bluuebunny/crossref_metadata_2025_split huggingface dataset:
Prediction of the three‐dimensional structures of the nerve growth factor and epidermal growth factor binding proteins (kallikreins) and an hypothetical structure of the high molecular weight complex of epidermal growth factor with its binding protein
Authors: Bax Ben , Ferguson Geraldine , Blaber Michael , Sternberg Michael J. E. , Walls Peter H. 
Published: February 2009

AbstractWe have predicted the three‐dimensional structures of the serine protease subunits (γ‐NGF, α‐NGF, and EGF‐BP) of the high molecular weight complexes of nerve growth factor (7S NGF) and epidermal growth factor (HMW‐EGF) from the mouse submandibular gland (from the X‐ray crystal structures of two related glandular kallikreins). The conformations of three of the six loops surrounding the active site are relatively well defined in the models of γ‐NGF and EGF‐BP, but three other loops are likely to have flexible conformations. Although the amino acid sequence of α‐NGF is closely related to those of γ‐NGF and EGF‐BP, it is catalytically inactive. Model‐building studies on α‐NGF suggested that mutations (in α‐NGF) just prior to the active site serine (195) and an unusual N‐terminal sequence are consistent with α‐NGF having a zymogen‐like conformation (similar to that in chymotrypsinogen).An hypothetical model of the quaternary structure of HMW‐EGF has been constructed using this model of EGF‐BP and the NMR structure of murine EGF. The C‐terminal arm of EGF was modeled into the active site of EGF‐BP based on data indicating that the C‐terminal arginine of EGF occupies the S1 subsite of EGF‐BP. Data suggesting one of the surface loops of EGF‐BP is buried in the HMW‐EGF complex and symmetry constraints were important in deriving a schematic model. A molecular docking program was used to fit EGF to EGF‐BP.
Determining protein loop conformation using scaling‐relaxation techniques
Authors: Zheng Qiang , Rosenfeld Rakefet , Vajda Sandor , Delisi Charles 
Published: February 2009

AbstractWe recently developed a rapid loop closure algorithm in which bond lengths are scaled to constrain the ends of a segment to match a known distance and then gradually relaxed to their standard values, with boundary constraints maintained. Although the algorithm predicted the Zif286 zinc‐finger loop to within approximately 2 Å, it had a serious limitation that made its more general use tentative: it omitted the atomic environment of the loop. Here we report an extension of the algorithm to take into account the protein environment surrounding a given loop from the outset of the conformational search and show that it predicts structure with an efficiency and accuracy that could not be achieved without continuous environmental inclusion. The algorithm should be widely applicable to structure determination when complete experimental information is unavailable.
Hydrophobic core repacking and aromatic–aromatic interaction in the thermostable mutant of T4 lysozyme ser 117 → phe
Authors: Anderson D. Eric , Hurley James H. , Nicholson Hale , Baase Walter A. , Matthews Brian W. 
Published: February 2009

AbstractThe T4 lysozyme mutant Ser 117 → Phe was isolated fortuitously and found to be more thermostable than wild‐type by 1.1–1.4 kcal/mol. In the wild‐type structure, the side chain of Ser 117 is in a sterically restricted region near the protein surface and forms a short hydrogen bond with Asn 132. The crystal structure of the S117F mutant shows that the introduced Phe side chain rotates by about 150° about the Cα–Cβ bond relative to wild type and is buried in the hydrophobic core of the protein. Burial of Phe 117 is accommodated by rearrangements of the surrounding side chains of Leu 121, Leu 133, and Phe 153 and by main‐chain shifts, which result in a minimal increase in packing density. The benzyl rings of Phe 117 and Phe 153 form a near‐optimal edge–face interaction in the mutant structure. This aromatic–aromatic interaction, as well as increased hydrophobic stabilization and elimination of a close contact in the wild‐type protein, apparently compensate for the loss of a hydrogen bond and the possible cost of structural rearrangements in the mutant. The structure illustrates the ability of a protein to accommodate a surprisingly large structural change in a manner that actually increases thermal stability. The mutant has activity about 10% that of wild‐type, supportive of the prior hypothesis (Grütter, M.G. & Matthews, B.W., 1982, J. Mol. Biol. 154, 525–535) that the peptidoglycan substrate of T4 lysozyme makes extended contacts with the C‐terminal domain in the vicinity of Ser 117.
Effect of pH and denaturants on the folding and stability of murine interleukin‐6
Authors: Ward Larry D. , Zhang Jian Guo , Simpson Richard J. , Checkley Greg , Preston Barry 
Published: February 2009

