bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparison of the immunogenicity of a pair of enantiomeric proteins
Authors: Dintzis Howard M. , Symer David E. , Dintzis Renee Z. , Zawadzke Laura E. , Berg Jeremy M.
Published: May 2005

The immunogenicity of a folded, all D‐amino acid protein‐ rubredoxin, has been compared with that for the corresponding L‐protein enantiomer. Following multiple administrations with alum adjuvant, the L‐protein induced a strong, specific lgG antibody response, whereas the D‐protein did not. This relative lack of responsiveness to the D‐protein cannot be attributed to rapid excretion, since it is retained at least 4 times longer than the natural L‐protein. These observations provide the first direct evidence that a folded D‐amino acid protein has low immunogenicity and is long lived in vivo. Proteins with such properties may be useful as molecular platforms in a variety of chemical and pharmaco‐logical applications. © 1993 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray crystallographic analysis of chitinase from barley seeds
Authors: Song Hyun Kyu , Hwang Kwang Yeon , Kim Kyeong Kyu , Suh Se Won
Published: May 2005

Chitinase from barley seeds has been crystallized at room temperature using polyethylene glycol as precipitant. The crystal is monoclinic, belonging to the space group P21, with unit cell parameters of a = 69.43 Å, b = 44.55 Å, c = 81.41 Å, and β = 111.95 Å. The asymmetric unit seems to contain two molecules of chitinase with a corresponding crystal volume per protein mass (VM) of 2.25 Å3/Da and a solvent content of 45% by volume. The crystal diffracts to at least 2.0 Å with X‐rays from a rotating anode source and is very stable in the X‐ray beam. X‐ray data have been collected to better than 2.2 Å Bragg spacing from a native crystal. © 1993 Wiley‐Liss, Inc.
Organization of turnip yellow mosaic virus investigated by neutron small angle scattering at 80 K: An intermediate state preceding decapsidation of the virion?
Authors: Witz Jean , Timmins Peter A. , Adrian Marc
Published: May 2005

The organization of turnip yellow mosaic virus has been investigated by neutron small angle scattering at 300 K and 80 K in buffers containing various amounts of D2O. We confirm that in native virions, no substantial part of the RNA is located at a radius larger than ca. 100‐110 Å, i.e., that there is very little interpretation of the RNA into the capsid. At 80 K, scattering curves do not depend much upon contrast, from 40% D2O to 100% D2O buffers, but are strongly affected by interparticle interference. We could, however, show that it is not the case for the subsidiary intensity maximum at q ∼ 0.06 Å−1. From the position of this maximum, we conclude that upon freezing, the radius of the capsid expands by c.a. 3.5% and the RNA penetrates deeply into the protein shell. Biological implications of this conformational change immediately preceding decapsidation are dicussed. © 1993 Wiley‐Liss, Inc.
Conformational analysis of protein structures derived from NMR data
Authors: MacArthur Malcolm W. , Thornton Janet M.
Published: May 2005

A study is presented of the conformational characteristics of NMR‐derived protein structures in the Protein Data Bank compared to X‐ray structures. Both ensemble and energy‐minimized average structures are analyzed. We have addressed the problem using the methods developed for crystal structures by examining the distribution of ϕ, Ψ, and χ angles as indicators of global conformational irregularity. All these features in NMR structures occur to varying degrees in multiple conformational states. Some measures of local geometry are very tightly constrained by the methods used to generate the structure, e.g., proline ϕ angles, α‐helix ϕ, Ψ angles, ω angles, and Cα chirality. The more lightly restrained torsion angles do show increasead clustering as the number of overall experimental observations increases. ϕ, Ψ, and χ1 angle conformational heterogeneity is strongly correlated with accessibility but shows additional differences which reflect the differing number of observations possible in NMR for the various side chains (e.g., many for Trp, few for Ser). In general, we find that the core is defined to a notional resolution of 2.0 to 2.3 Å. Of real interest is the behavior of surface residues and in particular the side chains where multiple rotameric states in different structures can vary from 10% to 88%. Later generation structures show a much tighter definition which correlates with increasing use of J‐coupling information, stereospecific assignments, and heteronumclear techniques. A suite of programs is being developed to address the special needs of NMR‐derived structures which will take into account the existence of increased mobility in solution. © 1993 Wiley‐Liss, Inc.
Recognition of errors in three‐dimensional structures of proteins
Authors: Sippl Manfred J.
Published: May 2005

A major problem in the determination of the three‐dimensional structure of proteins concerns the quality of the structural models obtained from the interpretation of experimental data. New developments in X‐ray crystallography and nuclear magnetic resonance spectroscopy have acceleratedd the process of structure determination and the biological community is confronted with a steadily increasing number of experimentally determined protein folds. However, in the recent past several experimentally determined protein structures have been proven to contain major errors, indicating that in some cases the interpretation of experimental data is difficult and may yield incorrect models. Such problems can be avoided when computational methods are employed which complement experimental structure determinations. A prerequisite of such computational tools is that they are independent of the parameters obtained from a particular experiment. In addition such techniques are able to support and accelerate experimental structure determinations. Here we present techniques based on knowledge based mean fields which can be used to judge the quality of protein folds. The methods can be used to identify misfolded structures as well as faulty parts of structural models. The techniques are even applicable in cases where only the Cα trace of a protein conformation is available. The capabilities of the technique are demonstrated using correct and incorrect protein folds. © 1993 Wiley‐Liss, Inc.
A comparison of 15N NMR relaxation measurements with a molecular dynamics simulation: Backbone dynamics of the glucocorticoid receptor DNA‐binding domain
Authors: Eriksson Mats A. L. , Berglund Helena , Härd Torleif , Nilsson Lennart
Published: May 2005

