bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modeling conformational changes in cyclosporin A
Authors: O'Donohue Mark F. , Burgess Antony W. , Treutlein Herbert R , Walkinshaw Malcolm D. 
Published: February 2009

AbstractNMR and X‐ray structures for the immunosuppressant cyclosporin A (CsA) reveal a remarkable difference between the unbound (free) conformation in organic solvents and the conformation bound to cyclophilin. We have performed computer simulations of the molecular dynamics of CsA under a variety of conditions and confirmed the stability of these two conformations at room temperature in water and in vacuum. However, when the free conformation was modeled in vacuum at 600 K, a transition pathway leading to the bound conformation was observed. This involved a change in the cis MeLeu‐9 peptide bond to a trans conformation and the movement of the side chains forming the dominant hydrophobic cluster (residues MeBmt‐1, MeLeu‐4, MeLeu‐6, and MeLeu‐10) to the opposite side of the plane formed by the backbone atoms in the molecular ring. The final conformation had a backbone RMS deviation from the bound conformation of 0.53 Å and was as stable in dynamics simulations as the bound conformation. Our calculations allowed us to make a detailed analysis of a transition pathway between the free and the bound conformations of CsA and to identify two distinct regions of coordinated movement in CsA, both of which underwent transitions independently.
Molecular modeling of the GM‐CSF and IL‐3 receptor complexes
Authors: Lyne P.D. , Bamborough P. , Duncan D. , Richards W.G. 
Published: February 2009

AbstractA model for the structure of the cytokine interleukin‐3 (IL‐3) is presented based on the structural homology of the hematopoietic cytokines and utilizing the crystal structures of interleukin‐5 and granulocyte macrophage colony stimulating factor (GM‐CSF). In addition, models of the receptor complexes of GM‐CSF and IL‐3 are presented based on the structural homology of the hematopoietic receptors to growth hormone. Several key interactions between the ligands and their receptors are discovered, some in agreement with previous mutagenesis studies and others that have not yet been the subject of mutagenesis studies. The models provide insights into the binding of GM‐CSF and IL‐3 to their receptors.
Urease activity in the crystalline state
Authors: Moncrief Mary Beth C. , Hausinger Robert P. , Hom Louis G. , Jabri Evelyn , Andrew Karplus P. 
Published: February 2009

AbstractCrystalline Klebsiella aerogenes urease was found to have less than 0.05% of the activity observed for the soluble enzyme under standard assay conditions. Li2SO4, present in the crystal storage buffer at 2 M concentration, was shown to inhibit soluble urease by a mixed inhibition mechanism (Ki's of 0.38 ± 0.05 M for the free enzyme and 0.13 ± 0.02 M for the enzyme‐urea complex). However, the activity of crystals was less than 0.5% of the expected value, suggesting that salt inhibition does not account for the near absence of crystalline activity. Dissolution of crystals resulted in ˜43% recovery of the soluble enzyme activity, demonstrating that protein denaturation during crystal growth does not cause the dramatic diminishment in the catalytic rate. Finally, crushed crystals exhibited only a threefold increase in activity over that of intact crystals, indicating that the rate of substrate diffusion into the crystals does not significantly limit the enzyme activity. We conclude that urease is effectively inactive in this crystal form, possibly due to conformational restrictions associated with a lid covering the active site, and propose that the small amounts of activity observed arise from limited enzyme activity at the crystal surfaces or trace levels of enzyme dissolution into the crystal storage buffer.
Role of the N‐terminal region of the skeletal muscle myosin light chain kinase target sequence in its interaction with calmodulin
Authors: Findlay Wendy A. , Gradwell Michael J. , Bayley Peter M. 
Published: February 2009

