bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stabilization of creatinase from Pseudomonas putida by random mutagenesis
Authors: Schumann Judith , Böhm Gerald , Jaenicke Rainer , Schumacher Günter , Rudolph Rainer 
Published: February 2009

AbstractCreatinase (creatine amidinohydrolase, EC 3.5.3.3) from Pseudomonas putida is a homodimer of 45 kDa subunit molecular mass, the three‐dimensional structure of which is known at 1.9 Å resolution. Three point mutants, A109V, V355M, and V182I, as well as one double mutant combining A109V and V355M, and the triple mutant with all three replacements, were compared with wild‐type creatinase regarding their physical and enzymological properties. High‐resolution crystal data for wild‐type creatinase and the first two mutants suggest isomorphism at least for these three proteins (R. Huber, pers. comm.). Physicochemical measurements confirm this prediction, showing that the mutations have no effect either on the quaternary structure and gross conformation or the catalytic properties as compared to wild‐type creatinase. The replacement of V182 (at the solvent‐exposed end of the first helix of the C‐terminal domain) does not cause significant differences in comparison with the wild‐type enzyme. The other point mutations stabilize the first step in the biphasic denaturation transition without affecting the second one. In sum, the enhanced stability seems to reflect slight improvements in the local packing without creating new well‐defined bonds. The increase in hydrophobicity generated by the introduction of additional methyl groups (A109V, V1821) must be compensated by minor readjustments of the global structure. Secondary or quaternary interactions are not affected. In going from single to double and triple mutants, to a first approximation, the increments of stabilization are additive.
Knowledge‐based model building of proteins: Concepts and examples
Authors: Bajorath Jürgen , Stenkamp Ronald , Aruffo Alejandro 
Published: February 2009

AbstractWe describe how to build protein models from structural templates. Methods to identify structural similarities between proteins in cases of significant, moderate to low, or virtually absent sequence similarity are discussed. The detection and evaluation of structural relationships is emphasized as a central aspect of protein modeling, distinct from the more technical aspects of model building. Computational techniques to generate and complement comparative protein models are also reviewed. Two examples, P‐selectin and gp39, are presented to illustrate the derivation of protein model structures and their use in experimental studies.
Toward computational determination of peptide‐receptor structure
Authors: Sezerman Ugur , Vajda Sandor , Cornette James , Delisi Charles 
Published: February 2009

AbstractWe introduce a method for docking small flexible ligands of the size of dipeptides and phosphocholine and test it against crystallographic complexes. We then show how the method can be used as the basis for a strategy for solving the much more difficult problem of docking fully flexible peptides in the 8–10‐residue size range. After developing the method we apply it to peptide–MHC class I systems and find that the predictions are in accord with biological and crystallographic data.
Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants
Authors: Royer Catherine A. , Mann Craig J. , Matthews C. Robert 
Published: February 2009

AbstractSingle tryptophan mutants of the trp aporepressor, tryptophan 19 → phenylalanine (W19F) and tryptophan 99 → phenylalanine (W99F), were used in this study to resolve the individual steady‐state and time‐resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein. The wild‐type protein exhibits a large increase in fluorescence intensity and lifetime, as well as a large red shift in the steady‐state fluorescence emission spectrum, upon unfolding by urea (Lane, A.N. & Jardetsky, O., 1987, Eur. J. Biochem. 164, 389–396; Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011–7020; Fernando, T. & Royer, C.A., 1992, Biochemistry 31, 6683–6691). Unfolding of the W19F mutant demonstrated that Trp 99 undergoes a large increase in intensity and a red shift upon exposure to solvent. Lifetime studies revealed that the contribution of the dominant 0.5‐ns component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance. This lifting of the quenching of Trp 99 may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys. J. 63, 741–750). On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant. Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady‐state emission. Thus, changes in the fluorescence properties of the two intrinsic tryptophan residues in trp aporepressor upon unfolding are explained readily in terms of disruption of the dimer interface.
Kinetics of folding and association of differently glycosylated variants of invertase from saccharomyces cerevisiae
Authors: Kern Gunther , Kern Dorothee , Jaenicke Rainer , Seckler Robert 
Published: February 2009

