarily unreactive with succinimidyl‐7‐amino‐4‐methylcoumarin‐3‐acetic acid (AMCA‐NHS), a lysine modification agent, as shown by mass spectrometric analysis of tryptic digests. N‐terminal sequence analysis of polypeptides generated by limited digestion of HuIFNα2 with endoproteinase Lys‐C demonstrated rapid cleavage at K31, which is consistent with the presence of this residue in a loop in the proposed HuIFNα2 model. Based on this model structure potential receptor binding sites are identified. © 1993 Wiley‐Liss, Inc.
Structural analysis of the zinc hydroxide–Thr‐199–Glu‐106 hydrogen‐bond network in human carbonic anhydrase II
Authors: Xue Yafeng , Liljas Anders , Jonsson Bengt‐Harald , Lindskog Sven
Published: May 2005

The singnificance of the zinc hydroxide–Thr‐199–Glu‐106 hydrogen‐bond network in the active site of human carbonic anhydrase II has been examined by X‐ray crystallographic analyses of site‐specific mutants. Mutants with Ala‐199 and Ala‐106 or Gln‐106 have low catalytic activities, while a mutant with Asp‐106 has almost full CO2 hydration activity. The structures of these four mutants, as well as that of the bicarbonate complex of the mutant with Ala‐199, have been determined at 1.7 to 2.2 Å resolution. Removal of the γ atoms of residue 199 leads to distorted tetrahedral geometry at the zine ion, and a catalytically important zinc‐bound water molecule has moved towards Glu‐106. In the bicarbonate complex of the mutant with Ala‐199 one oxygen atom from bicarbonate binds to zinc without displacing this water molecule. Tetrahedral coordination geometries are retained in the mutants at position 106. The mutants with Ala‐106 and Gln‐106 have a zinc‐bound sulfate ion, whereas this sulfate site is only partially occupied in the mutant with Asp‐106. The hydrogen‐bond network seems to be “reversed” in the mutants with Ala‐106 and Gln‐106. The network is preserved as in native enzyme in the mutant with Asp‐106 but the side chain of Asp‐106 is more extended than that of Glu‐106 in the native enzyme. These results illustrate the importance of Glu‐106 and Thr‐199 for controlling the precise coordination geometry of the zinc ion and its ligand preferences with results in an optimal orientation of a zine‐bound hydroxide ion for an attack on the CO2 substrate. © 1993 Wiley‐Liss, Inc.
Crystal structure of TGF‐β2 refined at 1.8 Å resolution
Authors: Daopin Sun , Li Mi , Davies David R.
Published: May 2005

The crystal structure of TGF‐β2 has been refined using data collected with synchrotron radiation (CHESS) to 1.8 Å resolution with a residual R (= ∑ | |Fo| − |Fc| | /∑ |Fo|) factor of 17.3%. The model consists of 890 protein atoms from all 112 residues and 59 water molecules. The monomer of TGF‐β2 assumes a rather extended conformation and lacks a well‐defined hydrophobic core. Surface accessibility calculations show only 44% of the nonpolar surface is buried in the monomer. In contrast, 55.8% of the nonpolar surface area is buried when the two monomers from a dimer, a typical value for globular proteins. This includes a 1300 Å2 buried interface area that is largely hydrophobic. Sequence comparisons using a profile derived from the refined TGF‐β2 structure suggest that the cluster of four disulfides (three intramonomeric disulfide bonds 15–78, 44–109, 48–111 forming a disulfide knot, and one intermonomeric disulfide 77–77) together with the extended β strand region constitutes the conserved structural motif for the TGF‐β superfamily. This structural motif, without the 77–77 disulfide bond, defines also the common fold for a general family of growth factors, including the nerve growth factor and platelet‐derived growth factor families. The fold is conserved only at the monomer level, while the active forms are dimers, suggesting that dimerization plays an important role in regulating the binding of these cytokines to their receptors and in modulating the biological responses. © 1993 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Crystallographic studies and primary structure of the antitumor monoclonal CC49 Fab′
Authors: Abergel Chantal , Padlan Eduardo A. , Kashmiri Syed V. S. , Milenic Diane , Calvo Benjamin , Schlom Jeffrey
Published: May 2005