AbstractThe binding of calmodulin (CaM) to four synthetic peptide analogues of the skeletal muscle myosin light chain kinase (sk‐MLCK) target sequence has been studied using 1H‐NMR. The 18‐residue peptide WFF is anchored to CaM via the interaction of the Trp 4 side chain with the C‐domain and the Phe 17 side chain with the N‐domain of the protein. A peptide corresponding to the first 10 residues (WF10) does not provide the second anchoring residue and is not long enough to span both domains of CaM. 1H‐NMR spectroscopy indicates that the WF10 peptide interacts specifically with the C‐domain of CaM, and the chemical shifts of the bound Trp side chain are very similar in the CaM:WF10 and CaM:WFF complexes. Binding of the C‐domain of CaM to the strongly basic region around Trp 4 of this MLCK sequence may be an important step in target recognition. Comparison of 1H‐NMR spectra of CaM bound to WFF, a Trp 4 → Phe analogue (FFF), or a Trp 4 → Phe/Phe 17 → Trp analogue (FFW) suggests that all three peptides bind to CaM in the same orientation, i.e., with the peptide side chain in position 4 interacting with the C‐domain and the side chain in position 17 interacting with the N‐domain. This indicates that a Trp residue in position 4 is not an absolute requirement for binding this target sequence and that interchanging the Trp 4 and Phe 17 residues does not reverse the orientation of the bound peptide, in confirmation of the deduction from previous indirect studies using circular dichroism (Findlay WA, Martin SR, Beckingham K, Bayley PM, 1995, Biochemistry 34: 2087–2094). Molecular modeling/energy minimization studies indicate that only minor local changes in the protein structure are required to accommodate binding of the bulkier Trp 17 side chain of the FFW peptide to the N‐domain of CaM.
Addition of side chain interactions to modified Lifson‐Roig helix‐coil theory: Application to energetics of Phenylalanine‐Methionine interactions
Authors: Stapley Benjamin J. , Rohl Carol A. , Doig Andrew J. 
Published: February 2009

AbstractWe introduce here i, i + 3 and i, i + 4 side chain interactions into the modified Lifson‐Roig helix‐coil theory of Doig et al. (1994, Biochemistry 33:3396–3403). The helix/coil equilibrium is a function of initiation, propagation, capping, and side chain interaction parameters. If each of these parameters is known, the helix content of any isolated peptide can be predicted. The model considers every possible conformation of a peptide, is not limited to peptides with only a single helical segment, and has physically meaningful parameters. We apply the theory to measure the i, i + 4 interaction energies between Phe and Met side chains. Peptides with these residues spaced i, i + 4 are significantly more helical than controls where they are spaced i, i + 5. Application of the model yields ΔG for the Phe‐Met orientation to be −0.75 kcal·mol−1, whereas that for the Met‐Phe orientation is −0.54 kcal·mol−1. These orientational preferences can be explained, in part, by rotamer preferences for the interacting side chains. We place Phe‐Met i, i + 4 at the N‐terminus, the C‐terminus, and in the center of the host peptide. The model quantitatively predicts the observed helix contents using a single parameter for the side chain‐side chain interaction energy. This result indicates that the model works well even when the interaction is at different locations in the helix.
Crystallization and preliminary X‐ray crystallographic analysis of phosphoribulokinase from Rhodobacter sphaeroides
Authors: Roberts David L. , Runquist Jennifer A. , Miziorko Henry M. , Kim Jung‐Ja P. 
Published: February 2009

AbstractA recombinant form of Rhodobacter sphaeroides phosphoribulokinase (PRK), expressed in Escherichia coli and isolated by affinity chromatography, was crystallized by the sitting drop vapor diffusion technique using NH4H2PO4 (pH 5.6) as the precipitating agent. PRK crystallizes in the cubic space group P432, with unit cell parameters a = b = c = 129.55 Å. Based on the assumption of one 32‐kDa monomer per asymmetric unit, the Vm value is 2.83 Å3/Da. The octameric molecular symmetry is consistent with two planar tetramers stacked in a nearly eclipsed arrangement. A native data set has been collected to 2.6 Å resolution.
Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor
Authors: Van Doren Steven R. , Kurochkin Alexander V. , Hu Weidong , Ye Qi‐Zhuang , Johnson Linda L. , Hupe Donald J. , Zuiderweg ERIK R.P. 
Published: February 2009

