bluuebunny/crossref_metadata_2025 huggingface dataset:
Helix packing in proteins: Prediction and energetic analysis of dimeric, trimeric, and tetrameric GCN4 coiled coil structures
Authors: Delano Warren L. , Brünger Axel T.
Published: May 2005

A simulated annealing method for atomic resolution structure prediction of α‐helical coiled coil proteins is described which draws upon knowledge of the oligomerization state, the helix directionality, and the properties of heptad repeat sequences. Unknown structural parameters, such as the coiled coil twist angle and the side chain conformations, are heavily sampled while allowing for flexibility in the helix backbone geometry. Structures of the wild‐type GCN4 dimer [O'Shea et al., Science 254:539–544, 1991] and a mutant tetramer [Harbury et al., Science 292:1401–1407, 1993] have been generated and compared with the X‐ray crystal structures. The wild‐type dimer model has a root mean square coordinate deviation from the crystal structure of 0.73 Å for nonhydrogen atoms in the dimerization interface. Structures of a mutant dimer and a mutant trimer have been predicted. Packing energetics were analyzed for core leucine and isoleucine side chains in dimeric and tetrameric coiled coils. Strong packing preferences were found in the dimers but not in the tetramers. Thus, packing in the dimer may be responsible for the switch from a two‐stranded to a four‐stranded coiled coil caused by the GCN4 leucine zipper mutations. © 1994 Wiley‐Liss, Inc.
Learning about protein folding via potential functions
Authors: Maiorov V. N. , Crippen G. M.
Published: May 2005

Over the last few years we have developed an empirical potential function that solves the protein structure recognition problem: given the sequence for an n‐residue globular protein and a collection of plausible protein conformations, including the native conformation for that sequence, identify the correct, native conformation. Having determined this potential on the basis of only some 6500 native/nonnative pairs of structures for 58 proteins, we find it recognizes the native conformation for essentially all compact, soluble, globular proteins having known native conformations in comparisons with 104 to 106 reasonable alternative conformations apiece. In this sense, the potential encodes nearly all the essential features of globular protein conformational preference. In addition it “knows” about many additional factors in protein folding, such as the stabilization of multimeric proteins, quaternary structure, the role of disulfide bridges and ligands, proproteins vs. processed proteins, and minimal strand lengths in globular proteins. Comparisons are made with other sorts of protein folding problems, and applications in protein conformational determination and prediction are discussed. © 1994 Wiley‐Liss, Inc.
Conformational analysis of hemopexin by fourier‐transform infrared and circular dichroism spectroscopy
Authors: Wu Ming‐Lei , Morgan William T.
Published: May 2005

Hemopexin is a serum glyco‐protein that binds heme with the highest known affinity of any characterized heme‐binding protein and plays an important role in receptormediated cellular heme uptake. Complete understanding of the function of hemopexin will require the elucidation of its molecular structure. Previous analysis of the secondary structure of hemopexin by far‐UV circular dichroism (CD) failed due to the unusual positive ellipticity of this protein at 233 nm. In this paper, we present an examination of the structure of hemopexin by both Fourier‐transform infrared (FTIR) and circular dichroism spectroscopy. Our studies show that hemopexin contains about 55% β‐structure, 15% α‐helix, and 20% turns. The two isolated structural domains of hemopexin each have secondary structures similar to hemopexin. Although there are significant tertiary conformational changes indicated by the CD spectra, the overall secondary structure of hemopexin is not affected by binding heme. However, moderate changes in secondary structure do occur when the heme‐binding domain of hemopexin associates with heme. In spite of the exceptionally tight binding at neutral pH, heme is released from the bis‐histidyl heme–hemopexin complex at pH 5.0. Under this acidic condition, hemopexin maintains the same overall secondary structure as the native protein and is able to resume the heme‐binding function and the native structure of the hemeprotein (as indicated by the CD spectra) when returned to neutral pH. We propose that the state of hemopexin identified in vitro at pH 5.0 resembles that of this protein in the acidic environment of the endosomes in vivo when hemopexin releases heme during receptor‐mediated endocytosis. © 1994 Wiley‐Liss, Inc.
Protein simulations using techniques suitable for very large systems: The cell multipole method for nonbond interactions and the Newton‐Euler inverse mass operator method for internal coordinate dynamics
Authors: Mathiowetz Alan M. , Jain Abhinandan , Karasawa Naoki , Goddard William A.
Published: May 2005

Two new methods developed for molecular dynamics simulations of very large proteins are applied to a series of proteins ranging up to the protein capsid of tomato bushy stunt virus (TBSV).

For molecular dynamics of very large proteins and polymers, it is useful to carry out the dynamics using internal coordinates (say, torsions only) rather than Cartesian coordinates. This allows larger time steps, eliminates problems with the classical description of high energy modes, and focuses on the important degrees of freedom. The resulting equation of motion has the form

equation image

where for T is the vector of generalized forces, ℳ︁(θ) is the moments of inertia tensor, is the vector of torsions, and 𝒞 is a vector containing Coriolis forces and nonbond forces. The problem is that to calculate the acceleration vector from ℳ︁, 𝒞, and Trequires inverting. ℳ︁(θ), an order 𝒩3calculation. Since the number of degrees of freedom might be 300,000 for a million atom system, solving these equations every time step is impractical, restricting internal coordinate methods to small systems. The new method, Newton‐Euler Inverse Mass Operator (NEIMO) dynamics, constructs the torsional accelerations vector directly by an order 𝒩 process, allowing internal‐coordinate dynamics to be solved for super larger (million atom) systems, The first use of the NEIMO method for molecular dynamics of proteins is presented here.