AbstractThe conformation and stability of a recombinant mouse interleukin‐6 (mIL‐6) has been investigated by analytical ultracentrifugation, fluorescence spectroscopy, urea‐gradient gel electrophoresis, and near‐ and far‐ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4.0, the tryptophan fluorescence of mIL‐6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence quantum yield was not due to unfolding of the molecule because the conformation of mIL‐6, as judged by both urea‐gradient gel electrophoresis and CD spectroscopy, was stable over the pH range 2.0–10.0. Sedimentation equilibrium experiments indicated that mIL‐6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicochemical studies. Quenching of tryptophan fluorescence (20%) also occurred in the presence of 6 M guanidine hydrochloride upon going from pH 7.4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp‐36 and Trp‐160, may be partially involved in the quenching of endogenous fluorescence. In this regard, similar results were obtained for a 17‐residue synthetic peptide, peptide H1, which corresponds to an N‐terminal region of mIL‐6 (residues Val‐27–Lys–43). The pH‐dependent acid quenching of endogenous tryptophan fluorescence of peptide H1 was 30% in the random coil conformation and 60% in the presence of α‐helix‐promoting solvents. Replacement of His‐33 with Ala‐33 in peptide H1 alleviated a significant portion of the pH‐dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His‐33 with the indole ring of Trp‐36 is a major determinant responsible for the quenching of the endogenous protein fluorescence of mIL‐6.
Defensins promote fusion and lysis of negatively charged membranes
Authors: Fujii Gary , Eisenberg David , Selsted Michael E. 
Published: February 2009

AbstractDefensins, a family of cationic peptides isolated from mammalian granulocytes and believed to permeabilize membranes, were tested for their ability to cause fusion and lysis of liposomes. Unlike α‐helical peptides whose lytic effects have been extensively studied, the defensins consist primarily of β‐sheet. Defensins fuse and lyse negatively charged liposomes but display reduced activity with neutral liposomes. These and other experiments suggest that fusion and lysis is mediated primarily by electrostatic forces and to a lesser extent, by hydrophobic interactions. Circular dichroism and fluorescence spectroscopy of native defensins indicate that the amphiphilic β‐sheet structure is maintained throughout the fusion process. Taken together, these results support the idea that protein‐mediated membrane fusion depends not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form a three‐dimensional amphiphilic structure, which promotes the efficient mixing of the lipids between membranes. A molecular model for membrane fusion by defensins is presented, which takes into account the contributions of electrostatic forces, hydrophobic interactions, and structural amphiphilicity.
Site‐specific mutations in the N‐terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor
Authors: Carney David F. , Hugli Tony E. 
Published: February 2009

AbstractC5a is an inflammatory mediator that evokes a variety of immune effector functions including chemotaxis, cell activation, spasmogenesis, and immune modulation. It is well established that the effector site in C5a is located in the C‐terminal region, although other regions in C5a also contribute to receptor interaction. We have examined the N‐terminal region (NTR) of human C5a by replacing selected residues in the NTR with glycine via site‐directed mutagenesis. Mutants of rC5a were expressed as fusion proteins, and rC5a was isolated after factor Xa cleavage. The potency of the mutants was evaluated by measuring both neutrophil chemotaxis and degranulation (β‐glucuronidase release). Mutants that contained the single residue substitutions Ile‐6 → Gly or Tyr‐13 → Gly were reduced in potency to 4–30% compared with wild‐type rC5a. Other single‐site glycine substitutions at positions Leu‐2, Ala‐10, Lys‐4, Lys‐5, Glu‐7, Glu‐8, and Lys‐14 showed little effect on C5a potency. The double mutant, Ile‐6 → Gly/Tyr‐13 → Gly, was reduced in potency to <0.2%, which correlated with a correspondingly low binding affinity for neutrophil C5a receptors. Circular dichroism studies revealed a 40% reduction in α‐helical content for the double mutant, suggesting that the NTR contributes stabilizing interactions that maintain local secondary or tertiary structure of C5a important for receptor interaction. We conclude that the N‐terminal region in C5a is involved in receptor binding either through direct interaction with the receptor or by stabilizing a binding site elsewhere in the intact C5a molecule.
Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein
Authors: Chao Heman , Davies Peter L. , Sykes Brian D. , Sönnichsen Frank D. 
Published: February 2009

AbstractTo help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two‐dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase‐directed expression vector. The 67‐amino acid‐long gene product differed from the natural AFP by inclusion of an N‐terminal methionine but was indistinguishable in activity. The NMR spectra of this AFP were complicated by cis‐trans proline isomerization from the C‐terminal sequence YPPA. Substitution of this sequence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP. This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination using 2D 1H NMR spectroscopy. Nine β‐strands are paired to form two triple‐stranded antiparallel sheets and one double‐stranded antiparallel sheet. Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP. These mutants were valuable in clarifying ambiguous NMR spectral assignments amongst the remaining six prolines of rQAE m1.1. In contrast to the replacement of the C‐terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein solubility and antifreeze activity.
Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase
Authors: Daubner S. Colette , Lohse Daniel L. , Fitzpatrick Paul F. 
Published: February 2009

AbstractPhenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin‐dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that the C‐terminal two‐thirds are homologous, while the N‐terminal thirds are not. This is consistent with a model in which the C‐terminal two‐thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains. To test such a model, two mutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized. One protein contains the first 158 amino acids of rat tyrosine hydroxylase. The second lacks the first 155 amino acid residues of this enzyme. The spectral properties of the two domains suggest that their three‐dimensional structures are changed only slightly from intact tyrosine hydroxylase. The N‐terminal domain mutant binds to heparin and is phosphorylated by cAMP‐dependent protein kinase at the same rate as the holoenzyme but lacks any catalytic activity. The C‐terminal domain mutant is fully active, with Vmax and Km values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C‐terminal 330 amino acids. The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, one regulatory and one catalytic.
Bacterial expression and characterization of the CREB bZip module: Circular dichroism and 2D 1H‐NMR studies
Authors: Santiago‐Rivera Zulma I. , Gorenstein David G. , Williams John S. , Andrisani Ourania M. 
Published: February 2009

AbstractIn this paper we describe the expression and purification from bacteria of the recombinant basic leucine zipper (bZip) domain of the cAMP response element binding protein, CREB327. The bZip peptide, CREB259–327, purified to near homogeneity, maintains the sequence‐specific CRE site recognition demonstrated by in vitro competition assays. Alkylation of the three cysteine residues of CREB259–327 was employed to prevent aggregation of the peptide due to cysteine oxidation. The Kd of the purified native and modified CREB259–327 for the CRE site was determined by gel retardation assays to be on the order of 10−7 M. We employed CD spectroscopy to study the folding properties of the native and modified CREB259–327. The CD analyses of the native/modified CREB259–327 peptide demonstrated a 20% increase in the α‐helical content upon binding to the cAMP response‐element. Only a 5% increase in the α‐helical content of CREB259–327 is observed upon binding to the AP‐1 site. This observation contrasts with CREB from the GCN4 protein (Weiss, M.A., et al., 1990, Nature 347, 575–578). In addition, the two‐dimensional (2D) 1H‐NMR studies of the bZip CREB peptide further support the distinct features of the CREB protein, in comparison to GCN4. Analysis by CD and 2D NMR of the dimerization domain of CREB suggests that the distinct DNA binding characteristics of CREB reside in the basic portion of the bZip module.
Role of the C‐terminus in the activity, conformation, and stability of interleukin‐6
Authors: Ward Larry D. , Hammacher Annet , Zhang Jian‐Guo , Morton Craig J. , Simpson Richard J. , Weinstock Janet , Yasukawa Kiyoshi , Norton Raymond S. 
Published: February 2009

AbstractTwo murine interleukin‐6 (mIL‐6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183–187) at the C‐terminus (pMC5) and another with the last five residues of mIL‐6 substituted by the corresponding residues of human IL‐6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was <0.05% of mIL‐6, whereas pMC5H and mIL‐6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL‐6. Both pMC5 and pMC5H, like mIL‐6, failed to interact with recombinant soluble human IL‐6 receptor when assayed by surface plasmon resonance‐based biosensor analysis. These studies suggest that the C‐terminal seven amino acids of human IL‐6, alone, do not define species specificity for receptor binding.A variety of biophysical techniques, as well as the binding of a conformational‐specific monoclonal antibody, indicated that the global fold of the mIL‐6 variants was similar to that of mIL‐6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr‐22 were influenced by the C‐terminal amino acids suggesting that the N‐ and C‐termini of mIL‐6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL‐6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C‐terminal amino acids of IL‐6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.
Modeling of protein loops by simulated annealing
Authors: Collura V. , Higo Junichi , Garnier J. 
Published: February 2009