The Fab′ of CC49, a murine monoclonal antibody directed against the human tumor‐associated antigen TAG‐72 has been crystallized. The crystals are monoclinic, space group P21 with cell parameters a = 115.6 Å, b = 116.4 Å, and c = 70.3 Å; β = 97.8°. The size of the unit cell is compatible with four Fab′ molecules in the asymmetric unit. The Fab molecules are related by two approximately perpendicular pseudo‐2‐fold axes. One pseudo‐2‐fold axis is parallel to the crystallographic 2‐fold axis and was found by inspection of the Harker section of the native Patterson map; the other was found by a self rotation function. The primary structures of the variable regions of the CC49 antibody light and heavy chains have been determined and are compared with those of the related antitumor antibody B72.3. © 1993 Wiley‐Liss, Inc.
The enzymatic mechanism of carboxypeptidase: A molecular dynamics study
Authors: Banci Lucia , Bertini Ivano , La Penna Giovanni
Published: May 2005

An MD simulation of the system carboxypeptidase A (CPA) with the tetrapeptide Val‐Leu‐Phe‐Phe has been performed in order to learn about the substrate disposition just prior to nucleophilic attack. We have explored the model in which the substrate does not substitute the zinc‐coordinated water (the “water” mechanism). The simulations do suggest as feasible that the Zn‐OH2 group performs a nucleophilic attack on the Phe‐Phe peptidic bond. We have also investigated the model in which the carbonyl oxygen displaces the zinc‐coordinated water. In this case the substrate and Glu‐270 orient themselves to allow an anhydride intermediate during the peptidic bond cleavage (the “anhydride” mechanism). Based on the results of the simulations, both “water” and “anhydride” mechanisms are structurally feasible, although the former model seems more probable on chemical grounds. © 1994 John Wiley & Sons, Inc.
Solution structure of a core peptide derived from scyllatoxin
Authors: Pagel Mark D. , Wemmer David E.
Published: May 2005

An analysis of the sequences of scyllatoxin and charybdotoxin suggested that it would be possible to design a core peptide sequence which would still fold to give the β‐hairpin and helix seen in the toxins, but which would eliminate one disulfide and connecting residues. The core sequence was modeled, then synthesized and purified. The cysteines oxidize in air to give the same disulfide pairings as seen in the parent toxins as the major product. The three‐dimensional structure of the core sequence peptide, termed Max, was determined using proton NMR spectroscopy and found to be identical in secondary structure to the toxins. However differences were found in the relative orientation of the β‐hairpin and helix. The use of this structural motif, found in many insect toxins, as a disulfide framework for exploring sequence/structure/activity relationships is discussed. © 1994 John Wiley & Sons, Inc.
A simplified amino acid potential for use in structure predictions of proteins
Authors: Wallqvist A. , Ullner M.
Published: May 2005

ABSTRACT A simplified description and a corresponding force field for polypeptides is introduced. Each amino acid residue is reduced to one interaction site, representing the backbone, and one or two side chain sites depending on its size and complexity. Site–site interactions are parameterized after a hydrophobicity criterium. The treatment of backbone sites is in addition designed to reproduce typical polypeptide hydrogen bonding patterns, as well as yielding conformations in accord with the allowed ϕ and ψ angles through an effective angle potential. There are no explicit charges in the model. The derived energy functions, which are based on thermodynamic data and sterical consideration of allowed backbone conformations, correspond to the introduction of an effective potential. The model is tested on two small proteins, avian pancreatic polypeptide and a parathyroid hormone‐related protein, by simulating folding from an initially extended state using Monte Carlo methods. The reduced amino acid description is able to satisfactorily reproduce the experimentally determined native structures. © 1994 John Wiley & Sons, Inc.
Monte carlo simulations of protein folding. II. Application to protein A, ROP, and crambin
Authors: Kolinski Andrzej , Skolnick Jeffrey
Published: May 2005