A second serious difficulty for large proteins is calculation of the nonbond forces. We report here the first application to proteins of the new Cell Multipole Method (CMM) to evaluate the Coulomb and van der Waals interactions. The cost of CMM scales linearly with the number of particles while retaining an accuracy significantly better than standard non bond methods (involving cutoffs).

Results for NEIMO and CMM are given for simulations of a wide range of peptide and protein systems, including the protein capsid of TBSV with 488,000 atoms. The computational times for NEIMO and CMM are demonstrated to scale linearly with size. With NEIMO the dynamics time steps can be as large as 20 fs (for small peptides), much larger than possible with standard Cartesian coordinate dynamics.

For TBSV we considered both the normal form and the high pH form, in which the Ca2+ ions are removed. These calculations lead to a contraction of the protein for both forms (probably because of ignoring the RNA core not observed in the X‐ray). © 1994 Wiley‐Liss, Inc.
Polar and nonpolar atomic environments in the protein core: Implications for folding and binding
Authors: Koehl Patrice , Delarue Marc
Published: May 2005

Hydrophobic interactions are believed to play an important role in protein folding and stability. Semi‐empirical attempts to estimate these interactions are usually based on a model of solvation, whose contribution to the stability of proteins is assumed to be proportional to the surface area buried upon folding. Here we propose an extension of this idea by defining an environment free energy that characterizes the environment of each atom of the protein, including solvent, polar or nonpolar atoms of the same protein or of another molecule that interacts with the protein. In our model, the difference of this environment free energy between the folded state and the unfolded (extended) state of a protein is shown to be proportional to the area buried by nonpolar atoms upon folding. General properties of this environment free energy are derived from statistical studies on a database of 82 well‐refined protein structures. This free energy is shown to be able to discriminate misfolded from correct structural models, to provide an estimate of the stabilization due to oligomerization, and to predict the stability of mutants in which hydrophobic residues have been substituted by site‐directed mutagenesis, provided that no large structural modifications occur. © 1994 Wiley‐Liss, Inc.
Crystallization and preliminary crystallographic analysis of the N‐terminal two domain fragment of vascular cell adhesion molecule‐1 (VCAM‐1)
Authors: Wang Jia‐huai , Pepinsky Blake , Karpusas Michael , Liu Jin‐huan , Osborn Laurelee
Published: May 2005

A molecular fragment comprising the first two domains of the human vascular cell adhesion molecule‐l (VCAM‐l) has been crystallized by the vapor diffusion method. Two crystal forms have been examined by X‐ray analysis: One crystal form belongs to the space group C2 with two molecules in the asymmetric unit and cell parameters: a = 122.1 Å, b = 48.9 Å, c = 73.4 Å, and β = 117.4°. The other crystal form belongs to the space group P21 with one molecule in the asymmetric unit and cell parameters: a = 40.4 Å, b = 45.7 Å, c = 54.7 Å, and β = 100.5°. Diffraction data up to 1.9 Å resolution have been collected for the C2 crystal form. © 1994 Wiley‐Liss, Inc.
Homology models of two isozymes of manganese peroxidase: Prediction of a Mn(II) binding site
Authors: Johnson Forrester , Loew Gilda H. , Du Ping
Published: May 2005

The three‐dimensional structures of two isozymes of manganese peroxidase (MnP) have been predicted from homology modeling using lignin peroxidase as a template. Although highly homologous, MnP differs from LiP by the requirement of Mn(II) as an intermediate in its oxidation of substrates. The Mn(II) site is absent in LiP and unique to the MnP family of peroxidases. The model structures were used to identify the unique Mn(II) binding sites, to determine to what extent they were conserved in the two isozymes, and to provide insight into why this site is absent in LiP. For each isozyme of MnP, three candidate Mn(II) binding sites were identified. Energy optimizations of the three possible Mn(II) enzyme complexes allowed the selection of the most favorable Mn(II) binding site as one with the most anionic oxygen moieties best configured to act as ligands for the Mn(II). At the preferred site, the Mn(II) is coordinated to the carboxyl oxygens of Glu‐35, Glu‐39, and Asp‐179, and a propionate group of the heme. The predicted Mn(II) binding site is conserved in both isozymes. Comparison between the residues at this site in MnP and the corresponding residues in LiP shows that two of the three anionic residues in MnP are replaced by neutral residues in LiP, explaining why LiP does not bind Mn(II). © 1994 Wiley‐Liss, Inc.
Structural analysis of two crystal forms of lentil lectin at 1.8 Å resolution
Authors: Loris Remy , van Overberge David , Dao‐Thi Minh‐Hoa , Poortmans Freddy , Maene Norbert , Wyns Lode
Published: May 2005