The rapid motions of the backbone of the DNA‐binding domain of the glucocorticoid receptor (GR DBD) have been investigated using proton‐detected heteronuclear NMR experiments on 15N‐labeled protein at pH 6.0 and with a 200 psec molecular dynamics simulation of hydrated GR DBD. The experimental data were interpreted in terms of a generalized order parameter (S2) and an effective correlation time (τe) for the internal motion of each amide bond. A back calculation, using the same model, yielded the {1H}‐15N nuclear Overhauser effects (NOEs) and the 15N spin‐lattice relaxation times (T1) from the simulated data. The rapid motions of the backbone turned out to be rather limited and uniform throughout the protein, with a somewhat reduced mobility in the two major α‐helical regions and a slightly enhanced flexibility for some residues in the first zinc coordinating region. The agreement between the experimental and simulated S2‐values was as good as quantitative for most of the residues, except for some residues that were subject to a more large‐scale, and in the simulation thus poorly sampled, motion. Examples of such motions that were found in the simulation include jumps of the amide bond of Ile‐487 between the charged oxygens of the side chain of Asp‐485 and less distinct large scale motions for some of the residues in the extended regions, that were shown to give rise to noisy and/or fast decaying internal reorientational correlation functions. For these residues large differences in the simulated and experimental τe‐values were found, indicating that motions on different time scales were dominating in the experimental and simulated values. The lower (<0.7) experimental NOEs for these residues could not be reproduced in the simulation and were shown to be a consequence of the lower τe‐values estimated in the simulation. By combining information from the simulation and the experiment a more complete picture of the motions for these residues can be obtained as is illustrated with an estimation of the jump angle and jump frequency for the amide bond of Ile‐487. © 1993 Wiley‐Liss, Inc.
X‐ray structures of fragments from binding and nonbinding versions of a humanized anti‐CD18 antibody: Structural indications of the key role of VH residues 59 to 65
Authors: Eigenbrot Charles , Gonzalez Tania , Mayeda Julia , Carter Paul , Werther Winifred , Hotaling Timothy , Fox Judy , Kessler Jeremy
Published: May 2005

X‐ray crystal structures of fragments from two different humanized antiCD18 antibodies are reported. The Fv fragment of the nonbinding version has been refined in space group C2 with a=64.2 Å, b=61.3 Å, c=51.8 Å, and β=99° to an R‐value of 18.0% at 1.9 Å, and the Fab fragment of the tight‐binding version has been refined in space group P3 with a=101. Å and c=45.5 Å to an R‐value of 17.8% at 3.0 Å resolution. The very large difference in their binding affinity (>1000‐fold) is attributed to large and local structural differences in the C‐terminal part of CDR‐H2, and from this we conclude there is direct contact between this region and antigen when they combine. X‐ray structures of antibody–antigen complexes available in the literature have yet to show this part of CDR‐H2 in contact with antigen, despite its hypervariable sequence. Implications of this result for antibody humanization are discussed. © 1994 John Wiley & Sons, Inc.
Homologous modeling of the lysosomal protective protein/carboxypeptidase L: Structural and functional implications of mutations identified in galactosialidosis patients
Authors: Elsliger Marc‐André , Potier Michel
Published: May 2005

The deficiency of the lysosomal protective protein/carboxypeptidase L (CARB L) causes the lysosomal storage disorder, galactosialidosis, characterized by neuraminidase and β‐galactosidase deficiencies in patients' cells. The three enzymes form a complex inside the lysosome, and the neuraminidase and β‐galactosidase deficiencies are secondary to CARB L deficiency. Sequence similarity and common enzymological properties suggest that the protomeric tertiary structure of CARB L is conserved within a family of serine carboxypeptidases which includes the yeast carboxypeptidase Y, killer expression I gene product and several plant carboxypeptidases. We used this homology to build a model of the CARB L structure based on the recently published X‐ray atomic coordinates of the wheat carboxypeptidase II (CPDW‐II) which shares 32% primary structure identity with CARB L. Small insertions and deletions were accommodated into the model structure by energy minimization using the DREIDING II force field. The Cα atomic‐coordinates of the final CARB L model have a RMS shift of 1.01 Å compared to the corresponding conserved residues in the CPDW‐II template structure. The correct orientation of the homologous catalytic triad residues Ser150, His429 and Asp392, the potential energy calculations and the distribution of hydrophobic and hydrophillic residues in the structure all support the validity of the CARB L model. Most missense mutations identified in galactosialidosis patients were located in secondary structural elements except for the Tyr211→Asn mutation which is in a loop. The other mutant residues have their side chains deeply buried in the central β‐sheet of the model structure except for the Phe412→Val mutation which is located in the dimer interface. The predicted effects of specific mutations on CARB L structural stability correlates well with recently published transient expression studies of mutant CARB L (Shimmoto, M. et al., J. Clin. Invest., 91:2393–2399, 1993). © 1994 John Wiley & Sons, Inc.
Molecular surface representations by sparse critical points
Authors: Lin Shuo Liang , Nussinov Ruth , Fischer Daniel , Wolfson Haim J.
Published: May 2005