The hierarchy of lattice Monte Carlo models described in the accompanying paper (Kolinski, A., Skolnick, J. Monte Carlo simulations of protein folding. I. Lattice model and interaction scheme. Proteins 18:338–352, 1994) is applied to the simulation of protein folding and the prediction of 3‐dimensional structure. Using sequence information alone, three proteins have been successfully folded: the B domain of staphylococcal protein A, a 120 residue, monomeric version of ROP dimer, and crambin. Starting from a random expanded conformation, the model proteins fold along relatively well‐defined folding pathways. These involve a collection of early intermediates, which are followed by the final (and rate‐determining) transition from compact intermediates closely resembling the molten globule state to the native‐like state. The predicted structures are rather unique, with native‐like packing of the side chains. The accuracy of the predicted native conformations is better than those obtained in previous folding simulations. The best (but by no means atypical) folds of protein A have a coordinate rms of 2.25 Å from the native Cα trace, and the best coordinate rms from crambin is 3.18 Å. For ROP monomer, the lowest coordinate rms from equivalent Cαs of ROP dimer is 3.65 Å. Thus, for two simple helical proteins and a small α/β protein, the ability to predict protein structure from sequence has been demonstrated. © 1994 John Wiley & Sons, Inc.
Lobster enolase crystallized by serendipity
Authors: Duquerroy S. , Le Bras G. , Janin J.
Published: May 2005

An unknown protein crystallized from a lobster muscle preparation in which arginine kinase was the majority component. It was identified as enolase by peptide sequencing and activity testing, and a SIRAS electron density map showed its three‐dimensional structure to be very similar to that of yeast enolase. © 1994 John Wiley & Sons, Inc.
Preliminary x‐ray analysis of Escherichia coli GMP synthetase: Determination of anomalous scattering factors for a cysteinyl mercury derivative
Authors: Tesmer John J. G. , Stemmler Tim L. , Penner‐Hahn James E. , Jo Davisson V. , Smith Janet L.
Published: May 2005

We have initiated a project to determine the three‐dimensional structure of GMP synthetase (GMPS) from Escherichia coli. GMPS catalyzes the conversion of XMP to GMP in the final step of de novo guanine nucleotide biosynthesis, and is a member of the glutamine amidotransferase family: a group of enzymes responsible for the assimilation of nitrogen into compounds such as amino acids, purine and pyrimidine bases, amino sugars, and antibiotics. The E. coli guaA gene encoding GMPS was cloned into a tac expression vector, overexpressed, and its gene product purified. Conditions for the growth of protein crystals were developed using recombinant GMPS in the presence of MgCl2, ATP, and XMP. The crystals are monoclinic, space group P21, with cell parameters of a = 156.0 Å, b = 102.0 Å, c = 78.8 Å, β= 96.7°. Diffraction data to 2.8 Å spacings were collected on a Xuong‐Hamlin area detector with an overall Rsym of 5.2%. Both the volume of the unit cell and the peaks in the self‐rotation function are consistent with one GMPS tetramer of D2 symmetry in the crystallographic asymmetric unit. Previously, GMPS has been observed only as a dimer in solution. GMPS was covalently modified with p‐chloromercuribenzylsulfonic acid (PCMBS), and its X‐ray fluorescence spectrum was measured through the LIII absorption edge of mercury Anomalous scattering factors for cysteinyl mercury were derived from this spectrum, and the feasibility of structure determination by multi‐wavelength anomalous diffraction was evaluated. The optimal MAD dispersive signal is 4.5% of |F|, and the optimal MAD Bijvoet signal is 7.5% of |F| at a concentration of approximately 1 mercury per 10‐kDa protein. The anomalous scattering factors tabulated here should be transferable to cysteinyl mercury in other proteins. © 1994 John Wiley & Sons, Inc.
Expression, purification, and crystallization of restriction endonuclease PvuII with DNA containing its recognition site
Authors: Balendiran K. , Bonventre Joseph , Knott Roger , Jack William , Benner Jack , Schildkraut Ira , Anderson John E.
Published: May 2005

We have overexpressed the type II restriction endonuclease PvuII (R.PvuII) in E. coli, prepared large amounts of the homogeneous enzyme, and crystallized it with an oligonucleotide carrying a PvuII recognition site. The cocrystals are orthorhombic space group P212121 with cell constants a = 95.8 Å, b = 86.3 Å, c = 48.5 Å, and diffract X‐rays to at least 2.7 Å. There is a complex of two protein subunits and one oligonucleotide duplex in the asymmetric unit. © 1994 Wiley‐Liss, Inc.
Energy minimization method using automata network for sequence and side‐chain conformation prediction from given backbone geometry
Authors: Kono Hidetoshi , Doi Junta
Published: May 2005