The structures of two crystal forms of lentil lectin are determined and refined at high resolution. Orthorhombic lentil lectin is refined at 1.80 Å resolution to anR‐factor of 0.184 and monoclinic lentil lectin at 1.75 Å resolution to anR‐factor of 0.175. These two structures are compared to each other and to the other available legume lectin structures. The monosaccharide binding pocket of each lectin monomer contains a tightly bound phosphate ion. This phosphate makes hydrogen bonding contacts with Asp‐81β, Gly‐99β, and Asn‐125β, three residues that are highly conserved in most of the known legume lectin sequences and essential for monosaccharide recognition in all legume lectin crystal structures described thus far. A detailed analysis of the composition and properties of the hydrophobic contact network and hydrophobic nuclei in lentil lectin is presented. Contact map calculations reveal that dense clusters of nonpolar as well as polar side chains playa major role in secondary structure packing. This is illustrated by a large cluster of 24 mainly hydrophobic amino acids that is responsible for the majority of packing interactions between the two β‐sheets. Another series of four smaller and less hydrophobic clusters is found to mediate the packing of a number of loop structures upon the front sheet. A very dense, but not very conserved cluster is found to stabilize the transition metal binding site. The highly conserved and invariant nonpolar residues are distributed asymmetrically over the protein. © 1994 Wiley‐Liss, Inc.
Crystallization, molecular replacement solution, and refinement of tetrameric β‐amylase from sweet potato
Authors: Cheong Cheom Gil , Eom Soo Hyun , Chang Changsoo , Shin Dong Hae , Song Hyun Kyu , Min Kyeongsik , Moon Jin Ho , Kim Kyeong Kyu , Hwang Kwang Yeon , Suh Se Won
Published: May 2005

Sweet potato β‐amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P42212 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β‐amylase, with a crystal volume per protein mass (VM) of 2.57 Å3/Da and a solvent content of 52% by volume. The three‐dimensional structure of the tetrameric β‐amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R‐value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut‐off) with good stereochemistry. The subunit structure of sweet potato β‐amylase (crystallized in the absence of α‐cyclodextrin) is very similar to that of soybean β‐amylase (complexed with α‐cyclodextrin). The root‐mean‐square (RMS) difference for 487 equivalent Cα atoms of the two β‐amylases is 0.96 Å. Each subunit of sweet potato β‐amylase is composed of a large (α/β)8 core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C‐terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)8 barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)8 barrel. © 1995 Wiley‐Liss, Inc.
A model of the complex between interleukin‐4 and its receptors
Authors: Gustchina Alla , Zdanov Alexander , Schalk‐Hihi Céline , Wlodawer Alexander
Published: May 2005

A three‐dimensional model of interleukin‐4 (IL‐4) bound to one molecule each of the high‐ and low‐affinity receptors (IL‐4R and IL‐2Rγ) was built, using the crystal structure of the complex of human growth hormone (HGH) with its receptor (HGHR) as a starting model. The modeling of IL‐4 with its receptors was based on the conservation of the sequences and on the predicted structural organization for cytokine receptors, and assuming that the binding mode of the ligands would be similar. Analysis of the interface between IL‐4 and both receptor molecules was carried out to reveal which residues are important for complex formation. The modeling procedures showed that there were no major problems in maintaining a reasonable fit of IL‐4 with the two receptor molecules, in a manner analogous to the complex of HGH–HGHR. Many of the residues that appear by modeling to be important for binding between IL‐4 and the receptors have been previously implicated in that role by different methods. A striking motif of aromatic and positively charged residues on the surface of the C‐terminal domains of the receptors is highly conserved in the structure of HGH–HGHR and in the models of IL‐4 complexed with its receptors. © 1995 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray diffraction studies of an a1/α2/DNA ternary complex
Authors: Li Thomas , Wolberger Cynthia , Stark Martha , Johnson Alexander D.
Published: May 2005

Crystals have been obtained of a ternary complex containing the yeast a1/α2 homeodomain heterodimer bound to a 21‐base pair DNA site containing two 5′ overhanging bases at each end. The crystals are grown from cobaltic hexamine and form in space group P61 or P65 with a = b = 133 Å, c = 45.4 Å. Crystals that are flash‐frozen at −179°C diffract to 2.7 Å along the c‐axis and to 2.4 Å in perpendicular directions. The crystals contain one protein–DNA complex in the crystallographic asymmetric unit. © 1995 Wiley‐Liss, Inc.
Funnels, pathways, and the energy landscape of protein folding: A synthesis
Authors: Bryngelson Joseph D. , Onuchic José Nelson , Socci Nicholas D. , Wolynes Peter G.
Published: May 2005