AbstractA core‐glycosylated form of the dimeric enzyme invertase has been isolated from secretion mutants of Saccharomyces cerevisiae blocked in transport to the Golgi apparatus. This glycosylation variant corresponds to the form that folds and associates during biosynthesis of the protein in vivo. In the present work, its largely homogeneous subunit size and well‐defined quaternary structure were utilized to characterize the folding and association pathway of this highly glycosylated protein in comparison with the nonglycosylated cytoplasmic and the high‐mannose‐glycosylated periplasmic forms of the same enzyme encoded by the suc2 gene.Renaturation of core‐glycosylated invertase upon dilution from guanidinium‐chloride solutions follows a unibimolecular reaction scheme with consecutive first‐order subunit folding and second‐order association reactions. The rate constant of the rate‐limiting step of subunit folding, as detected by fluorescence increase, is k1 = 1.6 + 0.4 × 10−3 s−1 at 20 °C; it is characterized by an activation enthalpy of ΔH = 65 kJ/mol. The reaction is not catalyzed by peptidyl‐prolyl cis‐trans isomerase of the cyclophilin type. Reactivation of the enzyme depends on protein concentration and coincides with subunit association, as monitored by size‐exclusion high‐pressure liquid chromatography. The association rate constant, estimated by numerical simulation of reactivation kinetics, increases from 5 × 103 M−1 s−1 to 7 × 104 M−1 s−1 between 5 and 30 °C.Although reactivation kinetics and yields of the core‐glycosylated and high‐mannose‐glycosylated invertase are essentially identical between 5 and 40 °C, the nonglycosylated cytoplasmic form displays strongly reduced reactivation yields at the high end and significantly reduced reactivation rates at the low end of this temperature range.
Mechanism of phage P22 tailspike protein folding mutations
Authors: Danner Martina , Seckler Robert 
Published: February 2009

AbstractTemperature‐sensitive folding (tsf) and global‐tsf‐suppressor (su) point mutations affect the folding yields of the trimeric, thermostable phage P22 tailspike endorhamnosidase at elevated temperature, both in vivo and in vitro, but they have little effect on function and stability of the native folded protein. To delineate the mechanism by which these mutations modify the partitioning between productive folding and off‐pathway aggregation, the kinetics of refolding after dilution from acid‐urea solutions and the thermal stability of folding intermediates were analyzed. The study included five tsf mutations of varying severity, the two known su mutations, and four tsf/su double mutants.At low temperature (10°C), subunit‐folding rates, measured as an increase in fluorescence, were similar for wild‐type and mutants. At 25°C, however, tsf mutations reduced the rate of subunit folding. The su mutations increased this rate, when present in the tsf‐mutant background, but had no effect in the wild‐type background. Conversely, tsf mutations accelerated, and su mutations retarded the irreversible off‐pathway reaction, as revealed by temperature down‐shifts after varied times during refolding at high temperature (40°C). The kinetic results are consistent with tsf mutations destabilizing and su mutations stabilizing an essential subunit folding intermediate.In accordance with this interpretation, tsf mutations decreased, and su mutations increased the temperature resistance of folding intermediates, as disclosed by temperature up‐shifts during refolding at 25°C. The stabilizing and destabilizing effects were most pronounced early during refolding. However, they were not limited to subunit‐folding intermediates and were also observable during thermal unfolding of the native protein.
Macromolecular solvation energies derived from small molecule crystal morphology
Authors: Rees Douglas C. , Wolfe Gershon M. 
Published: February 2009

AbstractThe morphology of small molecule crystals provides a model for evaluating surface solvation energies in a system with similar packing density to that observed for amino acid residues in proteins. The solvation energies associated with the transfer of methylene and carboxyl groups between vacuum and aqueous phases are estimated to be approx. +40 and ‐260 cal/Å2, respectively, from an analysis of the morphology of succinic acid crystals. These solvation energies predict values for contact angles in reasonable agreement with measurements determined from macroscopic monolayer surfaces. Transfer free energies between vapor and water phases for a series of carboxylic acids are also predicted reasonably well by these solvation energies, provided the surface exposure of different groups is quantitated with the molecular surface area rather than the more traditional accessible surface area. In general, molecular surfaces and molecular surface areas are seen to have important advantages for characterizing the structure and energetics of macromolecular surfaces. Crystal faces of succinic acid with the lowest surface energies in aqueous solution are characteristically smooth. Increasing surface roughness and apolarity are associated with higher surface energies, which suggests an approach for modifying the surface properties of proteins and other macromolecules.
Aldehyde dehydrogenases: Widespread structural and functional diversity within a shared framework
Authors: Hempel John , Nicholas Hugh , Lindahl Ronald 
Published: February 2009

AbstractSequences of 16 NAD and/or NADP‐linked aldehyde oxidoreductases are aligned, including representative examples of all aldehyde dehydrogenase forms with wide substrate preferences as well as additional types with distinct specificities for certain metabolic aldehyde intermediates, particularly semialdehydes, yielding pairwise identities from 15 to 83%. Eleven of 23 invariant residues are glycine and three are proline, indicating evolutionary restraint against alteration of peptide chain‐bending points. Additionally, another 66 positions show high conservation of residue type, mostly hydrophobic residues. Ten of these occur in predicted β‐strands, suggesting important interior‐packing interactions.A single invariant cysteine residue is found, further supporting its catalytic role. A previously identified essential glutamic acid residue is conserved in all but methyl malonyl semialdehyde dehydrogenase, which may relate to formation by that enzyme of a CoA ester as a product rather than a free carboxylate species. Earlier, similarity to a GXGXXG segment expected in the NAD‐binding site was noted from alignments with fewer sequences. The same region continues to be indicated, although now only the first glycine residue is strictly conserved and the second (usually threonine) is not present at all, suggesting greater variance in coenzyme‐binding interactions.
Import of cytochrome b2 to the mitochondrial intermembrane space: The tightly folded heme‐binding domain makes import dependent upon matrix ATP
Authors: Glick Benjamin S. , Wachter Clemens , Reid Graeme A. , Schatz Gottfried 
Published: February 2009

