bluuebunny/crossref_metadata_2025_split huggingface dataset:
Simulated annealing with restrained molecular dynamics using a flexible restraint potential: Theory and evaluation with simulated NMR constraints
Authors: Bassolino‐Klimas Donna , Tejero Roberto , Krystek Stanley R. , Metzler William J. , Montelione Gaetano T. , Bruccoleri Robert E. 
Published: July 2010

AbstractA new functional representation of NMR‐derived distance constraints, the flexible restraint potential, has been implemented in the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137–168) for molecular structure generation. In addition, flat‐bottomed restraint potentials for representing dihedral angle and vicinal scalar coupling constraints have been introduced into CONGEN. An effective simulated annealing (SA) protocol that combines both weight annealing and temperature annealing is described. Calculations have been performed using ideal simulated NMR constraints, in order to evaluate the use of restrained molecular dynamics (MD) with these target functions as implemented in CONGEN. In this benchmark study, internuclear distance, dihedral angle, and vicinal coupling constant constraints were calculated from the energy‐minimized X‐ray crystal structure of the 46‐amino acid polypeptide crambin (1CRN). Three‐dimensional structures of crambin that satisfy these simulated NMR constraints were generated using restrained MD and SA. Polypeptide structures with extended backbone and side‐chain conformations were used as starting conformations. Dynamical annealing calculations using extended starting conformations and assignments of initial velocities taken randomly from a Maxwellian distribution were found to adequately sample the conformational space consistent with the constraints. These calculations also show that loosened internuclear constraints can allow molecules to overcome local minima in the search for a global minimum with respect to both the NMR‐derived constraints and conformational energy. This protocol and the modified version of the CONGEN program described here are shown to be reliable and robust, and are applicable generally for protein structure determination by dynamical simulated annealing using NMR data.
Thermosensitive mutants of the MPTP and hPTP1B protein tyrosine phosphatases: Isolation and structural analysis
Authors: Muise Eric S. , Vrielink Alice , Ennis Maurice A. , Lemieux Nancy H. , Tremblay Michel L. 
Published: July 2010

AbstractA PCR‐based random mutagenesis procedure was employed to identify several thermosensitive mutants of the MPTP enzyme, the murine homologue of the human T‐cell PTPase and rat PTP‐S enzymes. Four mutants with varying degrees of thermosensitivity were characterized for their thermostability and refolding properties following incubation at the nonpermissive temperature. Structure analysis of these mutations based on the hPTP1B coordinate structure demonstrates a clear relationship between the position of each mutated amino acid relative to the catalytic cysteine residue and their thermostability. Introduction of two of these mutations in the related enzyme hPTP1B suggests that the structural defects and the resulting thermosensitivity of these mutations may represent an intrinsic property of all PTPase catalytic domains.
Expression, characterization, and crystallization of the catalytic core of rat phosphatidylinositide‐specific phospholipase C δ1
Authors: Grobler Jay A. , Hurley James H. 
Published: July 2010

AbstractPhosphatidylinositide‐specific phospholipase Cs (PI‐PLCs) catalyze the calcium‐dependent hydrolysis of phosphatidylinositides in response to diverse stimuli in higher eukaryotes. Mammalian PI‐PLCs contain divergent regulatory regions, but all share three conserved regions: an N‐terminal pleckstrin homology (PH) domain, X, and Y. We report the high‐level expression and characterization of a recombinant “catalytic core” of rat PI‐PLC δ1 that contains the catalytically essential X and Y regions, but not the PH domain. The expressed protein, PI‐PLC δδ1–134, is catalytically active versus phosphatidylinositol 4, 5‐bisphosphate in deoxycholate micelles with a Km of 182 μM and a Vmax of 27 μmol/min/mg. PI‐PLC δδ1–134 is monomeric and monodisperse as judged by dynamic light scattering. Far‐UV CD indicates a structure with approximately 35% α‐helix. A reversible change in the near‐ UV CD spectrum is observed on addition of calcium, suggesting that calcium can bind PI‐PLC δδ1–134 in the absence of phospholipid. Triclinic crystals of PI‐PLC δδ1–134 have been obtained that diffract beyond 2.4 A resolution under cryogenic conditions. Based on Vm = 2.72 Da/Å3 and on the self‐rotation function, there are two PI‐PLC δδ1–134 molecules per asymmetric unit that are related to each other by a noncrystallographic axis of approximate twofold symmetry parallel to a.
Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: Implications for domain switching
Authors: Murata Lauren B. , Schachman H. K. 
Published: July 2010

