bluuebunny/crossref_metadata_2025_split huggingface dataset:
Second‐site suppression of regulatory phosphorylation in Escherichia coli isocitrate dehydrogenase
Authors: Chen Ridong , Dean Antony M. , Grobler Jay A. , Hurley James H. 
Published: July 2010

AbstractInactivation of Escherichia coli isocitrate dehydrogenase upon phosphorylation at S113 depends upon the direct electrostatic repulsion of the negatively charged γ‐carboxylate of isocitrate by the negatively charged phosphoserine. The effect is mimicked by replacing S113 with aspartate or glutamate, which reduce performance (kcat/Ki‐isocitrat/KmNADP) by a factor of 107. Here, we demonstrate that the inactivating effects of the electrostatic repulsion are completely eliminated by a second‐site mutation, and provide the structural basis for this striking example of intragenic suppression. N115 is adjacent to S113 on one face of the D‐helix, interacts with isocitrate and NADP+, and has been postulated to serve in both substrate binding and in catalysis. The single N115L substitution reduces affinity for isocitrate by a factor of 50 and performance by a factor of 500. However, the N115L substitution completely suppresses the inactivating electrostatic effects of S113D or S113E: the performance of the double mutants is 105 higher than the S113D and S113E single mutants. These mutations have little effect on the kinetics of alternative substrates, which lack the charged γ‐carboxylate of isocitrate. Both glutamate and aspartate at site 113 remain fully ionized in the presence of leucine. In the crystal structure of the N115L mutant, the leucine adopts a different conformer from the wild‐type asparagine. Repacking around the leucine forces the amino‐terminus of the D‐helix away from the rest of the active site. The hydrogen bond between E113 and N115 in the S113E single mutant is broken in the S113E/N115L mutant, allowing the glutamate side chain to move away from the γ‐carboxylate of isocitrate. These movements increase the distance between the carboxylates, diminish the electrostatic repulsion, and lead to the remarkably high activity of the S113E/N115L mutant.
Reconstitution of active octameric mitochondrial creatine kinase from two genetically engineered fragments
Authors: Gross Martin , Wallimann Theo , Wyss Markus , Furter‐Graves Elizabeth M. , Furter Rolf 
Published: July 2010

AbstractCreatine kinase (CK) has been postulated to consist of two flexibly hinged domains. A previously demonstrated protease‐sensitive site in M‐CK (Morris & Jackson, 1991) has directed our attempts to dissect mitochondrial CK (Mi‐CK) into two protein fragments encompassing amino acids [1–167] and [168–380]. When expressed separately in Escherichia coli, the two fragments yielded large amounts of insoluble inclusion bodies, from which the respective polypeptides could be purified by a simple two‐step procedure. In contrast, co‐expression of the two fragments yielded a soluble, active, and correctly oligomerizing enzyme. This discontinuous CK showed nearly full specific activity and was virtually indistinguishable from native Mi‐CK by far‐ and near‐UV CD. However, the positive cooperativity of substrate binding was abolished, suggesting a role of the covalent domain linkage in the crosstalk between the substrate binding sites for ATP and creatine. The isolated C‐terminal fragment refolded into a native‐like conformation in vitro, whereas the N‐terminal fragment was largely unfolded. Prefolded [168–380] interacted in vitro with [1–167] to form an active enzyme. Kinetic analysis indicated that the fragments associate rapidly and with high affinity (1/K1, = 17 μM) and then isomerize slowly to an active enzyme (k2 = 0.12 min−1; k‐2 = 0.03 min−1). Our data suggest that the C‐terminal fragment of Mi‐CK represents an autonomous folding unit, and that the folding of the C‐terminal part might precede the conformational stabilization of the N‐terminal moiety in vivo.
Computational method for relative binding energies of enzyme‐substrate complexes
Authors: Zhang Tao , Koshland Daniel E. 
Published: July 2010