The understanding, and even the description of protein folding is impeded by the complexity of the process. Much of this complexity can be described and understood by taking a statistical approach to the energetics of protein conformation, that is, to the energy landscape. The statistical energy landscape approach explains when and why unique behaviors, such as specific folding pathways, occur in some proteins and more generally explains the distinction between folding processes common to all sequences and those peculiar to individual sequences. This approach also gives new, quantitative insights into the interpretation of experiments and simulations of protein folding thermodynamics and kinetics. Specifically, the picture provides simple explanations for folding as a two‐state first‐order phase transition, for the origin of metastable collapsed unfolded states and for the curved Arrhenius plots observed in both laboratory experiments and discrete lattice simulations. The relation of these quantitative ideas to folding pathways, to uniexponential vs. multiexponential behavior in protein folding experiments and to the effect of mutations on folding is also discussed. The success of energy landscape ideas in protein structure prediction is also described. The use of the energy landscape approach for analyzing data is illustrated with a quantitative analysis of some recent simulations, and a qualitative analysis of experiments on the folding of three proteins. The work unifies several previously proposed ideas concerning the mechanism protein folding and delimits the regions of validity of these ideas under different thermodynamic conditions. © 1995 Wiley‐Liss, Inc.
Modeling the quinone‐B binding site of the photosystem‐II reaction center using notions of complementarity and contact‐surface between atoms
Authors: Sobolev Vladimir , Edelman Marvin
Published: May 2005

Functional identity and significant similarities in cofactors and sequence exist between the L and M reaction center proteins of the photosynthetic bacteria and the D1 and D2 photosystem‐II reaction center proteins of cyanobacteria, algae, and plants. A model of the quinone (QB) binding site of the D1 protein is presented based upon the resolved structure of the QB binding pocket of the L subunit, and introducing novel quantitative notions of complementarity and contact surface between atoms. This model, built ‐without using traditional methods of molecular mechanics and restricted to residues in direct contact with QB, accounts for the experimentally derived functional state of mutants of the Dl protein in the region of QB. It predicts the binding of both the classical and phenol‐type PSII herbicides and rationalizes the relative levels of tolerance of mutant phenotypes. © 1995 Wiley‐Liss, Inc.
Dramatic differences in the motions of the mouth of open and closed cytochrome P450BM‐3 by molecular dynamics simulations
Authors: Paulsen Mark D. , Ornstein Rick L.
Published: May 2005

Molecular dynamics trajectories were calculated separately for each of the two molecules in the asymmetric unit of the crystal structure of the hemoprotein domain of cytochrome P450BM‐3. Each simulation was 200 ps in length and included a 10 Å layer of explicit solvent. The simulated time‐average structure of each P450BM‐3 molecule is closer to its crystal structure than the two molecular dynamics time‐averaged structures are to each other. In the crystal structure, molecule 2 has a more accessible substrate binding pocket than molecule 1, and this difference is maintained throughout the simulations presented here. In particular, the substrate docking regions of molecule 1 and molecule 2 diverge in the solution state simulations. The mouth of the substrate binding pocket is significantly more mobile in the simulation of molecule 2 than in the simulation of molecule 1. For molecule 1, the width of the mouth is only slightly larger than its X‐ray value of 8.7 Å and undergoes fluctuations of about 1 Å. However, in molecule 2, the mouth of the substrate binding pocket is dramatically more open in the time‐average molecular dynamics structure (14.7 Å) than in the X‐ray structure (10.9 Å). Furthermore, this region of the protein undergoes large amplitude motions during the trajectory that are not seen in the trajectory of molecule 1, repeatedly opening and closing up to 7 Å. Presumably, the binding of different substrates will induce the mouth region to adopt different conformations from within the wide range of structures that are accessible. © 1995 Wiley‐Liss, Inc.
Cocrystallization of Lysyl–tRNA synthetase from Thermus thermophilus with its cognate tRNAlys and with Escherichia coli tRNAlys
Authors: Yaremchuk A. D. , Cusack S. , Tukalo M. A. , Krikliviy I. A.
Published: May 2005

Lysyl‐tRNA synthetase from Thermus thermophilus has been cocrystallized with either its cognate tRNAlYS or Escherichia coli tRNAlys using ammonium sulfate as precipitant. The crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA in 18–22% ammonium sulfate in 50 mM Tris‐maleate buffer at pH 7.5. Both complexes form square prismatic, tetragonal crystals with very similar unit cell parameters (a = b = 233 Å, c = 119 Å) and diffract to at least 2.7 Å resolution. However the homocomplex is of space group P4212 and the heterocomplex of space group I422. © 1995 Wiley‐Liss, Inc.
Purification, crystallization, and preliminary X‐ray diffraction analysis of even‐skipped homeodomain complexed to DNA
Authors: Hirsch Joel A. , Aggarwal Aneel K.
Published: May 2005

Embryonic development in metazoa, to a significant extent, is directed by genes which contain a conserved sequence motif named the homeobox. This sequence encodes a polypeptide called the homeodomain which has sequence specific DNA‐binding activity. We report the purification, crystallization, and preliminary diffraction analysis of the Drosophila Even‐skipped homeodomain (Eve HD) bound to two different oligonucleotides. Crystals of Eve HD complexed with an AT‐rich sequence belong to space group P21, a = 34.06, b = 61.61, c = 39.99 Å, b=90.0°. These crystals diffract to at least 2.0 Å and both native and derivative data sets have been collected. Crystals of Eve HD complexed with a GC‐rich sequence belong to space group P63, a = b = 124.52, c = 66.78 Å and diffract to 3.5 Å resolution. A native data set has been collected. © 1995 Wiley‐Liss, Inc.
Investigation of the folding pathway of the TEM‐1 β‐lactamase
Authors: Vanhove Marc , Raquet Xavier , Frère Jean‐Marie
Published: May 2005