AbstractStromelysin, a representative matrix metalloproteinase and target of drug development efforts, plays a prominent role in the pathological proteolysis associated with arthritis and secondarily in that of cancer metastasis and invasion. To provide a structural template to aid the development of therapeutic inhibitors, we have determined a medium‐resolution structure of a 20‐kDa complex of human stromelysin's catalytic domain with a hydrophobic peptidic inhibitor using multinuclear, multidimensional NMR spectroscopy. This domain of this zinc hydrolase contains a mixed β‐sheet comprising one antiparallel strand and four parallel strands, three helices, and a methionine‐containing turn near the catalytic center. The ensemble of 20 structures was calculated using, on average, 8 interresidue NOE restraints per residue for the 166‐residue protein fragment complexed with a 4‐residue substrate analogue. The mean RMS deviation (RMSD) to the average structure for backbone heavy atoms is 0.91 A and for all heavy atoms is 1.42 Å. The structure has good stereochemical properties, including its backbone torsion angles. The β‐sheet and α‐helices of the catalytic domains of human stromelysin (NMR model) and human fibroblast collagenase (X‐ray crystallographic model of Lovejoy B et al., 1994b, Biochemistry 55:8207‐8217) superimpose well, having a pairwise RMSD for backbone heavy atoms of 2.28 Å when three loop segments are disregarded. The hydroxamate‐substituted inhibitor binds across the hydrophobic active site of stromelysin in an extended conformation. The first hydrophobic side chain is deeply buried in the principal S1' subsite, the second hydrophobic side chain is located on the opposite side of the inhibitor backbone in the hydrophobic S2' surface subsite, and a third hydrophobic side chain (P3') lies at the surface.
Crystallization and preliminary X‐ray analysis of L‐2‐haloacid dehalogenase from xanthobacter autotrophicus GJ10
Authors: Ridder Ivo S. , Rozeboom Henriëtte J. , Dijkstra Bauke W. , Kingma Jaap , Janssen Dick B. 
Published: February 2009

AbstractHaloacid dehalogenases are enzymes that cleave carbon‐chlorine or carbon‐bromine bonds of 2‐haloalkanoates. X‐ray‐quality crystals of L‐2‐haloacid dehalogenase from the 1, 2‐dichloroethane‐degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 20% PEG 8000, 200 mM sodium formate at pH 6.8‐7.0, using macroseeding techniques. The crystals, which diffract in the X‐ray beam up to 2.0 Å resolution, belong to the spacegroup C2221. Cell parameters are a = 58.8 Å, b = 93.1 Å, c = 84.2 Å. A native data set to 2.3 Å has been collected, with a completeness of 97% and an Rsym of 6.0%.
Ethanol utilization regulatory protein: Profile alignments give no evidence of origin through aldehyde and alcohol dehydrogenase gene fusion
Authors: Nicholas Hugh B. , Persson Bengt , Jörnvall Hans , Hempel John 
Published: February 2009

AbstractThe suggestion that the ethanol regulatory protein from Aspergillus has its evolutionary origin in a gene fusion between aldehyde and alcohol dehydrogenase genes (Hawkins AR, Lamb HK, Radford A, Moore JD, 1994, Gene 746:145‐158) has been tested by profile analysis with aldehyde and alcohol dehydrogenase family profiles. We show that the degree and kind of similarity observed between these profiles and the ethanol regulatory protein sequence is that expected from random sequences of the same composition. This level of similarity fails to support the suggested gene fusion.
The lipocalin XLCpl1 expressed in the neural plate ofXenopus laevisembryos is a secreted retinaldehyde binding protein
Authors: Lepperdinger Günter , Strobl Birgit , Weber Anita , Mollay Christa , Jilek Alexander , Thalhamer Josef , Flöckner Hannes 
Published: February 2009

AbstractThe cellular and structural properties and binding capabilities of a lipocalin expressed in the early neural plate ofXenopus laevisembryos and the adult choroid plexus have been investigated. It was found that this lipocalin, termed Xlcpll, binds retinal at a nanomolar concentration, retinoic acid in the micromolar range, but does not show binding to retinol. Furthermore, this protein also binds D/L thyroxine. TheXlcpl1cDNA was expressed in cell culture using the vaccinia virus expression system. In AtT20 cells, Xlcpl1 was secreted via the constitutive secretory pathway. We therefore assume that cpl1 binds retinaldehyde during the transport through the compartments of the secretory pathway that are considered to be the storage compartments of retinoids. Therefore, cpll‐expressing cells will secrete the precursors of active retinoids such as retinoic acid isomers. These retinoids may enter the cytosol by diffusion or receptor‐controlled mechanisms, as has been shown for exogenously applied retinoids. Based on these data, it is suggested that cpl1 is an integral member of the retinoid signaling pathway and, therefore, it plays a key role in pattern formation in early embryonic development.
Free energy mapping of class I MHC molecules and structural determination of bound peptides
Authors: Sezerman Ugur , Vajda Sandor , Delisi Charles 
Published: February 2009