AbstractEach catalytic (c) polypeptide chain of Escherichia coli aspartate transcarbamoylase (ATCase) is composed of two globular domains connected by two interdomain helices. Helix 12, near the C‐terminus, extends from the second domain back through the first domain, bringing the two termini close together. This helix is of critical importance for the assembly of a stable enzyme. The trimeric E. coli enzyme ornithine transcarbamoylase (OTCase) is proposed to be similar in tertiary and quaternary structure to the ATCase trimer and has a predicted α‐helical segment near its C‐terminus. In our companion paper, we have shown that this putative helix is essential for OTCase folding and assembly (Murata L, Schachman HK, 1996, Protein Sci 5:709–718). Here, the similarity between OTCase and the ATCase trimer, which are 32% identical in sequence, was tested further by the construction of several chimeras in which various structural elements were switched between the enzymes by genetic techniques. These elements included the two globular domains and regions containing the C‐terminal helices. In contrast to results reported previously (Houghton J, O'Donovan G, Wild J, 1989, Nature 338:172–174), none of the chimeric proteins exhibited in vivo activity and all were insoluble when overexpressed. Attempts to make hybrid trimers composed of c chains from ATCase and OTCase were also unsuccessful. These results underscore the complexities of specific intrachain and interchain side‐chain interactions required to maintain tertiary and quaternary structures in these enzymes.
4‐Oxalocrotonate tautomerase, a 41‐kDa homohexamer: Backbone and side‐chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy
Authors: Stivers James T. , Abeygunawardana Chitrananda , Whitman Christian P. , Mildvan Albert S. 
Published: July 2010

Abstract4‐Oxalocrotonate tautomerase (4‐OT), a homohexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated α‐keto acids using Pro‐1 as a general base (Stivers et al., 1996a, 1996b). We report the backbone and side‐chain 1H, 15N, and 13C NMR assignments and the solution secondary structure for 4‐OT using 2D and 3D homonuclear and heteronuclear NMR methods. The subunit secondary structure consists of an α‐helix (residues 13–30), two β‐strands (β1, residues 2–8; β2, residues 39–45), a β‐hairpin (residues 50–57), two loops (1, residues 9–12; II, 34–38), and two turns (I, residues 30–33; II, 47–50). The remaining residues form coils. The β1 strand is parallel to the β2 strand of the same subunit on the basis of cross strand NHi‐NHj NOEs in a 2D 15N‐edited 1H‐NOESY spectrum of hexameric 4‐OT containing two 15N‐labeled subunits/hexamer. The β1 strand is also antiparallel to another β1 strand from an adjacent subunit forming a subunit interface. Because only three such pairwise interactions are possible, the hexamer is a trimer of dimers. The diffusion constant, determined by dynamic light scattering, and the rotational correlation time (14.5 ns) estimated from 15N T1/T2 measurements, are consistent with the hexameric molecular weight of 41 kDa. Residue Phe‐50 is in the active site on the basis of transferred NOEs to the bound partial substrate 2‐oxo‐l, 6‐hexanedioate. Modification of the general base, Pro‐1, with the active site‐directed irreversible inhibitor, 3‐bromopyruvate, significantly alters the amide 15N and NH chemical shifts of residues in the β‐hairpin and in loop II, providing evidence that these regions change conformation when the active site is occupied.
Destabilizing effect of proline substitutions in two helical regions of T4 lysozyme: Leucine 66 to proline and leucine 91 to proline
Authors: Gray Terry M. , Arnoys Eric J. , Blankespoor Stephen , Born Tim , Jagar Rebekah , Everman Rebecca , Plowman Darla , Stair Angela , Zhang Daisy 
Published: July 2010