Globular proteins have high packing densities as a result of residue side chains in the core achieving a tight, complementary packing. The internal packing is considered the main determinant of native protein structure. From that point of view, we present here a method of energy minimization using an automata network to predict a set of amino acid sequences and their side‐chain conformations from a desired backbone geometry for de novo design of proteins. Using discrete side‐chain conformations, that is, rotamers, the sequence generation problem from a given backbone geometry becomes one of combinatorial problems. We focused on the residues composing the interior core region and predicted a set of amino acid Sequences and their side‐chain conformations only from a given backbone geometry. The kinds of residues were restricted to six hydrophobic amino acids (Ala, Ile, Met, Leu, Phe, and Val) because the core regions are almost always composed of hydrophobic residues. The obtained sequences were well packed as was the native sequence. The method can be used for automated sequence generation in the de novo design of proteins. © 1994 Wiley‐Liss, Inc.
Molecular basis of cooperativity in protein folding. V. Thermodynamic and structural conditions for the stabilization of compact denatured states
Authors: Xie Dong , Freire Ernesto
Published: May 2005

The heat‐denatured state of proteins has been usually assumed to be a fully hydrated random coil. It is now evident that under certain solvent conditions or after chemical or genetic modifications, the protein molecule may exhibit a hydrophobic core and residual secondary structure after thermal denaturation. This state of the protein has been called the “compact denatured” or “molten globule” state. Recently is has been shown that α‐lactalbumin at pH < 5 denatures into a molten globule state upon increasing the temperature (Griko, Y., Freire, E., Privalov, P. L. Biochemistry 33:1889–1899, 1994). This state has a lower heat capacity and a higher enthalpy at low temperatures than the unfolded state. At those temperatures the stabilization of the molten globule state is of an entropic origin since the enthalpy contributes unfavorably to the Gibbs free energy. Since the molten globule is more structured than the unfolded state and, therefore, is expected to have a lower configurational entropy, the net entropic gain must originate primarily from solvent related entropy arising from the hydrophobic effect, and to a lesser extent from protonation or electrostatic effects. In this work, we have examined a large ensemble of partly folded states derived from the native structure of α‐lactalbumin in order to identify those states that satisfy the energetic criteria of the molten globule. It was found that only few states satisfied the experimental constraints and that, furthermore, those states were part of the same structural family. In particular, the regions corresponding to the A, B, and C helices were found to be folded, while the β sheet and the D helix were found to be unfolded. At temperatures below 45°C the states exhibiting those structural characteristics are enthalpically higher than the unfolded state in agreement with the experimental data. Interestingly, those states have a heat capacity close to that observed for the acid pH compact denatured state of α‐lactalbumin [980 cal (mol.K)−l]. In addition, the folded regions of these states include those residues found to be highly protected by NMR hydrogen exchange experiments. This work represents an initial attempt to model the structural origin of the thermodynamic properties of partly folded states. The results suggest a number of structural features that are consistent with experimental data. © 1994 Wiley‐Liss, Inc.
Helix‐capping interaction in λ cro protein: A free energy simulation analysis
Authors: Tidor Bruce
Published: May 2005

The stability mutant Tyr‐26 → Asp was studied in the Cro protein from bacteriophage λ using free energy molecular dynamics simulations. The mutant was calculated to be more stable than the wild type by 3.0 ± 1.7 kcal/mol/monomer, in reasonable agreement with experiment (1.4 kcal/mol/monomer). Moreover, the aspartic acid in the mutant was found to form a capping interation with the amino terminus of the third α‐helix of Cro. The simulations were analyzed to understand better the source of the stability of this helix‐capping interaction and to examine the results in light of previous explanations of stabilizing helix caps‐namely, a model of local unsatisfied hydrogen bonds at the helix termini and the helix macro dipole model. Analysis of the simulations shows that the stabilizing effect of this charged helical cap is due both to favorable hydrogen bonds with backbone NH groups at the helix terminus and to favorable electrostatic interactions (but not hydrogen bonds) with their carbonyls (effectively the next row of local dipoles in the helix). However, electrostatic interactions are weak or negligible with backbone dipolar groups in the helix further away from the terminus. Moreover, the importance of other local electrostatic interactions with polar side chains near the helix terminus, which are neglected in most treatments of this effect, are shown to be important. Thus, the results support a model that is intermediate between the two previous explanations: both unsatisfied hydrogen bonds at the helix terminus and other, local preoriented dipolar groups stabilize the helix cap. These findings suggest that similar interactions with preoriented dipolar groups may be important for cooperativity in other charge–dipole interactions and may be employed to advantage for molecular design. © 1994 Wiley‐Liss, Inc.
Crystallization of a scRIP—gelonin isolated from plant seeds Gelonium multiforum
Authors: Satyamurty P. , Hosur M. V. , Misquith S. , Surolia A. , Kannan K. K.
Published: May 2005