AbstractFree energy maps of the binding site are constructed for class I major histocompatibility complex (MHC) proteins, by rotating and translating amino acid probes along the cleft, and performing a side‐chain conformational search at each position. The free energy maps are used to determine favorable residue positions that are then combined to form docked peptide conformations. Because the generic backbone structural motif of peptides bound to class I MHC is known, the mapping is restricted to appropriate regions of the site, but allows for the sometimes substantial variations in backbone and side‐chain conformations. In a test demonstrating the quality of predictions for a known MHC site using only a rotational and conformational search, we started from the crystal structure of the HIV‐1 gp120/HLA‐A2 complex, and predicted the HLA‐A2 bound structures of peptides from the influenza matrix protein, the HIV‐1 reverse transcriptase, and the human T cell leukemia virus. The calculated peptides are at 1.6, 1.3, and 1.4 Å all‐atom RMSDs from their respective crystal structures (Madden DR, Garboczi DN, Wiley DC, 1993). A further test, which also included a local translational search, predicted structures across MHCs. In particular, we obtained the Kb/SEV‐9 complex (Fremont DH et al., 1992, Science 257:919‐927) starting with the complex between HLA‐B27 and a generic peptide (Madden DR, Gorga JC, Strominger JL, Wiley DC, 1991, Nature (Lond) 555:321‐325), with an all‐atom RMSD of 1.2 Å, indicating that the docking procedure is essentially as effective for predictions across MHCs as it is for determinations within the same MHC, although at substantially greater computational cost. The requirements for further improvement in accuracy are identified and discussed briefly.
In vivo formation of allosteric aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains: Implications for protein folding and assembly
Authors: Zhang Pinghui , Schachman H.K. 
Published: February 2009

AbstractBecause the N‐ and C‐terminal amino acids of the catalytic (c) polypeptide chains of Escherichia coli aspartate transcarbamoylase (ATCase) are in close proximity to each other, it has been possible to form in vivo five different active ATCase variants in which the terminal regions of the wild‐type c chains are linked in a continuous polypeptide chain and new termini are introduced elsewhere in either of the two structural domains of the c chain. These circularly permuted (cp) chains were produced by constructing tandem pyrB genes, which encode the c chain of ATCase, followed by application of PCR. Chains expressed in this way assemble efficiently in vivo to form active, stable ATCase variants. Three such variants have been purified and shown to have the kinetic and physical properties characteristic of wild‐type ATCase composed of two catalytic (C) trimers and three regulatory (R) dimers. The values of Vmax for cpATCase122, cpATCase222, and cpATCase281 ranged from 16‐21 μmol carbamoylaspartate per μg per h, compared with 15 for wild‐type ATCase, and the values for K0.5 for the variants were 4‐17 mM aspartate, whereas wild‐type ATCase exhibited a value of 6 mM. Hill coefficients for the three variants varied from 1.8 to 2.1, compared with 1.4 for the wild‐type enzyme. As observed with wild‐type ATCase, ATP activated the variants containing the circularly permuted chains, as shown by the lowering of K0.5 for aspartate and a decrease in the Hill coefficient (nH). In contrast, CTP caused both an increase in K0.5 and nH for the variants, just as observed with wild‐type ATCase. Thus, the enzyme containing the permuted chains with widely diverse N‐ and C‐termini exhibited the homotropic and heterotopic effects characteristic of wild‐type ATCase. The decrease in the sedimentation coefficient of the variants caused by the binding of the bisubstrate ligand N‐(phosphonacetyl)‐L‐aspartate (PALA) was also virtually identical to that obtained with wild‐type ATCase, thereby indicating that these altered ATCase molecules undergo the analogous ligand‐promoted allosteric transition from the taut (T) state to the relaxed (R) conformation. These ATCase molecules with new N‐ and C‐termini widely dispersed throughout the c chains are valuable models for studying in vivo and in vitro folding of polypeptide chains.
A recipe for designing water‐soluble, β‐sheet‐forming peptides
Authors: Mayo Kevin H. , Ilyina Elena , Park Henry 
Published: February 2009