The TEM‐1 β‐lactamase is a globular protein containing 12 proline residues. The folding mechanism of this enzyme was investigated by kinetic and equilibrium experiments with the help of fluorescence spectroscopy and circular dichroism. The equilibrium denaturation of the protein induced by guanidine hydrochloride occurs in two discrete steps, indicating the existence of a thermodynamically stable intermediate state. Thisstate is 5.2 ± 0.4 kcal/mol less stable than the native conformation and 5.7 ± 0.2 kcal/mol more stable than the fully denaturedprotein. This intermediate state exhibits a high content of native secondary structure elements but is devoid of specific tertiary organization; its relation to the “molten globule” is discussed. Refolding kinetic experimentsrevealed the existence of a transient intermediate conformation between thethermodynamically stable intermediate and the native protein. This transient intermediate appears rapidly during the folding reaction. It exhibits a secondary structure content very similar to that of the native protein and has also recovered a significant amount of tertiary organisation. The final refolding step of the TEM‐1 β‐lactamase, leading to the native enzyme, is dominated by two major slow kinetic phases which probablyreflect a very complex process kinetically limited by proline cis/transisomerization. © 1995 Wiley‐Liss, Inc.
Insulin and epidermal growth factor receptors contain the cysteine repeat motif found in the tumor necrosis factor receptor
Authors: Ward C. W. , Hoyne P. A. , Flegg R. H.
Published: May 2005

The insulin receptor (INSR) and epidermal growth factor receptor (EGFR) are representatives of two structurally related subfamilies of tyrosine kinase receptors. Using the Wisconsin GCG sequence analysis programs, we have demonstrated that the cysteinerich regions of INSR and EGFR conform to the structural motif found in the tumor necrosis factorreceptor (TNFR) family. The study also revealed that these regions were not composed of simple repeats of eight cysteine residues as previously proposed and that the second Cysrich region of EGFR contained one fewer TNFR repeat than the first. The sequence alignments identified two cysteineresidues in INSR that could be responsible for the additional disulfide bonds known to be involved in dimer formation. The published data on the alignments for the fibronectin type III repeat region of the INSR together with previous cysteine mutagenesis studies indicated that there were two disulfide bonds linking the α and β chains of the INSR, but only one α‐β linkage in the insulin‐like growth factor 1 receptor (IG 1R). Database searches and sequence alignments showed that the TNFR motif is also found in the cysteine‐rich repeats of laminins and the noncatalytic domains of furin‐like proteases. If the starting position of the repeat is altered the characteristic laminin repeat of eight cysteine residues can be shown to consist of a TNFR‐like motif fused to the last half of an EGF‐like repeat. The overlapping regions of these two motifs are known to have identical disulfide bonding patterns and similar protein folds. © 1995 Wiley‐Liss, Inc.
Molecular dynamics simulations of rubredoxin from Clostridium pasteurianum: Changes in structure and electrostatic potential duringredox reactions
Authors: Yelle Robert B. , Park Noh‐Sum , Ichiye Toshiko
Published: May 2005

Molecular dynamics simulations of Clostridium pasteurianum rubredoxin in the oxidized and reduced forms have been performed. Good agreement between both forms and crystal data has been obtained (rms deviation of backbone atoms of 1.06 and 1.42 Å, respectively), which was due in part to the use of explicit solvent and counterions. The reduced form exhibits an unexpected structural change: the redox site becomes much more solvent‐accessible, so that water enters a channel between the surface and the site, but with little actual structural rearrangement (the rms deviation of backbone atoms between the oxidized and reduced is 0.77 Å). The increase in solvent accessibility is also seen, although to a much lesser extent, between the oxidized and reduced crystal structures of Pyrococcus furiosus rubredoxin, but no high resolution crystal or nuclear magnetic resonance solution data exist for reduced C. pasteurianum rubredoxin. The electrostatic potential at the iron site and fluctuations in the potential, which contribute to both the redox and electron transfer properties, have also been evaluated for both the oxidized and the reduced simulations. These results show that the backbone plays a significant role (62–70 kcall/mol/e) and the polar sidechains contribute relatively little (0–4 kcal/mol/e) to the absolute electrostatic potential at the iron of rubredoxin for both forms. However, both groups contribute significantly to the change in redox state by becoming more polarized and more densely packed around the redox site upon reduction. Furthermore, these results show that the solvent becomes much more polarized in the reduced form than in the oxidized form, even excluding the penetrating water. Finally, the simulation indicates that the contribution of the charged side chains to the electrostatic potential is largely canceled by that of the counterions. © 1995 Wiley‐Liss, Inc.
The cytidylyltransferase superfamily: Identification of the nucleotide‐binding site and fold prediction
Authors: Bork Peer , Holm Liisa , Koonin Eugene V. , Sander Chris
Published: May 2005