We have defined a molecular surface representation that describes precisely and concisely the complete molecular surface. The representation consists of a limited number of critical points disposed at key locations over the surface. These points adequately represent the shape and the important characteristics of the surface, despite the fact that they are modest in number. We expect the representation to be useful in areas such as molecular recognition and visualization. In particular, using this representation, we are able to achieve accurate and efficient protein–protein and protein–small molecule docking. © 1994 John Wiley & Sons, Inc.
Crystallization and structural analysis of bullfrog red cell L‐subunit ferritins
Authors: Trikha J. , Waldo G. S. , Lewandowski F. A. , Ha Y. , Theil E. C. , Weber P. C. , Allewell N. M.
Published: May 2005

Ferritin is a 24 subunit protein that controls biomineralization of iron in animals, bacteria, and plants. Rates of mineralization vary among members of the ferritin family, particularly between L and H type subunits of animal ferritins which are differentially expressed in various cell types. To examine ferritin from a highly differentiated cell type and to clarify the relationship between ferritin structure and function, bullfrog red cell L ferritin has been cloned, overexpressed in E. coli, and crystallized under two conditions. Crystals were obtained at high ionic strength in the presence of MnCl2 at a concentration comparable to that of the protein and in the presence of MgCl2 at a concentration much higher than that of the protein. Under both crystallization conditions, the crystals are tetragonal bipyramids in the space group F432 with unit cell dimensions a=b=c= 182 ± 0.5 Å. Crystals obtained in the presence of manganese and ammonium sulfate diffract to 1.9 Å, while those obtained in the presence of magnesium and sodium tartrate diffract to 1.6 Å. Isomorphous crystals have been obtained under similar conditions for a site‐directed mutant with a reduced mineralization rate in which Glu−57, −58, −59, and −61 are all replaced by Ala. The structure of wild type L‐subunit with magnesium has been solved by molecular replacement using the calcium salt of human liver H subunit (Lawson et al., Nature (London) 349:541–544, 1991) as the model. The crystallographic R factor for the 6–2.2 Å shell is 0.21. The overall fold of human H and bullfrog L ferritins is similar with an rms difference in backbone atomic positions of 0.97 Å. The largest structural differences occur in the D helix and the loop connecting the D and E helices of the four helix bundle. Because red cell L ferritin and liver H ferritin show differences in both rates of mineralization and three‐dimensional structure, more detailed comparisons of these structures are likely to shed new light on the relationship between conformation and function. © 1994 John Wiley & Sons, Inc.
Evaluation of the conformational free energies of loops in proteins
Authors: Smith Kenneth C. , Honig Barry
Published: May 2005

In this paper we discuss the problem of including solvation free energies in evaluating the relative stabilities of loops in proteins. A conformational search based on a gas‐phase potential function is used to generate a large number of trial conformations. As has been found previously, the energy minimization step in this process tends to pack charged and polar side chains against the protein surface, resulting in conformations which are unstable in the aqueous phase. Various solvation models can easily identify such structures. In order to provide a more severe test of solvation models, gas phase conformations were generated in which side chains were kept extended so as to maximize their interaction with the solvent. The free energies of these conformations were compared to that calculated for the crystal structure in three loops of the protein E. coli RNase H, with lengths of 7, 8, and 9 residues. Free energies were evaluated with a finite difference Poisson‐Boltzmann (FDPB) calculation for electrostatics and a surface area‐based term for nonpolar contributions. These were added to a gas‐phase potential function. A free energy function based on atomic solvation parameters was also tested. Both functions were quite successful in selecting, based on a free energy criterion, conformations quite close to the crystal structure for two of the three loops. For one loop, which is involved in crystal contacts, conformations that are quite different from the crystal structure were also selected. A method to avoid precision problems associated with using the FDPB method to evaluate conformational free energies in proteins is described. © 1994 John Wiley & Sons, Inc.
Crystallization and preliminary structural studies of a chorismate mutase catalytic antibody complexed with a transition state analog
Authors: Haynes Matthew R. , Stura Enrico A. , Hilvert Donald , Wilson Ian A.
Published: May 2005

The Fab′ fragment of a catalytic antibody with chorismate mutase activity has been crystallized as a complex with the transition‐state analog hapten. The complex was crystallized by the vapor diffusion method using ammonium sulfate as the precipitant. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions a = 37.1 Å, b = 63.3 Å, c = 178.5 Å, and there is one Fab' molecule per asymmetric unit. The crystals diffract X‐rays to at least 3.0 Å and are suitable for X‐ray crystallographic studies. © 1994 John Wiley & Sons, Inc.
Thermodynamics of ubiquitin unfolding
Authors: Wintrode Patrick L. , Makhatadze George I. , Privalov Peter L.
Published: May 2005