Single crystals of the protein gelonin isolated from the seeds of Gelonium multiforum have been grown at room temperature by vapor diffusion method. The crystals are monclinic with a = 49.4 Å, b = 44.9 Å, c = 137.4 Å, and β = 98.3°. The space group is P21, with two molecules in the asymmetric unit which are related by a noncrystallographic 2‐fold axis along ψ =13° and ϕ =88°. The crystals diffract X‐rays to high resolution, making it possible to obtain an accurate structure of this single chain ribosome inactivating protein. © 1994 Wiley‐Liss, Inc.
Predicting molecular interactions and inducible complementarity: Fragment docking of fab‐peptide complexes
Authors: Friedman Alan R. , Roberts Victoria A. , Tainer John A.
Published: May 2005

Antibody‐antigen interactions are representative of a broad class of receptor‐ligand interactions involving both specificity and potential inducible complementarity. To test possible mechanisms of antigenantibody recognition and specificity computationally, we have used a Metropolis Monte Carlo algorithm to dock fragments of the epitope Glu‐Val‐Val‐Pro‐His‐Lys‐Lys to the X‐ray structures of both the free and the complexed Fab of the antibody B13I2 (raised against the C‐helix of myohemerythri). The fragments Pro‐His and Val‐Pro‐His, which contain residues experimentally identified as important for binding, docked correctly to both structures, but all tetrapeptide and larger fragments docked correctly only to the complexed Fab, even when torsional flexibility was added to the ligand. However, only tetrapeptide and larger fragments showed significantly more favorable energies when docked to the complexed Fab coordinates than when docked to either the free Fab or a non‐specific site remote from the combining site. Comparison of the free and complexed B13I2 structures revealed that atoms within 5 Å of Val‐Pro‐His showed little movement upon peptide binding, but atoms within 5 Å of the other four epitope residues showed greater movements. These results computationally distinguish recognition and binding processes with practical implications for drug design strategies. Overall, this new fragment docking approach establishes distinct roles for the “lock‐and‐key” (recognition) and the “handshake” (binding) paradigms in antibody‐antigen interaction, suggests an incremental approach to incorporating flexibility in computational docking, and identifies critical regions within receptor binding sites for ligand recognition. © 1994 Wiley‐Liss, Inc.
Cold adaption of enzymes: Structural comparison between salmon and bovine trypsins
Authors: Smalås Arne O. , Heimstad Eldbjørg S. , Hordvik Asbjørn , Willassen Nils P. , Male Rune
Published: May 2005

The crystal structure of an anionic form of salmon trypsin has been determined at 1.82 Å resolution. We report the first structure of a trypsin from a phoikilothermic organism in a detailed comparison to mammalian trypsins in order to look for structural rationalizations for the cold‐adaption features of salmon trypsin. This form of salmon trypsin (T II) comprises 222 residues, and is homologous to bovine trypsin (BT) in about 65% of the primary structure. The tertiary structures are similar, with an overall displacement in main chain atomic positions between salmon trypsin and various crystal structures of bovine trypsin of about 0.8 Å. Intramolecular hydrogen bonds and hydrophobic interactions are compared and discussed in order to estimate possible differences in molecular flexibility which might explain the higher catalytic efficiency and lower thermostability of salmon trypsin compared to bovine trypsin. No overall differences in intramolecular interactions are detected between the two structures, but there are differences in certain regions of the structures which may explain some of the observed differences in physical properties. The distribution of charged residues is different in the two trypsins, and the impact this might have on substrate affinity has been discussed. © 1994 Wiley‐Liss, Inc.
Crystallization and characterization of the recombinant human Clara cell 10‐kDa protein
Authors: Matthews John H. , Pattabiraman N. , Ward Keith B. , Mantile Giuditta , Miele Lucio , Mukherjee Anil B.
Published: May 2005