The crystal structure of glycerol‐3‐phosphate cytidylyltransferase from B. subtilis (TagD) is about to be solved. Here, we report a testable structure prediction based on the identification by sequence analysis of a superfamily of functionally diverse but structurally similar nucleotide‐binding enzymes. We predict that TagD is a member of this family. The most conserved region in this superfamily resembles the ATP‐binding HiGH motif of class I aminoacyI‐tRNA synthetases. The predicted secondary structure of cytidylyltransferase and its homologues is compatible with the α/β topography of the class I aminoacyl‐tRNA synthetases. The hypothesis of similarity of fold is strengthened by sequence‐structure alignment and 3D model building using the known structure of tyrosyl tRNA synthetase as template. The proposed 3D model of TagD is plausible both structurally, with a well packed hydrophobic core, and functionally, as the most conserved residues cluster around the putative nucleotide binding site. If correct, the model would imply a very ancient evolutionary link between class I tRNA synthetases and the novel cytidylyltransferase superfamily. © 1995 Wiley‐Liss, Inc.
Size‐independent comparison of protein three‐dimensional structures
Authors: Maiorov Vladimir N. , Crippen Gordon M.
Published: May 2005

Protein structures are routinely compared by their root‐mean‐square deviation (RMSD) in atomic coordinates after optimal rigid body superposition. What is not so clear is the significance of different RMSD values, particularly above the customary arbitrary cutoff for obvious similarity of 2–3 Å. Our earlier work argued for an intrinsic cutoff for protein similarity that varied with the number of residues in the polypeptide chains being compared. Here we introduce a new measure, ρ, of structural similarity based on RMSD that is independent of the sizes of the molecules involved, or of any other special properties of molecules. When ρ is less than 0.4–0.5, protein structures are visually recognized to be obviously similar, but the mathematically pleasing intrinsic cutoff of ρ>1.0 corresponds to overall similarity in folding motif at a level not usually recognized until smoothing of the polypeptide chain path makes it striking. When the structures are scaled to unit radius of gyration and equal principle moments of inertia, the comparisons are even more universal, since they are no longer obscured by differences in overall size and ellipticity. With increasing chain length, the distribution of ρ for pairs of random structures is skewed to higher values, but the value for the best 1% of the comparisons rises only slowly with the number of residues. This level is close to an intrinsic cutoff between similar and dissimilar comparisons, namely the maximal scaled ρ possible for the two structures to be more similar to each other than one is to the other's mirror image. The intrinsic cutoff is independent of the number of residues or points being compared. For proteins having fewer than 100 residues, the 1% ρ falls below the intrinsic cutoff, so that for very small proteins, geometrically significant similarity can often occur by chance. We believe these ideas will be helpful in judging success in NMR structure determination and protein folding modeling. © 1995 Wiley‐Liss, Inc.
Crystallization and preliminary crystallographic analysis of a 2,3‐dihydroxybiphenyl dioxygenase from Pseudomonas sp. strain KKS102 having polychlorinated biphenyl (PCB)‐degrading activity
Authors: Sugiyama Kazuyuki , Narita Hiroki , Yamamoto Takeshi , Senda Toshiya , Kimbara Kazuhide , Inokuchi Norio , Iwama Masanori , Irie Masachika , Fukuda Masao , Yano Keiji , Mitsui Yukio
Published: May 2005

Crystals have been obtained for a 2,3‐dihydroxybiphenyl dioxygenase (conventionally called BphC) from a polychlorinated biphenyl (PCB)‐degrader, Pseudomonas sp. strain KKS1O2. The crystals were grown using both ammonium sulfate and MPD as the precipitating agents. The crystals belonged to a tetragonal space group (I422) and diffracted to 2.5 Å. © 1995 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray crystallographic studies of nonhistone region of macroH2A.1
Authors: Senadhi Vijay‐Kumar , Chandra Naveen , Dharia Chhaya , Pehrson John R.
Published: May 2005

Histone macroH2A has a novel hybrid structure consisting of a large nonhistone region and a region that closely resembles a full‐length histone H2A. One key to understanding macroH2A function is determining the structure and function of its nonhistone region. The nonhistone region of one of the two known macroH2A subtypes was expressed in Escherichia coli and purified using affinity and molecular sieve chromatography. Crystals of the protein suitable for structural studies were grown from polyethylene glycol solutions by vapor equilibration techniques. The crystals belong to the hexagonal space group P64 (or its enantiomorph P62) with unit cell parameters: a = b = 106.2 Å, c = 125.9 Å, α = β = 90°, and γ = 120°. There are four molecules in the asymmetric unit. Self‐rotation function studies revealed three twofold noncrystallographic rotation axes related approximately by 222 symmetry. These crystals have 47% solvent content and diffract to 3.8 Å resolution. © 1995 Wiley‐Liss, Inc.
Automatic identification of discrete substates in proteins: Singular value decomposition analysis of time‐averaged crystallographic refinements
Authors: Romo T. D. , Clarage J. B. , Sorensen D. C. , Phillips G. N.
Published: May 2005

The singular value decomposition (SVD) provides a method for decomposing a molecular dynamics trajectory into fundamental modes of atomic motion. The right singular vectors are projections of the protein conformations onto these modes showing the protein motion in a generalized low‐dimensional basis. Statistical analysis of the right singular vectors can be used to classify discrete configurational substates in the protein. The configuration space portraits formed from the right singular vectors can also be used to visualize complex high‐dimensional motion and to examine the extent of configuration space sampling by the simulation. © 1995 Wiley‐Liss, Inc.
Phe‐46(CD4) orients the distal histidine for hydrogen bonding to bound ligands in sperm whale myoglobin
Authors: Lai Henry H. , Li Tiansheng , Lyons Daniel S. , Phillips George N. , Olson John S. , Gibson Quentin H.
Published: May 2005