AbstractBased on observations of solubility and folding properties of peptide 33‐mers derived from the β‐sheet domains of platelet factor‐4 (PF4), interleukin‐8 (IL‐8), and growth related protein (Gro‐α), as well as other β‐sheet‐forming peptides, general guidelines have been developed to aid in the design of water soluble, self‐association‐induced β‐sheet‐forming peptides. CD, 1H‐NMR, and pulsed field gradient NMR self‐diffusion measurements have been used to assess the degree of folding and state of aggregation. PF4 peptide forms native‐like β‐sheet tetramers and is sparingly soluble above pH 6. IL‐8 peptide is insoluble between pH 4.5 and pH 7.5, yet forms stable, nativelike β‐sheet dimers at higher pH. Gro‐α peptide is soluble at all pH values, yet displays no discernable β‐sheet structure even when diffusion data indicate dimer‐tetramer aggregation. A recipe used in the de novo design of water‐soluble β‐sheet‐forming peptides calls for the peptide to contain 40‐50% hydrophobic residues, usually aliphatic ones (I, L, V, A, M) (appropriately paired and mostly but not always alternating with polar residues in the sheet sequence), a positively charged (K, R) to negatively charged (E, D) residue ratio between 4/2 and 6/2, and a noneharged polar residue (N, Q, T, S) composition of about 20% or less. Results on four de novo designed, 33‐residue peptides are presented supporting this approach. Under near physiologic conditions, all four peptides are soluble, form β‐sheet structures to varying degrees, and self‐associate. One peptide folds as a stable, compact β‐sheet tetramer, whereas the others are transient β‐sheet‐containing aggregates.
A theoretical study of the active sites of papain and S195C rat trypsin: Implications for the low reactivity of mutant serine proteinases
Authors: Beveridge Allan J. 
Published: February 2009

AbstractThe serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp … His … Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn … His+ … Cys−) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp … His … Cys triad, are almost catalytically inert. In this study, ab initio Hartree‐Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys− … His+ … Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site‐directed mutagenesis.
Differences in hydropathic properties of ligand binding at four independent sites in wheat germ agglutinin‐oligosaccharide crystal complexes
Authors: Wright Christine Schubert , Kellogg Glen E. 
Published: February 2009

AbstractThe binding interactions of N‐acetyl‐D‐neuraminic acid and N,N' diacetyl‐chitobiose (GlcNAc‐β‐1,4‐GIcNAc), observed in crystal complexes of wheat germ agglutinin (WGA) at four independent sites/monomer, were analyzed and compared with the modeling program HINT (Hydropathic INTeractions). This empirical method allows assessment of relative ligand binding strength and is particularly applicable to cases of weak binding where experimental data is absent. Although the four WGA binding sites are interrelated by a fourfold sequence repeat (eight sites/dimer), similarity extends only to the presence of an aromatic amino acid‐rich pocket and a conserved serine. Strong binding requires additional interactions from a contacting domain in the second subunit. Ligand positions were either derived from crystal structures and further optimized by modeling and molecular mechanics, or from comparative modeling. Analysis of the overall HINT binding scores for the two types of ligands are consistent with the presence of two high‐affinity and two low‐affinity sites per monomer. Identity of these sites correlates well with crystal structure occupancies. The high‐affinity sites are roughly equivalent, as predicted from solution binding studies. Binding scores for the low‐affinity sites are weaker by at least a factor of two. Quantitative estimates for polar, nonpolar, and ionic interactions revealed that H‐bonding makes the largest contribution to complex stabilization in the seven bound configurations, consistent with published thermodynamic data. Although the observed nonpolar interactions are small, they may play a critical role in orienting the ligand optimally.