AbstractCytochrome b2 is synthesized as a precursor in the cytoplasm and imported to the intermembrane space of yeast mitochondria. We show here that the precursor contains a tightly folded heme‐binding domain and that translocation of this domain across the outer membrane requires ATP. Surprisingly, it is ATP in the mitochondrial matrix rather than external ATP that drives import of the heme‐binding domain. When the folded structure of the heme‐binding domain is disrupted by mutation or by urea denaturation, import and correct processing take place in ATP‐depleted mitochondria. These results indicate that (1) cytochrome b2 reaches the intermembrane space without completely crossing the inner membrane, and (2) some precursors fold outside the mitochondria but remain translocation‐competent, and import of these precursors in vitro does not require ATP‐dependent cytosolic chaperone proteins.
NMR and protein folding: Equilibrium and stopped‐flow studies
Authors: Frieden Carl , Hoeltzli Sydney D. , Ropson Ira J. 
Published: February 2009

AbstractNMR studies are now unraveling the structure of intermediates of protein folding using hydrogen—deuterium exchange methodologies. These studies provide information about the time dependence of formation of secondary structure. They require the ability to assign specific resonances in the NMR spectra to specific amide protons of a protein followed by experiments involving competition between folding and exchange reactions. Another approach is to use 19F‐substituted amino acids to follow changes in side‐chain environment upon folding. Current techniques of molecular biology allow assignments of 19F resonances to specific amino acids by site‐directed mutagenesis. It is possible to follow changes and to analyze results from 19F spectra in real time using a stopped‐flow device incorporated into the NMR spectrometer.
Sequence‐specific
Authors: Fletcher C.M. , Harrison R.A. , Lachmann P.J. , Neuhaus D. 
Published: February 2009

AbstractCD59 is a recently discovered cell‐surface glycoprotein that restricts lysis by homologous complement and has limited sequence similarity to snake venom neurotoxins. This paper describes the first results of a two‐dimensional NMR study of CD59 prepared from human urine. Nearly complete 1H‐NMR assignments were obtained for the 77 amino acid residues and partial assignments for the N‐glycan and the glycosylphosphatidylinositol (GPI) anchor. These results together confirm that the C‐terminal residue of the mature protein is Asn 77 and that the urine‐derived form retains the nonlipid part of the GPI anchor. The data further indicate that the GPI anchor and possibly the N‐glycan are structurally inhomogeneous and suggest that the phospholipid present in the intact GPI anchor was removed by phosphatidylinositol‐specific phospholipase‐D. The folding topology of the protein was determined from NOE enhancements and slowly exchanging backbone amide protons and consists primarily of five extended strands (denoted β1–β5 in sequence order), arranged into separate two‐stranded (β1 and β2) and three‐stranded (β3–β5) antiparallel β‐sheets. The same folding topology is found in all of the snake venom neurotoxins whose structures have been determined. The region between the β4 and β5 strands has helical character, a feature that is not present in the neurotoxins but that is seen in the topologically similar wheat germ agglutinin.
Characterization of the NR1, NR2A, and NR2C receptor proteins
Authors: Köpke Andreas K.E. , Bonk Ines , Sydow Sabine , Menke Hildegard , Spiess Joachim 
Published: February 2009