The role of Phe‐46(CD4) in modulating the functional properties of sperm whale myoglobin was investigated by replacing this residue with Leu, Ile, Val, Ala, Trp, Tyr, and Glu. This highly conserved amino acid almost makes direct contact with the distal histidine and has been postulated to affect ligand binding. The overall association rate constants for CO, O2, and NO binding were little affected by decreasing the size of residue 46 step‐wise from Phe to Leu to Val to Ala. In contrast, the rates of CO, O2, and NO dissociation increased 4‐, 10‐, and 25‐fold, respectively, for the same series of mutants, causing large decreases in the affinity of myoglobin for all three diatomic gases. The rates of autooxidation at 37°C, pH 7.0 increased dramatically from ∼0.1–0.3 h−1 for wild‐type, Tyr‐46, and Trp‐46 myoglobins to 1.5, 5.2, 4.9, and 5.0 h−1 for the Leu‐46, Ile‐46, Val‐46, and Ala‐46 mutants, respectively. Rates of NO and O2 geminate recombination were measured using 35 ps and 9 ns laser excitation pulses. Decreasing the size of residue 46 causes significant decreases in the extent of both picosecond and nanosecond rebinding processes. High resolution structures of Leu‐46 and Val‐46 metmyoglobins, Val‐46 CO‐myoglobin, and Val‐46 deoxymyoglobin were determined by X‐ray crystallography. When Phe‐46 is replaced by Val, the loss of internal packing volume is compensated by (1) contraction of the CD corner toward the core of the protein, (2) movement of the E‐helix toward the mutation site, (3) greater exposure of the distal pocket to intruding solvent molecules, and (4) large disorder in the position of the side chain of the distal histidine (His‐64). In wild‐type myoglobin, the van der Waals contact between Cζ of Phe‐46 and Cβ of His‐64 appears to restrict rotation of the imidazole side chain. Insertion of Val at position 46 relieves this steric restriction, allowing the imidazole side chain to rotate about the Cα–Cβ bond toward the surface of the globin and about the Cβ–Cγ bond toward the space previously occupied by the native Phe‐46 side chain. This movement disrupts hydrogen bonding with bound ligands, causing significant decreases in affinity, and opens the distal pocket to solvent water molecules, causing marked increases in the rate of autooxidation. The upward movement of the imidazole side chain also creates new space for photodissociated ligands and for incoming water molecules to approach the iron atom. Both of these phenomena inhibit geminate recombination and can be correlated with molecular dynamics calculations. All of these results show that this mutation in the second shell of amino acids around the distal pocket can influence ligand binding significantly. © 1995 Wiley‐Liss, Inc.
Empirical correlation for the replacement of Ala by Gly: Importance of amino acid secondary intrinsic propensities
Authors: López‐Hernández Eva , Serrano Luis
Published: May 2005

A series of Ala vs. Gly mutations at different helical and nonhelical positions of the chemotactic protein CheY, fromE. coli, has been made. We have used this information to fit a general analytical equation that describes the free energy changes of an Ala to Gly mutation within ±0.45 kcal mol−1with 95% confidence. The equation includes three terms: (1) the change in solvent‐accessible hydrophobic surface area, corrected for the possible closure of the cavity left by deleting the Cβof the Ala; (2) the change in hydrophilic area of the nonintramolecularly hydrogen‐bonded groups; and (3) the dihedral angles of the position being mutated. This last term extends the calculation to any conformation, not only α‐helices. The general applicability of the equation for Ala vs. Gly mutations, when Ala or a small solvent‐exposed polar residue is the wild‐type residue, has been tested using data from other proteins: barnase, CI2 trypsin inhibitor, T4 lysozyme, andStaphylococcusnuclease. The predictive power of this simple approach offers the possibility of extending it to more complex mutations. © 1995 Wiley‐Liss, Inc.
Interaction stabilizing tertiary structure of bacteriorhodopsin studied by denaturation experiments
Authors: Mitaku Shigeki , Suzuki Keiko , Odashima Seiji , Ikuta Kazuya , Suwa Makiko , Kukita Fumio , Ishikawa Mitsuru , Itoh Hiroyasu
Published: May 2005

The structural stability of bacteriorhodopsin was studied by denaturation experiments, using aliphatic alcohol as denaturants. The disappearance of a positive peak at 285 nm of the circular dichroism spectra, the change in the intrinsic fluorescence decay time, and the decrease of the regeneration activity bacteriorhodopsin indicated the denaturation of the tertiary structure of this protein at a methanol concentration of about 3 M. The circular dichroism band at 222 nm was unchanged by the denaturation. It was concluded that the alcohol‐denatured state in water was similar to the molten globule state of soluble proteins, in which only the tertiary structure was destroyed. Solvent substitution from water to hexane did not cause denaturation of bacteriorhodopsin. However, further addition of alcohol destroyed the secondary as well as the tertiary structures. Comparing the alcohol effects on bacteriorhodopsin in water to that in hexane, the dominant interactions for the structure formation of this protein could be revealed: the hydrophobic interaction that arose from the structure of water is essential for the stability of membrane spanning helices, while the interaction which binds the helices is polar in nature. © 1995 Wiley‐Liss, Inc.
Multiple modes of ligand recognition: Crystal structures of cyclin‐dependent protein kinase 2 in complex with ATP and two inhibitors, olomoucine and isopentenyladenine
Authors: Schulze‐Gahmen Ursula , Brandsen Jeroen , Jones Heather D. , Morgan David O. , Meijer Laurent , Vesely Jaroslav , Kim Sung‐Hou
Published: May 2005