AbstractA class of temperature‐sensitive (ts) mutants of T4 lysozyme with reduced activity at 30 °C and no activity at 43 °C has been selected. These mutants, designated “tight” ts mutants, differ from most other T4 lysozyme mutants that are active at 43 °C, but only manifest their ts lesion by a reduced halo size around phage plaques after exposure of the growth plates to chloroform vapors. For example, in the series of T4 lysozyme mutants at position 157, the original randomly selected mutant, T157I, is the least stable of the series, yet, apart from the halo assay and subsequent in vitro protein stability measurements, this mutant is indistinguishable from wild type (WT) even at 43 °C.Two mutants were identified: L91P and L66P. Both insert proline residues into α‐helical regions of the WT protein structure. The stabilities (δδG) as determined by urea denaturation are 8.2 kcal/mol for L91P and 7.1 kcal/mol for L66P. CD spectra indicate that no major conformational changes have occurred in the mutant structures. The structures of the mutants were modeled with a 40‐ps molecular dynamics simulation using explicit solvent. For L91P, the reduction of stability appears to be due to an unsatisfied hydrogen bond in the α‐helix and to a new buried cavity. For L66P, the reduction of stability appears to be due to a disruption of the interdomain α‐helix, at least two unsatisfied hydrogen bonds, and a newly formed solvent‐filled pocket that protrudes into the hydrophobic core, possibly reducing the stabilizing contribution of a partially buried intrachain salt bridge.
The importance of larger data sets for protein secondary structure prediction with neural networks
Authors: Chandonia John‐Marc , Karplus Martin 
Published: July 2010

AbstractA neural network algorithm is applied to secondary structure and structural class prediction for a database of 318 nonhomologous protein chains. Significant improvement in accuracy is obtained as compared with performance on smaller databases. A systematic study of the effects of network topology shows that, for the larger database, better results are obtained with more units in the hidden layer. In a 32‐fold cross validated test, secondary structure prediction accuracy is 67.0%, relative to 62.6% obtained previously, without any evolutionary information on the sequence. Introduction of sequence profiles increases this value to 72.9%, suggesting that the two types of information are essentially independent. Tertiary structural class is predicted with 80.2% accuracy, relative to 73.9% obtained previously. The use of a larger database is facilitated by the introduction of a scaled conjugate gradient algorithm for optimizing the neural network. This algorithm is about 10–20 times as fast as the standard steepest descent algorithm.
Extremely thermostable L(+)‐lactate dehydrogenase from thermotoga maritima: Cloning, characterization, and crystallization of the recombinant enzyme in its tetrameric and octameric state
Authors: Ostendorp Ralf , Auerbach Günter , Jaenicke Rainer 
Published: July 2010

AbstractL(+)‐lactate dehydrogenase (LDH; E.C.1.1.1.27) from the hyperthermophilic bacterium Thermotoga maritima has been shown to represent the most stable LDH isolated so far (Wrba A, Jaenicke R, Huber R, Stetter KO, 1990, Eur J Biochem 188:195–201). In order to obtain the enzyme in amounts sufficient for physical characterization, and to analyze the molecular basis of its intrinsic stability, the gene was cloned and expressed functionally in Escherichia coli. Growth of the cells and purification of the enzyme were performed aerobically at 26 °C, i.e., ca. 60° below the optimal growth temperature of Thermotoga. Two enzyme species with LDH activity were purified to homogeneity. Crystals of the enzyme obtained at 4 °C show satisfactory diffraction suitable for X‐ray analysis up to a resolution of 2.8 Å. As shown by gel‐permeation chromatography, chemical crosslinking, light scattering, analytical ultracentrifugation, and electron microscopy, the two LDH species represent homotetramers and homooctamers (i.e., dimers of tetramers), with a common subunit molecular mass of 35 kDa. The spectroscopic characteristics (UV absorption, fluorescence emission, near‐ and far‐UV CD) of the two species are indistinguishable. The calculated α‐helix content is 45%, in accordance with the result of homology modeling. Compared to the tetrameric enzyme, the octamer exhibits reduced specific activity, whereas KM is unaltered. The extreme intrinsic stability of the protein is reflected by its unaltered catalytic activity over 4 h at 85 °C; irreversible thermal denaturation becomes significant at ∼95 °C. The anomalous resistance toward chemical denaturation using guanidinium chloride and urea confirms this observation. Both the high optimal temperature and the pH optimum of the catalytic activity correspond to the growth conditions of T. maritima in its natural habitat.
Protein fold recognition using sequence‐derived predictions
Authors: Fischer Daniel , Eisenberg David 
Published: July 2010