The energetics of ubiquitin unfolding have been studied using differential scanning microcalorimetry. For the first time it has been shown directly that the enthalpy of protein unfolding is a nonlinear function of temperature. Thermodynamic parameters of ubiquitin unfolding were correlated with the structure of the protein. The enthalpy of hydrogen bonding in ubiquitin was calculated and compared to that obtained for other proteins. It appears that the energy of hydrogen bonding correlates with the average length of the hydrogen bond in a given protein structure. © 1994 John Wiley & Sons, Inc.
Hydrogen bond strength and β‐sheet propensities: The role of a side chain blocking effect
Authors: Bai Yawen , Englander S. Walter
Published: May 2005

Amino acid side chains can enhance peptide group hydrogen bond strength in protein structures by obstructing the competing hydrogen bond to solvent in the unfolded state. Available data indicate that the steric blocking effect contributes an average of 0.5 kJ per residue to protein hydrogen bond strength and accounts for the intrinsic α‐sheet propensities of the amino acids. In available data for helical models, the contribution to α‐helix propensities is obscured especially by large context‐dependent effects. These issues are all related by a common side chain‐dependent steric clash which disfavors peptide to water H‐bond formation, peptide to catalyst complexation in hydrogen exchange reactions (Bai et al., Proteins 17:75–86, 1993), and peptide to peptide H‐bonding in the helical main chain conformation (Creamer and Rose, Proc. Natl. Acad. Sci. U.S.A. 89:5937–5941, 1992) but not in α‐strands. © 1994 John Wiley & Sons, Inc.
A method for α‐helical integral membrane protein fold prediction
Authors: Taylor William R. , Jones David T. , Green N. Michael
Published: May 2005

Integral membrane proteins (of the α‐helical class) are of central importance in a wide variety of vital cellular functions. Despite considerable effort on methods to predict the location of the helices, little attention has been directed toward developing an automatic method to pack the helices together. In principle, the prediction of membrane proteins should be easier than the prediction of globular proteins: there is only one type of secondary structure and all helices pack with a common alignment across the membrane. This allows all possible structures to be represented on a simple lattice and exhaustively enumerated. Prediction success lies not in generating many possible folds but in recognizing which corresponds to the native. Our evaluation of each fold is based on how well the exposed surface predicted from a multiple sequence alignment fits its allocated position. Just as exposure to solvent in globular proteins can be predicted from sequence variation, so exposure to lipid can be recognized by variable‐hydrophobic (variphobic) positions. Application to both bacteriorhodopsin and the eukaryotic rhodopsin/opsin families revealed that the angular size of the lipid‐exposed faces must be predicted accurately to allow selection of the correct fold. With the inherent uncertainties in helix prediction and parameter choice, this accuracy could not be guaranteed but the correct fold was typically found in the top six candidates. Our method provides the first completely automatic method that can proceed from a scan of the protein sequence databanks to a predicted three‐dimensional structure with no intervention required from the investigator. Within the limited domain of the seven helix bundle proteins, a good chance can be given of selecting the correct structure. However, the limited number of sequences available with a corresponding known structure makes further characterization of the method difficult. © 1994 John Wiley & Sons, Inc.
Crystallization and preliminary crystallographic studies of the precursor and mature forms of a neutral lipase from the fungus Rhizopus delemar
Authors: Swenson Lora , Green Ruth , Joerger Rolf , Haas Michael , Scott Karen , Wei Yunyi , Derewenda Urszula , Lawson David M. , Derewenda Zygmunt S.
Published: May 2005

A neutral lipase from the filamentous fungus Rhizopus delemar has been crystallized in both its proenzyme and mature forms. Although the latter crystallizes readily and produces a variety of crystal forms, only one was found to be suitable for X‐ray studies. It is monoclinic (C2, a = 92.8 Å, b = 128.9 Å, c = 78.3 Å, β = 135.8) with two molecules in the asymmetric unit related by a noncrystallographic diad. The prolipase crystals are orthorhombic (P212121, with a = 79.8 Å, b = 115.2 Å, c = 73.0 Å) and also contain a pair of molecules in the asymmetric unit. Initial results of molecular replacement calculations using the refined coordinates of the related lipase from Rhizomucor miehei identified the correct orientations and positions of the protein molecules in the unit cells of crystals of both proenzyme and the mature form. © 1994 John Wiley & Sons, Inc.
Investigation of the functional interplay between the primary site and the subsite of RNase T1: Kinetic analysis of single and multiple mutants for modified substrates
Authors: Steyaert Jan , Haikal Abdel Fattah , Wyns Lode
Published: May 2005

We report on the functional cooperativity of the primary site and the sub‐site of ribonuclease T1 (RNase T1; EC 3.1.27.3). The kinetic properties of the single Tyr‐38‐Phe and Asn‐98‐Ala mutants have been compared with those of the corresponding double mutant. The Tyr‐38‐Phe mutation has been used to probe enzyme‐substrate interactions at the primary site; the Asn‐98‐Ala mutation monitors subsite interactions.1 In addition to the dinucleoside phosphate substrate GpC, we measured the kinetics for GpMe, a synthetic substrate in which the leaving nucleoside cytosine has been replaced by methanol. All data were combined in a triple mutant box to analyze the interplay between Tyr‐38, Asn‐98, and the leaving group. The free energy barriers to kcat, introduced by the single Tyr‐38‐Phe and Asn‐98‐Ala mutations are not additive in the corresponding double mutant. The energetic coupling between both mutations is independent of the binding of the leaving cytosine at the subsite. We conclude that the coupling of the Tyr‐38‐Phe and Asn‐98‐Ala mutations arises through distortion or reorientation of the 3′‐guanylic acid moiety bound at the primary site. The experimental data indicate that the enzyme–substrate interactions beyond the scissile phosphodiester bond contribute to catalysis through the formation of new or improved contacts in going from ground state to transition state, which are functionally independent of primary site interactions. © 1994 John Wiley & Sons, Inc.
Analysis of Cα geometry in protein structures
Authors: Oldfield T. J. , Hubbard R. E.
Published: May 2005