AbstractA method is presented to model loops of protein to be used in homology modeling of proteins. This method employs the ESAP program of Higo et al. (Higo, J., Collura, V., & Garnier, J., 1992, Biopolymers 32, 33–43) and is based on a fast Monte Carlo simulation and a simulated annealing algorithm. The method is tested on different loops or peptide segments from immunoglobulin, bovine pancreatic trypsin inhibitor, and bovine trypsin. The predicted structure is obtained from the ensemble average of the coordinates of the Monte Carlo simulation at 300 K, which exhibits the lowest internal energy. The starting conformation of the loop prior to modeling is chosen to be completely extended, and a closing harmonic potential is applied to N, CA, C, and O atoms of the terminal residues. A rigid geometry potential of Robson and Platt (1986, J. Mol. Biol. 188, 259–281) with a united atom representation is used. This we demonstrate to yield a loop structure with good hydrogen bonding and torsion angles in the allowed regions of the Ramachandran map. The average accuracy of the modeling evaluated on the eight modeled loops is 1 Å root mean square deviation (rmsd) for the backbone atoms and 2.3 Å rmsd for all heavy atoms.
Confidence limits on the branching order of phylogenetic trees
Authors: Shearer Andrew C. , Johnson Mark S. 
Published: February 2009

AbstractWe describe a confidence test for branching order that can aid protein phylogeny reconstruction as well as the evaluation of the optimal tree. It is proposed that the process resulting in the observed amino acid residue differences, which is the basis for the identification of the order and relative times of divergence events, is appropriately described by a modification of the negative binomial distribution. The relative total numbers of mutations (accepted and nonaccepted), which result in a given number of amino acid differences, may be obtained as the expectation of this distribution. The associated variances enable significant differences in tree branching order to be established. If the total rates of mutation of the genes encoding the compared proteins are equal, the expected total mutations and their associated variances map identically to their relative times of divergence. In addition, significantly different rates of change (due to differences in total mutation rate and/or acceptance rate) may be identified without the requirement of an outlying reference group. The method is equally applicable to phylogenies derived from DNA or RNA sequence information.
Calculation of protein backbone geometry from α‐carbon coordinates based on peptide‐group dipole alignment
Authors: Liwo A. , Wawak R. J. , Scheraga H. A. , Pincus M. R. , Rackovsky S. 
Published: February 2009

AbstractAn algorithm is proposed for the conversion of a virtual‐bond polypeptide chain (connected Cα atoms) to an all‐atom backbone, based on determining the most extensive hydrogen‐bond network between the peptide groups of the backbone, while maintaining all of the backbone atoms in energetically feasible conformations. Hydrogen bonding is represented by aligning the peptide‐group dipoles. These peptide groups are not contiguous in the amino acid sequence. The first dipoles to be aligned are those that are both sufficiently close in space to be arranged in approximately linear arrays termed dipole paths. The criteria used in the construction of dipole paths are: to assure good alignment of the greatest possible number of dipoles that are close in space; to optimize the electrostatic interactions between the dipoles that belong to different paths close in space; and to avoid locally unfavorable amino acid residue conformations. The equations for dipole alignment are solved separately for each path, and then the remaining single dipoles are aligned optimally with the electrostatic field from the dipoles that belong to the dipole‐path network. A least‐squares minimizer is used to keep the geometry of the α‐carbon trace of the resulting backbone close to that of the input virtual‐bond chain. This procedure is sufficient to convert the virtual‐bond chain to a real chain; in applications to real systems, however, the final structure is obtained by minimizing the total ECEPP/2 (empirical conformational energy program for peptides) energy of the system, starting from the geometry resulting from the solution of the alignment equations. When applied to model α‐helical and β‐sheet structures, the algorithm, followed by the ECEPP/2 energy minimization, resulted in an energy and backbone geometry characteristic of these α‐helical and β‐sheet structures. Application to the α‐carbon trace of the backbone of the crystallographic 5PTI structure of bovine pancreatic trypsin inhibitor, followed by ECEPP/2 energy minimization with C
α
‐distance constraints, led to a structure with almost as low energy and root mean square deviation as the ECEPP/2 geometry analog of 5PTI, the best agreement between the crystal and reconstructed backbone being observed for the residues involved in the dipole‐path network.
Comparison of conformational characteristics in structurally similar protein pairs
Authors: Flores T.P. , Orengo C.A. , Moss D.S. , Thornton J.M. 
Published: February 2009

AbstractAlthough it is known that three‐dimensional structure is well conserved during the evolutionary development of proteins, there have been few studies that consider other parameters apart from divergence of the main‐chain coordinates. In this study, we align the structures of 90 pairs of homologous proteins having sequence identities ranging from 5 to 100%. Their structures are compared as a function of sequence identity, including not only consideration of Cα coordinates but also accessibility, Ooi numbers, secondary structure, and side‐chain angles. We discuss how these properties change as the sequences become less similar. This will be of practical use in homology modeling, especially for modeling very distantly related or analogous proteins. We also consider how the average size and number of insertions and deletions vary as sequences diverge. This study presents further quantitative evidence that structure is remarkably well conserved in detail, as well as at the topological level, even when the sequences do not show similarity that is significant statistically.