AbstractN‐Methyl‐d‐aspartate (NMDA) receptor subunits were characterized with seven polyclonal antibodies. The antibodies were directed against NR1‐A, NR2A‐N1, and NR2C‐N1, representing N‐terminal sequences of the NR1, NR2A, and NR2C subunits, and against NR1‐E, NR2A‐C1, and NR2C‐C1, derived from C‐terminal sequences of these subunits. The anti‐NR1‐D antibody was raised against the putative internal loop of NR1.A size of 118 kDa was found in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for NR1 (from rat brain) detected by anti‐NR1‐D and ‐NR1‐E, but not anti‐NR1‐A. With the anti‐NR1‐A antibody, a 125‐kDa protein was discovered that may represent a glutamate receptor not yet characterized. NR2A and NR2C were identified as proteins with sizes of 175 and 140 kDa, respectively.Enzymatic N‐deglycosylation generated a 97‐kDa protein from NR1, a 105‐kDa protein from the 125‐kDa protein, a 162‐kDa protein from NR2A, and a 127‐kDa protein from NR2C. In contrast to the deglycosylation product of the NR2A, the 97‐ and 127‐kDa proteins derived from NR1 and NR2C, respectively, were found significantly smaller than the molecular masses of 103 and 141 kDa, respectively, predicted on the basis of DNA data. These products may represent truncated proteins.The tissue content of the NR1 and NR2A was high in bovine hippocampus and cortex but lower in the cerebellum. In contrast, NR2C was solely found in the cerebellum. The 125‐kDa protein was highest in the cerebellum and cortex.
Fluorescence characterization of Trp 21 in rat glutathione S‐transferase 1–1: Microconformational changes induced by S‐hexyl glutathione
Authors: Wang Regina W. , Bird Andrew W. , Newton Deborah J. , Lu Anthony Y. H. , Atkins William M. 
Published: February 2009

AbstractThe glutathione S‐transferase (GST) isoenzyme A1–1 from rat contains a single tryptophan, Trp 21, which is expected to lie within α‐helix 1 based on comparison with the X‐ray crystal structures of the pi‐ and mu‐class enzymes. Steady‐state and multifrequency phase/modulation fluorescence studies have been performed in order to characterize the fluorescence parameters of this tryptophan and to document ligand‐induced conformational changes in this region of the protein. Addition of S‐hexyl glutathione to GST isoenzyme A1–1 causes an increase in the steady‐state fluorescence intensity, whereas addition of the substrate glutathione has no effect. Frequency‐domain excited‐state lifetime measurements indicate that Trp 21 exhibits three exponential decays in substrate‐free GST. In the presence of S‐hexyl glutathione, the data are also best described by the sum of three exponential decays, but the recovered lifetime values change. For the substrate‐free protein, the short lifetime component contributes 9–16% of the total intensity at four wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S‐hexyl glutathione. Steady‐state quenching experiments indicate that Trp 21 is insensitive to quenching by iodide, but it is readily quenched by acrylamide. Acrylamide‐quenching experiments at several emission wavelengths indicate that the long‐wavelength components become quenched more easily in the presence of S‐hexyl glutathione. Differential fluorescence polarization measurements also have been performed, and the data describe the sum of two anisotropy decay rates. The recovered rotational correlation times for this model are 26 ns and 0.81 ns, which can be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that regions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the “H‐site” is occupied.
MIF proteins are theta‐class glutathione S‐transferase homologs
Authors: Blocki Frank A. , Ellis LYNDA B.M. , Wackett Lawrence P. 
Published: February 2009

AbstractMIF proteins are mammalian polypeptides of approximately 13, 000 molecular weight. This class includes human macrophage migration inhibitory factor (MIF), a rat liver protein that has glutathione S‐transferase (GST) activity (TRANSMIF), and the mouse delayed early response gene 6 (DER6) protein. MIF proteins were previously linked to GSTs by demonstrating transferase activity and observing N‐terminal sequence homology with a mu‐class GST (Blocki, F.A., Schlievert, P.M., & Wackett, L.P., 1992, Nature 360, 269–270). In this study, MIF proteins are shown to be structurally related to the theta class of GSTs. This is established in three ways. First, unique primary sequence patterns are developed for each of the GST gene classes. The patterns identify the three MIF proteins as theta‐like transferase homologs. Second, pattern analysis indicates that GST members of the theta class contain a serine residue in place of the N‐terminal tyrosine that is implicated in glutathione deprotonation and activation in GSTs of known structure (Liu, S., et al., 1992, J. Biol. Chem. 267, 4296–4299). The MIF proteins contain a threonine at this position. Third, polyclonal antibodies raised against recombinant human MIF cross‐react on Western blots with rat theta GST but not with alpha and mu GSTs. That MIF proteins have glutathione‐binding ability may provide a common structural key toward understanding the varied functions of this widely distributed emerging gene family. Because theta is thought to be the most ancient evolutionary GST class, MIF proteins may have diverged early in evolution but retained a glutathione‐binding domain.
Classification of doubly wound nucleotide binding topologies using automated loop searches
Authors: Swindells Mark B. 
Published: February 2009

AbstractA classification is presented of doubly wound α/β nucleotide binding topologies, whose binding sites are located in the cleft formed by a topological switch point. In particular, the switch point loop nearest the N‐terminus is used to identify specific structural classes of binding protein. This yields seven structurally distinct loop conformations, which are subsequently used as motifs for scanning the Protein Data Bank. The searches, which are effective at identifying functional relationships within a large database of structures, reveal a remarkable and previously unnoticed similarity between the coenzyme binding sites of flavodoxin and tryptophan synthetase, even though there is no sequence or topological similarity between them.