The polypeptide of a protein molecule can be considered as a chain of Cα atoms linked by pseudobonds between the Cα atoms of successive amino acid residues. This paper presents an analysis of the angle and dihedral angles made by these pseudobonds in protein structures determined at high resolution by X‐ray crystallography. This analysis reveals a strong correlation between Cα geometry and the protein fold. The regular features of protein secondary structure such as α‐helix and α‐sheet are very clearly defined. In addition, it is possible to identify with some confidence the discrete populations of particular conformations of α‐turn. Comparison with the traditional Ramachandran type of plot demonstrates that an analysis of protein structure on the basis of Cα geometry provides a richer description of protein conformation. In addition, the characteristics of this geometry could be a useful guide in model building of protein structure. © 1994 John Wiley & Sons, Inc.
Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: Patterns revealed by three‐dimensional molecular modelling of mutations of the protease domain
Authors: Greengard Judith S. , Fisher Cindy L. , Villoutreix Bruno , Griffin John H.
Published: May 2005

Familial deficiency of protein C is associated with inherited thrombophilia. To explore how specific missense mutations might cause observed clinical phenotypes, known protein C missense mutations were mapped onto three‐dimensional homology models of the protein C protease domain, and the implications for domain folding and structure were evaluated. Most Type I missense mutations either replaced internal hydrophobic residues (I201T, L223F, A259V, A267T, A346T, A346V, G376D) or nearby interacting residues (I403M, T298M, Q184H), thus disrupting the packing of internal hydrophobic side chains, or changed hydrophilic residues, thus disrupting ion pairs (N256D, R178W). Mutations (P168L, R169W) at the activation site destabilized the region containing the activation peptide structure. Most Type II mutations involved solvent‐exposed residues and were clustered either in a positively charged region (R147W, R157Q, R229Q, R352W) or were located in or near the active site region (S252N, D359N, G381S, G391S, H211Q). The cluster of arginines 147, 157, 229, and 352 may identify a functionally important exosite. Identification of the spatial relationships of natural mutations in the protein C model is helpful for understanding manifestations of protein C deficiency and for identification of novel, functionally important molecular features and exosites. © 1994 John Wiley & Sons, Inc.
Ion pair formation involving methylated lysine side chains: A theoretical study
Authors: Mavri Janez , Vogel Hans J.
Published: May 2005

Lysine residues with one, two, or three methyl groups substituted on the ϵ‐nitrogen atom are found in many proteins. To evaluate the effect of the posttranslational methylation on ion‐pair formation we have performed semiempirical and ab initio molecular orbital calculations, using the AMI method and the 6‐31G* basis set, respectively. Combinations of various methylated forms of methylamine and ethylamine with formate, acetate, and dimethyl phosphate were studied as model compounds. This approach allowed us to obtain information relevant to the interaction of the modified Lys residues with carboxylate groups of proteins, and the backbone of nucleic acids. We have found that the interaction energy decreases with an increasing number of methyl groups. Inclusion of a solvent reaction field in the semiempirical calculations gave reasonable values for the interaction energy in aqueous solution, when formate and acetate were the counterions. These studies suggest that, in addition to other factors, a weakening of ionic interactions contributes to the various physiological effects of lysine methylation. © 1994 John Wiley & Sons, Inc.
Structure of trichosanthin at 1.88 Å resolution
Authors: Zhou Kangjing , Fu Zhuji , Chen Minghuang , Lin Yujuan , Pan Kezhen
Published: May 2005

Trichosanthin (TCS) is one of the single chain ribosome‐inactivating proteins (RIPs). The crystals of the orthorhombic form of trichosanthin have been obtained from a citrate buffer (pH 5.4) with KC1 as the precipitant. The crystal belongs to the space group P212121 with a = 38.31, b = 76.22, c = 79.21 Å. The structure was solved by molecular replacement method and refined using the programs XPLOR and PROLSQ to an R‐factor of 0.191 for the reflections within the 6–1.88 Å resolution range. The bond length and bond angle in the protein molecule have root‐mean‐square deviations from ideal value of 0.013 Å and 3.3°, respectively. The refined model includes 247 residues and 197 water molecules. The TCS molecule consists of two structural domains. The large domain contains six α‐helices, a six stranded sheet, and an antiparallel β‐sheet. The small domain has a largest α‐helix, which shows a distinct bend. The possible active site of the molecule located on the cleft between two domains was proposed. In the active site Arg‐163 and Glu‐160, Glu‐189 and Arg‐122 form two ion pairs, Glu‐189 and Gln‐156 are hydrogen bonded to each other. Three water molecules are bonded to the residues in the active site region. The structures of TCS molecule and ricin A‐chain (RTA) superimpose quite well, showing that the structures of the two protein molecules are homologous. Comparison of the structures of the TCS molecule in this orthorhombic crystal with that in the monoclinic crystal indicates that there are no essential differences of the structures between the two protein crystals. © 1994 Wiley‐Liss, Inc.
Helix‐forming tendencies of nonpolar amino acids predicted by Monte Carlo simulated annealing
Authors: Okamoto Yuko
Published: May 2005