Crystals of recombinant human Clara cell 10‐kDa protein were grown both from ammonium sulfate and polyethylene glycol (PEG) solutions. Crystals grown from ammonium sulfate solution have been characterized by X‐ray diffraction studies as monoclinic with the space group C2 and lattice constants a = 69.2 Å, b = 83.0 Å, c = 58.3 Å, and β = 99.7°. The monoclinic crystals diffract to beyond 2.5 Å. Some of the crystals grown from PEG were of a similar habit to those grown from ammonium sulfate, but others were triclinic with the space group P1 and cell constants a = 40.3 Å, b = 46.3 Å, c = 51.3 Å, α = 117.7°, β = 102.3°, and γ = 71.4°. These crystals diffract to beyond 3.2 Å. © 1994 Wiley‐Liss, Inc.
Crystallization of the reaction center from Rhodobacter sphaeroides in a new tetragonal form
Authors: Allen J. P.
Published: May 2005

The reaction center from the nonsulfur purple bacteriumRhodobacter sphaeroideshas been crystallized in a new form. The crystals grew in the presence of polyethylene glycol 4000, the detergent β‐octyl glucoside, and the amphiphiles heptane triol and benzamidine hydrochloride, using the sitting drop method. The space group of these crystals is tetragonal, P41(43)212, and the cell constants are a = b = 141.5 Å and c = 276.7 Å with probably 2 proteins per asymmetric unit. A native data set has been set collected to a resolution of 2.8 Å consisting of 56,332 unique reflections (50,731 with F > 2σ) with anRsym of 9.5%. Analysis of the diffraction data is underway using molecular and isomorphous replacement. © 1994 Wiley‐Liss, Inc.
A model for the structure of a homodimeric prohormone: The precursor to the locust neuropeptide AKH I
Authors: Horne T. J. , Doak D. G. , Rayne R. C. , Balacco G. , O'Shea M. , Campbell I. D.
Published: May 2005

We have determined the structure in solution of a homodimeric protein that is a precursor to the locust neuropeptide adipokinetic hormone I using nuclear magnetic resonance spectroscopy. This precursor, called P1, is comprised of two 41 residue strands joined by a single inter‐chain disulphide at Cys39. We have also determined the structure of an end product of P1 processing, called APRP1; this is a homodimer comprised of residues 14–41 of PI. Nuclear Overhauser Effect (nOe) data indicate that in both P1 and APRP1, residues 22–37 (numbered with respect to P1) form pairs of α‐helices, with no evidence for any other secondary structure. © 1994 Wiley‐Liss, Inc.
Quantum mechanical model assembly study on the energetics of binding of arabinose, fucose, and galactose to L‐arabinose‐binding protein
Authors: Peräkylä Mikael , Pakkanen Tapani A.
Published: May 2005

Binding energies ofL‐arabinose, D‐fucose, and D‐galactose to L‐arabinose‐binding protein was investigated theoretically. The calculated binding energies were composed of three contributions: (1) direct ligand‐active site interaction energies calculated using static ab initio model assemblies; (2) solvation energies of the ligands; and (3) long‐range electrostatic interaction energies between the ligands and the protein matrix. The calculated binding energies and the contributions of the energy components were used to analyze the experimental affinities of the ligands. © 1994 Wiley‐Liss, Inc.
Predicted secondary and supersecondary structure for the serine–threonine‐specific protein phosphatase family
Authors: Jenny Thomas F. , Gerloff Dietlind L. , Cohen Mark A. , Benner Steven A.
Published: May 2005

A bona fide consensus prediction for the secondary and supersecondary structure of the serine–threonine specific protein phosphatases is presented. The prediction includes assignments of active site segments, an internal helix, and a region of possible 310 helical structure. An experimental structure for a member of this family of proteins should appear shortly, allowing this prediction to be evaluated. © 1995 Wiley‐Liss, Inc.
Model assembly study of the ligand binding by p‐hydroxybenzoate hydroxylase: Correlation between the calculated binding energies and the experimental dissociation constants
Authors: Peräkylä Mikael , Pakkanen Tapani A.
Published: May 2005