AbstractIn protein fold recognition, one assigns a probe amino acid sequence of unknown structure to one of a library of target 3D structures. Correct assignment depends on effective scoring of the probe sequence for its compatibility with each of the target structures. Here we show that, in addition to the amino acid sequence of the probe, sequence‐derived properties of the probe sequence (such as the predicted secondary structure) are useful in fold assignment. The additional measure of compatibility between probe and target is the level of agreement between the predicted secondary structure of the probe and the known secondary structure of the target fold. That is, we recommend a sequence‐structure compatibility function that combines previously developed compatibility functions (such as the 3D‐1D scores of Bowie et al. [1991] or sequence‐sequence replacement tables) with the predicted secondary structure of the probe sequence.The effect on fold assignment of adding predicted secondary structure is evaluated here by using a benchmark set of proteins (Fischer et al., 1996a). The 3D structures of the probe sequences of the benchmark are actually known, but are ignored by our method. The results show that the inclusion of the predicted secondary structure improves fold assignment by about 25%. The results also show that, if the true secondary structure of the probe were known, correct fold assignment would increase by an additional 8–32%. We conclude that incorporating sequence‐derived predictions significantly improves assignment of sequences to known 3D folds.Finally, we apply the new method to assign folds to sequences in the SWISSPROT database; six fold assignments are given that are not detectable by standard sequence‐sequence comparison methods; for two of these, the fold is known from X‐ray crystallography and the fold assignment is correct.
Self‐consistently optimized statistical mechanical energy functions for sequence structure alignment
Authors: Koretke K. K. , Luthey‐Schulten Z. , Wolynes P. G. 
Published: July 2010

AbstractA quantitative form of the principle of minimal frustration is used to obtain from a database analysis statistical mechanical energy functions and gap parameters for aligning sequences to three‐dimensional structures. The analysis that partially takes into account correlations in the energy landscape improves upon the previous approximations of Goldstein et al. (1994, 1995) (Goldstein R, Luthey‐Schulten Z, Wolynes P, 1994, Proceedings of the 27th Hawaii International Conference on System Sciences. Los Alamitos, California: IEEE Computer Society Press. pp 306–315; Goldstein R, Luthey‐Schulten Z, Wolynes P, 1995, In: Elber R, ed. New developments in theoretical studies of proteins. Singapore: World Scientific). The energy function allows for ordering of alignments based on the compatibility of a sequence to be in a given structure (i.e., lowest energy) and therefore removes the necessity of using percent identity or similarity as scoring parameters. The alignments produced by the energy function on distant homologues with low percent identity (less than 21%) are generally better than those generated with evolutionary information. The lowest energy alignment generated with the energy function for sequences containing prosite signatures but unknown structures is a structure containing the same prosite signature, providing a check on the robustness of the algorithm. Finally, the energy function can make use of known experimental evidence as constraints within the alignment algorithm to aid in finding the correct structural alignment.
Direct evidence for a two‐state protein unfolding transition from hydrogen‐deuterium exchange, mass spectrometry, and NMR
Authors: Yi Qian , Baker David 
Published: July 2010

AbstractWe use mass spectrometry in conjunction with hydrogen‐deuterium exchange and NMR to characterize the conformational dynamics of the 62‐residue IgG binding domain of protein L under conditions in which the native state is marginally stable. Mass spectra of protein L after short incubations in D2O reveal the presence of two distinct populations containing different numbers of protected protons. NMR experiments indicate that protons in the hydrophobic core are protected in one population, whereas all protons are exchanged for deuterons in the other. As the exchange period is increased, molecules are transferred from the former population to the latter. The absence of molecules with a subset of the core protons protected suggests that exchange occurs in part via a highly concerted transition to an excited state in which all protons exchange rapidly with deuterons. A steady increase in the molecular weight of the population with protected protons, and variation in the exchange rates of the individual protected protons indicates the presence of an additional exchange mechanism. A simple model in which exchange results from rapid (>105/s) local fluctuations around the native state superimposed upon transitions to an unfolded excited state at ∼0.06/s is supported by qualitative agreement between the observed mass spectra and the mass spectra simulated according to the model using NMR‐derived estimates of the proton exchange rates.
The heme redox center of chloroplast cytochrome f is linked to a buried five‐water chain
Authors: Martinez S. E. , Huang D. , Ponomarev M. , Cramer W. A. , Smith J. L. 
Published: July 2010