Monte Carlo simulated annealing is applied to the study of the α‐helix‐forming tendencies of seven nonpolar amino acids, Ala, Leu, Met, Phe, Ile, Val, and Gly. Homooligomers of 10 amino acids are used and the helix tendency is calculated by folding α‐helicies from completely random initial conformations. The results of the simulation imply that Met, Ala, and Leu are helix formers and that Val, Ile, and Gly are helix breakers, while Phe comes in between the two groups. The differences between helix formers and breakers turned out to be large in agreement with the recent experiments with short peptides. It is argued from the energy distributions of the obtained conformations that the helix tendency is small for the helix breakers because of steric hindrance of side chains. Homoglycine is shown to favor a random coil conformation. The β‐strand tendencies of the same homooligomers are also considered, and they are shown to agree with the frequencies of amino acids in β‐sheet from the protein data base. © 1994 Wiley‐Liss, Inc.
Temperature‐induced complementarity as a mechanism for biomolecular assembly
Authors: Leikin S. , Parsegian V. A.
Published: May 2005

Recent advances in the measurement and theory of “hydration” interactions between biomolecules provide a basis on which to formulate mechanisms of biomolecular recognition. In this paper we have developed a mathematical formalism for analyzing specificity encoded in dynamic distributions of surface polar groups, a formalism that incorporates newly recognized properties of directly measured “hydration” forces. As expected, attraction between surfaces requires complementary patterns of surface polar groups. In contrast to usual expectations, thermal motion can create these complementary surface configurations. We have demonstrated that assembly can occur with an increase in conformational entropy of polar residues. Elevated temperature then facilitates recognition rather than hinders it. This mechanism might underlie some temperature‐favored assembly reactions common in biological systems that are usually associated with the “hydrophobic effect” only. © 1994 Wiley‐Liss, Inc.
Kinetic study on myoglobin refolding monitored by five optical probe stopped‐flow methods
Authors: Chiba Kaori , Ikai Atsushi , Kawamura‐Konishi Yasuko , Kihara Hiroshi
Published: May 2005

The refolding kinetics of horse cyanometmyoglobin induced by concentration jump of urea was investigated by five optical probe stopped‐flow methods: absorption at 422 nm, tryptophyl fluorescence at around 340 nm, circular dichroism (CD) at 222 nm, CD at 260 nm, and CD at 422 nm. In the refolding process, we detected three phases with rate constants of > 1 × 102 s−1, (4.5–9.3) S−1, and (2–5) × 10−3 s−1. In the fastest phase, a substantial amount of secondary structure (40%) is formed within the dead time of the CD stopped‐flow apparatus (10.7 ms). The kinetic intermediate populated in the fastest phase is shown to capture a hemindicyanide, suggesting that a “heme pocket precursor” recognized by hemindicyanide must be constructed within the dead time. In the middle phase, most of secondary and tertiary structures, especially around the captured hemindicyanide, have been constructed. In the slowest phase, we detected a minor structural rearrangement accompanying the ligand‐exchange reaction in the fifth coordination of ferric iron. We present a possible model for the refolding process of myoglobin in the presence of the heme group. © 1994 Wiley‐Liss, Inc.
Decoupling of melting domains in immobilized ribonuclease A
Authors: Rialdi Giovanni , Battistel Ezio
Published: May 2005

Ribonuclease A has been immobilized on silica beads through glutaraldeyde‐mediated chemical coupling in order to improve the stability of the protein against thermal denaturation. The thermodynamic and binding properties of the immobilized enzyme have been studied and compared with those of the free enzyme. The parameters describing the binding of the inhibitor 3′ ‐CMP (Ka and ΔH) as monitored by spectrophotometry and calorimetry were not significantly affected after immobilization. Conversely both the stability and unfolding mechanism drastically changed. Thermodynamic analysis of the DSC data suggests that uncoupling of protein domains has occurred as a consequence of the immobilization. The two state approximation of the protein unfolding process is not longer valid for the immobilized RNase. Protein stability strongly depends on the hydrophobicity properties of the support surface as well as on the presence of the inhibitor and pH. For example, after immobilization on a highly hydrophobic surface, the enzyme is partially in the unfolded state. The binding of a ligand is able to reorganize the protein structure into a native‐like conformation. The refolding rates are different for the two protein domains and vary as a function of pH and presence of the inhibitor 3′‐CMP. © 1994 Wiley‐Liss, Inc.
Characterization of two crystal forms of Clostridium thermocellum endoglucanase CelC
Authors: Dominguez Roberto , Souchon Hélène , Alzari Pedro M.
Published: May 2005