The energies of binding of seven ligands by p‐hydroxybenzoate hydroxylase (PHBH) were calculated theoretically. Direct enzyme–ligand interaction energies were calculated using the ab initio quantum mechanical model assembly of the active site at the 3‐21G level. Solvation energies of the ligands needed in the evaluation of the binding energies were calculated with the semiempirical AM1–SM2 method and the long‐range electrostatic interaction energies between the ligands and the protein matrix classically using the static charge distributions of the ligands and the protein. Energies for proton‐transfer between the ligands OH or SH substituent at position 4 and the active‐site tyrosine within the ab initio model assemblies were calculated and compared to the corresponding pKas in aqueous solution. Excluding 3,4‐dihydroxybenzoate, the natural product of PHBH, a linear relationship between the calculated binding energies and the experimental binding free energies was found with a correlation coefficient of 0.90. Contributions of the direct enzyme–ligand interaction energies, solvation energies and the long‐range electrostatic interaction energies to the calculated binding energies were analyzed. The proton‐transfer energies of the ligands with substituents ortho to the ionized OH were found to be perturbed less in the model calculations than the energies of their meta isomers as deduced from the corresponding pKas. © 1995 Wiley‐Liss, Inc.
Crystallization and preliminary structural studies of the ncd motor domain
Authors: Sablin Elena P. , Fletterick Robert J.
Published: May 2005

The motor domain of the kinesin homolog ncd has been crystallized in the presence of MgATP by the vapor diffusion method using polyethylene glycol as the precipitant. The crystals belong to the orthorhombic space group I222 with unit cell dimensions a = 127.1 Å, b = 122.3 Å, c = 68.0 Å, and there is one ncd molecule per asymmetric unit. The crystals diffract X‐ray to at least 2.3 Å and are appropriate for high‐resolution structure determination. © 1995 Wiley‐Liss, Inc.
Preliminary X‐ray diffraction analysis of crystals of turnip yellow mosaic virus (TYMV)
Authors: Canady Mary A. , Day John , McPherson Alexander
Published: May 2005

Turnip yellow mosaic virus (TYMV) was purified from Chinese cabbage and crystallized in a form that permits high resolution structural analysis using X‐ray diffraction. The crystals have a hexagonal bipyramidal morphology and often achieve dimensions of 1.0 × 1.0 × 0.5 mm. The crystals appear to be of hexagonal space group P6222 with a = b = 525 Å, c=315 Å, but we cannot strictly rule out the possibility that the space group is P622. They appear different than any crystals of TYMV previously reported. There are three T = 3 virus particles in the unit cell, which implies that one quarter of the particle, or 45 protein subunits, comprises the asymmetric unit of the crystal. Native data have been collected using synchrotron radiation to a resolution of 3.2 Å. © 1995 Wiley‐Liss, Inc.
Configurational effects in antibody–antigen interactions studied by microcalorimetry
Authors: Murphy Kenneth P. , Freire Ernesto , Paterson Yvonne
Published: May 2005

In this paper we study the binding of two monoclonal antibodies, E3 and E8, to cytochrome c using high‐sensitivity isothermal titration calorimetry. We combine the calorimetric results with empirical calculations which relate changes in heat capacity to changes in entropy which arise from the hydrophobic effect. The change in heat capacity for binding E3 is −350 ± 60 cal K−1 mol−1 while for E8 it is −165 ± 40 cal K−1 mol−1. This result indicates that the hydrophobic effect makes a much larger contribution for E3 than for E8. Since the total entropy change at 25°C is very similar for both antibodies, it follows that the configurational entropy cost for binding E3 is much larger than for binding E8 (−77 ± 15 vs. −34 ± 11 cal K−1 mol−1). These results illustrate a case of entropy compensation in which the cost of restricting conformational degrees of freedom is to a large extent compensated by solvent release. We also show that the thermodynamic data can be used to make estimates of the surface area changes that occur upon binding. The results of the present study are consistent with previous hydrogen–deuterium exchange data, detected using 2D NMR, on the two antibody–antigen interactions. The NMR study indicated that protection from exchange is limited to the binding epitope for E8, but extends beyond the epitope for E3. These results were interpreted as suggesting that a larger surface area was buried on cytochrome c upon binding to E3 than to E8, and that larger changes in configurational entropy occur upon binding of E3 than E8. These findings are confirmed by the present study using isothermal titration calorimetry. © 1995 Wiley‐Liss, Inc.
Affinities of phosphorylated substrates for the E. coli tryptophan synthase α‐subunit: Roles of Ser‐235 and helix‐8′ dipole
Authors: Sarker Krishna D. , Hardman John K.
Published: May 2005