Cyclin‐dependent kinases (CDKs) are conserved regulators of the eukaryotic cell cycle with different isoforms controlling specific phases of the cell cycle. Mitogenic or growth inhibitory signals are mediated, respectively, by activation or inhibition of CDKs which phosphorylate proteins associated with the cell cycle. The central role of CDKs in cell cycle regulation makes them a potential new target for inhibitory molecules with anti‐proliferative and/or anti‐neoplastic effects. We describe the crystal structures of the complexes of CDK2 with a weakly specific CDK inhibitor, N6‐(δ2‐isopentenyl)adenine, and a strongly specific inhibitor, olomoucine. Both inhibitors are adenine derivatives and bind in the adenine binding pocket of CDK2, but in an unexpected and different orientation from the adenine of the authentic ligand ATP. The N6‐benzyl substituent in olomoucine binds outside the conserved binding pocket and is most likely responsible for its specificity. The structural information from the CDK2‐olomoucine complex will be useful in directing the search for the next generation inhibitors with improved properties. © 1995 Wiley‐Liss, Inc.
Optimal local propensities for model proteins
Authors: Govindarajan Sridhar , Goldstein Richard A.
Published: May 2005

Lattice models of proteins were used to examine the role of local propensities in stabilizing the native state of a protein, using techniques drawn from spin‐glass theory to characterize the free‐energy landscapes. In thestrongevolutionary limit, optimal conditions for folding are achieved when the contributions from local interactions to the stability of the native state is small. Further increasing the local interactions rapidly decreases the foldability. © 1995 Wiley‐Liss, Inc.
Stability of TEM β‐lactamase mutants hydrolyzing third generation cephalosporins
Authors: Raquet X. , Vanhove M. , Lamotte‐Brasseur J. , Goussard S. , Courvalin P. , Frère J‐M.
Published: May 2005

The stability properties of six natural mutants of the TEM‐1 β‐lactamase have been studied. The glutamate to lysine substitution at positions 104 and 240 stabilize the enzyme. Conversely, the G238S mutant's decreased stability might reflect an altered conformation of the active site and thus be related to the modified substrate profile. The relative stability of the R164S and R164H mutants is explained by the formation of a hydrogen bond between these residues and Asp‐179 conferring a somewhat different structure to the omega loop and thus also explaining the extended substrate profile of these mutants. The loss of stability of the R164H mutant with increasing pH values can be explained by the titration of a hydrogen bond between the Nδ of His‐164 and the Oδ of Asp‐179. The properties of the G238S+E104K double mutant which is the most active against third‐generation cephalosporins result from a balance of destabilizing and stabilizing substitutions, and their effects seem to be additive. The behavior of the R164S + E240K mutant might be explained on the basis of a similar compensation phenomenon. © 1995 Wiley‐Liss, Inc.
Protein conformational landscapes: Energy minimization and clustering of a long molecular dynamics trajectory
Authors: Troyer John M. , Cohen Fred E.
Published: May 2005

Using energy minimization and cluster analysis, we have analyzed a 1020 ps molecular dynamics trajectory of solvated bovine pancreatic trypsin inhibitor. Elucidation of conformational sub states in this way both illustrates the degree of conformational convergence in the simulation and reduces the structural data to a tractable subset. The relative movement of structures upon energy minimization was used to estimate the sizes of features on the protein potential energy surface. The structures were analyzed using their pairwise root‐mean‐square Cα deviations, which gave a global measure of conformational changes that would not be apparent by monitoring single degrees of freedom. At time scales of 0.1 ps, energy minimization detected sharp transitions between energy minima separated by 0.1 Å rms deviation. Larger conformational clusters containing these smaller minima and separated by 0.25 Å were seen at 1 ps time scales. Both of these small features of the conformational landscape were characterized by movements in loop regions associated with small, correlated backbone dihedral angle shifts. On a nanosecond time scale, the main features of the protein energy landscape were clusters separated by over 0.7 Å rms deviation, with only seven of these sub states visited over the 1 ns trajectory. These substates, discernible both before and after energy minimization, differ mainly in a monotonic pivot of the loop residues 11–18 over the course of the simulation. This loop contains lysine 17, which specifically binds to trypsin in the active site. The trajectory did not return to previously visited clusters, indicating that this trajectory has not been shown to have completely sampled the conformational substates available to it. Because the apparent convergence to a single region of conformation space depends on both the time scale of observation and the size of the conformational features examined, convergence must be operationally defined within the context of the simulation. © 1995 Wiley‐Liss, Inc.