AbstractThe crystal structure of the 252‐residue lumen‐side domain of reduced cytochrome f, a subunit of the proton‐pumping integral cytochrome b6f complex of oxygenic photosynthetic membranes, was determined to a resolution of 1.96 Å from crystals cooled to —35°. The model was refined to an R‐factor of 15.8% with a 0.013‐Å RMS deviation of bond lengths from ideality. Compared to the structure of cytochrome f at 20°, the structure at —35° has a small change in relative orientation of the two folding domains and significantly lower isotropic temperature factors for protein atoms. The structure revealed an L‐shaped array of five buried water molecules that extend in two directions from the NΔ1 of the heme ligand His 25. The longer branch extends 11 Å within the large domain, toward Lys 66 in the prominent basic patch at the top of the large domain, which has been implicated in the interaction with the electron acceptor, plastocyanin. The water sites are highly occupied, and their temperature factors are comparable to those of protein atoms. Virtually all residues that form hydrogen bonds with the water chain are invariant among 13 known cytochrome f sequences. The water chain has many features that optimize it as a proton wire, including insulation from the protein medium. It is suggested that this chain may function as the lumen‐side exit port for proton translocation by the cytochrome b6f complex.
The proteins encoded by the rbs operon of escherichia coli: II. Use of chimeric protein constructs to isolate and characterize RbsC
Authors: Zaitseva Jelena , Zhang Huide , Binnie R. Alan , Hermodson Mark 
Published: July 2010

AbstractChimeric genes encoding full‐length copies of rbsA and rbsC connected by segments coding for short bridge peptides were constructed and expressed in Escherichia coli. Surprisingly, the chimeric genes complemented the strain in which rbsA and rbsC were deleted. The chimeric proteins were overproduced, and the products were purified by affinity chromatography. In order to obtain highly purified protein, a poly‐His leader peptide was incorporated so that Ni‐chelate affinity chromatography could be employed. The leader peptide and the bridge peptide were designed with factor Xa‐cleavable sites to permit recovery of the individual RbsA and RbsC protein. A rbsC gene encoding a poly‐His leader was also constructed and expressed. Both the chimeric RbsA‐C species and the poly‐HisRbsC were produced at levels that permitted isolation of the equivalent of milligram quantities of RbsC per liter of culture. This is a substantial increase in amounts from any previous RbsC production vectors. All proteins from the rbs operon have now been overproduced and substantially purified.
Secondary structure and NMR assignments ofbacillus circulansxylanase
Authors: Plesniak Leigh A. , Mcintosh Lawrence P. , Wakarchuk Warren W. 
Published: July 2010

AbstractBacillus circulansxylanase (BCX) is a member of the family of low molecular weight endo‐β‐(1,4)‐xylanases. The main‐chain1H,13C, and15N resonances of this 20.4‐kDa enzyme were assigned using heteronuclear NMR experiments recorded on a combination of selectively and uniformly labeled protein samples. Using chemical shift, NOE, J coupling, and amide hydrogen exchange information, 14 β‐strands, arranged in a network of three β‐sheets, and a single α‐helix were identified in BCX. The NMR‐derived secondary structure and β‐sheet topology agree closely with that observed in the crystal structure of this protein. The HNof Ile 118 has a strongly upfield‐shifted resonance at 4.03 ppm, indicative of a potential amide‐aromatic hydrogen bond to the indole ring of Trp 71. This interaction, which is conserved in all low molecular weight xylanases of known structure, may play an important role in establishing the active site conformation of these enzymes. Following hen egg white and bacteriophage T4 lysozymes,B. circulansxylanase represents the third family of β‐glycanases for which extensive NMR assignments have been reported. These assignments provide the background for detailed studies of the mechanism of carbohydrate recognition and hydrolysis by this bacterial xylanase.