The roles of Ser‐235 and helix‐8′ (residues 235–242) in the functional binding and turnover of phosphorylated substrates by the α‐subunit of the E. coli tryptophan synthase (TSase) α2β2‐holoenzyme complex are examined. Previous crystallographic analyses indicated that this region was one of several near the phosphate moiety of the physiological substrate, indole‐3‐glycerol phosphate (IGP). The peptidyl amido group of Ser‐235 was suggested to H‐bond to the phosphate group; a helix macrodipole binding role was suggested for helix‐8′. The activities and substrate Kms of mutant α‐subunits altered in this region by site‐specific mutagenesis are reported here. Substitutions at Ser‐235 by an acidic (glutamic acid, mutant SE235), basic (lysine, mutant SK235), or a nonpeptidyl amido‐containing residue (proline, mutant SP235) exhibit 40‐ to 180‐fold Km increases for IGP and D‐glyceraldehyde‐3‐phosphate; no Km defects for indole were observed. kcat values for SP235, SE235, and SK235 are 100, 70, and 40%, respectively, of the wild‐type value. Steric considerations may explain the results with the SE235 and SK235 mutant α‐subunits; however, the SP235 results are consistent with the suggested phosphate binding role for the Ser‐235 peptidyl amide group during catalysis. A helix‐8′ dipole role was explored following proline substitutions separately at the first six (of eight) residues. Proline substitutions at positions‐1 through ‐4 in helix‐8′ have normal indole Kms and catalytic activities in all four TSase reactions, suggesting no major global structural changes in these proteins. By these criteria, substitutions at positions‐5 and ‐6 lead to significant structural alterations. Km increases for phosphorylated substrates are substantial (up to 40‐fold) and are dependent upon the presence of L‐serine at the β‐subunit active site. In the absence of L‐serine, substitution only at the first position results in binding defects; in the presence of L‐serine, substitutions at the first, second and third positions show binding defects of decreasing magnitude, sequentially. Substitutions at the fourth and fifth position have no effect on substrate binding. It is suggested that during catalysis a helix dipole effect on binding may be exerted but only via inter‐subunit‐induced conformational changes due to ligand (L‐serine) binding to the β‐subunit. © 1995 Wiley‐Liss, Inc.
Empirical evaluation of the influence of side chains on the conformational entropy of the polypeptide backbone
Authors: Stites Wesley E. , Pranata Julianto
Published: May 2005

Changes in amino acid side chains have long been recognized to alterthe range and distribution of ϕ, ψ angles found in the main chain of polypeptides. Altering the range and distribution of ϕ, ψ angles also alters the conformational entropy of the flexible denatured state and may thus stabilize or destabilize it relative to the comparatively conformationally rigid native state. A database of 12,320 residues from 61 nonhomologous, high resolution crystal structures was examined to determine the ϕ, ψ conformational preferences of each of the 20 amino acids. These observed distributions in the native state of proteins are assumed to also reflect the distributions found in the denatured state. The distributionswere used to approximate the energy surface for each residue, allowing the calculation of relative conformational entropies for each residue relative to glycine. In the most extreme case, replacement of glycine by proline, conformational entropy changes will stabilize the native state relative to the denatured state by −0.82 ± 0.08 kcal/mol at 20°C. Surprisingly, alanine is found to be the most ordered residue other than proline. This unexpected result is a result of the high percentage of alanines found in helical conformations. This either indicates that the observed distributions in the native state do not reflect the distributions in the denatured state, or that alanine is much more likely to adopt a helical conformation in the denatured state than residues with longer side chains. Among those residues with ϕ, ψ angles compatible with helix incorporation the percentage of alanines actually in helices is very similar to other residues. This and the consistent ordering of alanine relative to other residues regardless of secondary structure are evidence that ϕ, ψ distributions in native states reflect those in the denatured states. © 1995 Wiley‐Liss, Inc.
Crystallization of UDP‐N‐acetylglucosamine O‐acyltransferase from Escherichia coli
Authors: Pfitzner Ute , Raetz Christian R. H , Roderick Steven L.
Published: May 2005

Crystals of UDP‐N‐acetylglucosamine O‐acyltransferase (lpxA) fromEscherichia coli have been obtained from solutions of sodium/potassium phosphate and dimethylsulfoxide. These crystals belong to the cubic space group P213 (a = 99.0 Å), diffract X‐raysto approximately 2.5 Å resolution and contain one subunit of the enzyme in the asymmetric unit. © 1995 Wiley‐Liss, Inc.