Endoglucanase CelC from Clostridium thermocellum expressed in Escherichia coli has been crystallized in two different crystal forms by the hanging drop method. Crystals of form I were grown with polyethylene glycol as a precipitant. They are orthorhombic, space group P212121, with cell dimensions a =51.4 Å, b =84.3 Å, and c =87.5 Å. Crystals of form II, obtained in ammonium sulfate solutions, belong to the tetragonal space group P41212 (or P43212) with cell dimensions of a = b = 130.7 Å and c = 69.6 Å. Diffraction data to 2.8 Å resolution were observed for both crystal forms with a rotating anode generator. Preliminary oscillation images of the orthorhombic form I crystals using a synchrotron radiation source show diffraction to 2.2 Å resolution, indicating that these crystals are suitable for high resolution crystallographic analysis. © 1994 Wiley‐Liss, Inc.
Crystallization of an enzyme‐inhibitor complex: Proteinase K with its protein inhibitor, PK13
Authors: Pal Gour P. , Jany Klaus D. , Tsernoglou Demetrius
Published: May 2005

Crystals of a complex of proteinase K (molecular mass, 28,790 Da) with its naturally occurring protein inhibitor PK13 (19,641 Da), have been prepared by a microdialysis technique and modified by hanging drop vapor diffusion against 25% ammonium sulfate in 50 mM Tris‐HCl, pH 7.8. The crystals are long prisms with diamond‐shaped cross sections of 0.2 × 0.4 × 1.5 mm3 and they diffract X‐rays to a resolution of 2.5 Å. They belong to the orthorhombic space group P212121 with cell dimensions a = 64.1 Å, b = 66.8 Å, and c = 133.8 Å. Assuming one whole complex in the asymmetric unit, one obtains VM = 2.95 Å3/Da and the solvent content, Vsolv = 58.3%. © 1994 Wiley‐Liss, Inc.
Searching protein structure databases has come of age
Authors: Holm Liisa , Sander Chris
Published: May 2005

The number of protein structures known in atomic detail has increased from one in 1960 (Kendrew, J. C., Strandberg, B. E., Hart, R. G., Davies, D. R., Phillips, D. C., Shore, V. C. Nature (London) 185:422–427, 1960) to more than 1000 in 1994. The rate at which new structures are being published exceeds one a day as a result of recent advances in protein engineering, crystallography, and spectroscopy. More and more frequently, a newly determined structure is similar in fold to a known one, even when no sequence similarity is detectable. A new generation of computer algorithms has now been developed that allows routine comparison of a protein structure with the database of all known structures. Such structure database searches are already used daily and they are beginning to rival sequence database searches as a tool for discovering biologically interesting relationships. © 1994 Wiley‐Liss, Inc.
HOOK: A program for finding novel molecular architectures that satisfy the chemical and steric requirements of a macromolecule binding site
Authors: Eisen Michael B. , Wiley Don C. , Karplus Martin , Hubbard Roderick E.
Published: May 2005

A program (HOOK) is described for generating potential ligands that satisfy the chemical and steric requirements of the binding region of a macromolecule. Functional group sites with defined positions and orientations are derived from known ligand structures or the multicopy simulation search (MCSS) method (Miranker, A., Karplus, M. Proteins 11:29–34, 1991). HOOK places molecular “skeletons” from a database into the protein binding region by making bonds between sites (“hooks”) on the skeleton and functional groups. The nonpolar interactions with the binding region of candidate molecules are assessed by use of a simplified van der Waals potential. The method is illustrated by constructing ligands for the sialic acid binding site of the hemagglutinin from the influenza A virus and the active site of chloramphenicol acetyltransferase. Aspects of the HOOK program that lead to a highly efficient search of 105 or more skeletons for binding to 102 or more functional group minima are outlined. © 1994 Wiley‐Liss, Inc.
Parser for protein folding units
Authors: Holm Liisa , Sander Chris
Published: May 2005

General patterns of protein structural organization have emerged from studies of hundreds of structures elucidated by X‐ray crystallography and nuclear magnetic resonance. Structural units are commonly identified by visual inspection of molecular models using qualitative criteria. Here, we propose an algorithm for identification of structural units by objective, quantitative criteria based on atomic interactions. The underlying physical concept is maximal interactions within each unit and minimal interaction between units (domains). In a simple harmonic approximation, interdomain dynamics is determined by the strength of the interface and the distribution of masses. The most likely domain decomposition involves units with the most correlated motion, or largest interdomain fluctuation time. The decomposition of a convoluted 3‐D structure is complicated by the possibility that the chain can cross over several times between units. Grouping the residues by solving an eigenvalue problem for the contact matrix reduces the problem to a one‐dimensional search for all reasonable trial bisections. Recursive bisection yields a tree of putative folding units. Simple physical criteria are used to identify units that could exist by themselves. The units so defined closely correspond to crystallographers' notion of structural domains. The results are useful for the analysis of folding principles, for modular protein design and for protein engineering. © 1994 Wiley‐Liss, Inc.
X‐ray crystal structure and molecular dynamics simulation of bovine pancreas phospholipase A2–n‐dodecylphosphorylcholine complex
Authors: Tomoo Koji , Ohishi Hirofumi , Ishida Toshimasa , Inoue Masatoshi , Ikeda Kiyoshi , Sumiya Shigeyuki , Kitamura Kunihiro
Published: May 2005

The crystal structure of n‐dodecylphosphorylcholine (n‐C12PC)–bovine pancreas phospholipase A2 (PLA2) complex provided the following structural.characteristics: (1) the dodecyl chain of n‐C12PC was located at the PLA2 N ‐terminal helical region by hydrophobic interactions, which corresponds to the binding pocket of 2‐acyl fatty acid chain (β‐chain) of the substrate phospholipid, (2) the region from Lys‐53 to Lys‐56 creates a cholinereceiving pocket of n‐C12PC and (3) the N‐termillal group of Ala‐1 shifts significantly toward the Tyr‐52 OH group by the binding of the n‐C12PC inhibitor. Since the accuracy of the X‐ray analysis (R = 0.275 at 2.3 Å resolution) was insufficient to establish these important X‐ray insights, the complex structure was further investigated through the molecular dynamics (M D) simulation, assuming a system in aqueous solution at 310K. The M D simulation covering 176 ps showed that the structural characteristics observed by X‐ray analysis are intrinsic and also stable in the dynamic state. Furthermore, the M D simulation made clear that the PLA2 binding pocket is large enough to permit the conformational fluctuation of the n‐C12PC hydrocarbon chain. © 1994 Wiley‐Liss, Inc. © 1994 Wiley‐Liss, Inc.
Structure and dynamics of the neutrophil defensins NP‐2, NP‐5, and HNP‐1: NMR studies of amide hydrogen exchange kinetics
Authors: Skalicky Jack J. , Selsted Michael E. , Pardi Arthur
Published: May 2005

The exchange kinetics for the slowly exchanging amide hydrogens in three defensins, rabbit NP‐2, rabbit NP‐5, and human HNP‐1, have been measured over a range of pH at 25°C using 1D and 2D NMR methods. These NHs have exchange rates 102 to 105 times slower than rates from unstructured model peptides. The observed distribution of exchange rates under these conditions can be rationalized by intramolecular hydrogen bonding of the individual NHs, solvent accessibility of the NHs, and local fluctuations in structure. The temperature dependencies of NH chemical shifts (NH temperature coefficients) were measured for the defensins and these values are consistent with the defensin structure. A comparison is made between NH exchange kinetics, NH solvent accessibility, and NH temperature coefficients of the defensins and other globular proteins. Titration of the histidine side chain in NP‐2 was examined and the results are mapped to the three‐dimensional structure. © 1994 Wiley‐Liss, Inc.
Estimation of changes in side chain configurational entropy in binding and folding: General methods and application to helix formation
Authors: Lee Kon Ho , Xie Dong , Freire Ernesto , Amzel L. Mario
Published: May 2005

Theoretical estimations of changes in side chain configurational entropy are essential for understanding the different contributions to the overall thermodynamic behavior of important biological processes like folding and binding. The configurational entropy of any given side chain in any particular protein can be evaluated from the complete energy profile of the side chain. Calculations of the energy profiles can be performed using the side chain single bond dihedrals as the only independent variables as long as the structures at each value of the dihedrals are allowed to relax through small changes in the valence bond angles. The probabilities of different side chain conformers obtained from these energy profiles are very similar to the conformer populations obtained by analysis of side chain preferences in the proteins of the Protein Data Bank. Also, side chain conformational entropies obtained from the energy profiles agree extremely well with those obtained from the Protein Data Bank conformer populations. Changes in side chain configurational entropy in binding and folding can be computed as differences in conformational entropy because, in most cases, the frequency of the rotational oscillation around the energy minimum of any given conformer does not appear to change significantly in the reaction. Changes of side chain conformational entropy calculated in this way were compared with experimental values. The only available experimental data–the effect of side chain substitution on the stability of α‐helices–were used for this comparison. The experimental values were corrected to subtract the solvent contributions. This comparison yields an excellent agreement between calculated and experimental values, validating not only the theoretical estimates but also the separability of the entropic contributions into configurational terms and solvation related terms. © 1994 Wiley‐Liss, Inc.
Intrinsic pKas of ionizable residues in proteins: An explicit solvent calculation for lysozyme
Authors: Del Buono Gabriela S. , Figueirido Francisco E. , Levy Ronald M.
Published: May 2005

Molecular dynamics simulations of triclinic hen egg white lysozyme in aqueous solution were performed to calculate the intrinsic pKas of 14 ionizable residues. An all‐atom model was used for both solvent and solute, and a single 180 ps simulation in conjunction with a Gaussian fluctuation analysis method was used. An advantage of the Gaussian fluctuation method is that it only requires a single simulation of the system in a reference state to calculate all the pKas in the protein, in contrast to multiple simulations for the free energy perturbation method. pKint shifts with respect to reference titratable residues were evaluated and compared to results obtained using a finite difference Poisson‐Boltzmann (FDPB) method with a continuum solvent model; overall agreement with the direction of the shifts was generally observed, though the magnitude of the shifts was typically larger with the explicit solvent model. The contribution of the first solvation shell to the total charging free energies of the titratable groups was explicitly evaluated and found to be significant. Dielectric shielding between pairs of titratable groups was examined and found to be smaller than expected. The effect of the approximations used to treat the long‐range interactions on the pKint shifts is discussed. © 1994 Wiley‐Liss, Inc.