AbstractA computational method for estimating the relative binding free energies of enzyme‐substrate complexes is described that combines electrostatic and solvation models and X‐ray crystallographic data. The polar contribution is evaluated by the Poisson‐Boltzman equation. The nonpolar contribution is evaluated by solvent transfer data and surface area calculations. This algorithm was used to calculate the relative binding energies of 63 pairs of nine different mutant proteins with seven different substituted R‐malate substrates of Escherichia coli isocitrate dehydrogenase. Comparison of calculated values with the experimentally observed values shows a high degree of correlation.
On the role of surface tension in the stabilization of globular proteins
Authors: Lin Tiao‐Yin , Timasheff Serge N. 
Published: July 2010

AbstractThe stabilization of proteins by a variety of co‐solvents can be related to their property of increasing the surface tension of water. It is demonstrated that, during the thermal unfolding of proteins, this increase of the surface tension can be overcome by the increase in the temperature of the solution at the midpoint of the transition, Tm, and the weak binding of co‐solvent molecules. Three such co‐solvents were studied: trehalose, lysine hydrochloride (LysHCl), and arginine hydrochloride (ArgHCl). Trehalose and LysHCl increase the midpoint of Tm. The increase of the surface tension by addition of trehalose is completely compensated by its decrease due to the increase in Tm. However, for LysHCl, the increase of the surface tension by the co‐solvent is partly reduced by its binding to the protein. For trehalose, preferential interaction measurements with RNaseA demonstrate that it is totally excluded from the protein. In contrast, LysHCl gives evidence of binding to RNaseA. ArgHCl also increases the surface tension of water. Nevertheless, Tm of RNaseA decreases on addition of ArgHCl to the solution. Preferential interaction measurements showed very small values of preferential hydration of the native protein, indicating extensive binding of ArgHCl to the protein. During unfolding, the amount of additional ArgHCl binding is sufficiently large to counteract the surface tension effects, and the protein is destabilized. Therefore, although surface tension appears to be a critical factor in the stabilization of proteins, its increase by co‐solvent does not ensure increased stabilization. The binding of ligands can reduce significantly, or even overwhelm, its effects.
Woodward's reagent K inactivation of Escherichia coli L‐threonine dehydrogenase: Increased absorbance at 340–350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups
Authors: Johnson Adam R. , Dekker Eugene E. 
Published: July 2010

AbstractL‐Threonine dehydrogenase (TDH) from Escherichia coli is rapidly inactivated and develops a new absorbance peak at 347 nm when incubated with N‐ethyl‐5‐phenylisoxazolium‐3′‐sulfonate (Woodward's reagent K, WRK). The cofactors, NAD+ or NADH (1.5 mM), provide complete protection against inactivation; L‐threonine (60 mM) is ∼50% as effective. Tryptic digestion of WRK‐modified TDH followed by HPLC fractionation (pH 6.2) yields four 340‐nm‐absorbing peptides, two of which are absent from enzyme incubated with WRK and NAD+. Peptide I has the sequence TAICGTDVH (TDH residues 35–43), whereas peptide II is TAICGTDVHIY (residues 35–45). Peptides not protected are TMLDTMNHGGR (III, residues 248–258) and NCRGGRTHLCR (IV, residues 98–108). Absorbance spectra of these WRK‐peptides were compared with WRK adducts of imidazole, 2‐hydroxy‐ethanethiolate, and acetate. Peptides III and IV have pH‐dependent λmax values (340–350 nm), consistent with histidine modification. Peptide I has a pH‐independent λmax (350 nm) indicating that a thiol is modified. WRK, therefore, does not react specifically with carboxyl groups in this enzyme, but rather modifies Cys‐38 in the active site of TDH; modification of His‐105 and His‐255 does not affect enzyme activity. These results are the first definitive proof of WRK modifying cysteine and histidine residues of a protein and show that enzyme inactivation by WRK associated with the appearance of new absorptivity at 340–350 nm does not establish modification of aspartate or glutamate residues, as has been assumed in numerous earlier reports.
pH‐Dependent self‐association of the Src homology 2 (SH2) domain of the Src homologous and collagen‐like (SHC) protein
Authors: Réty Stéphane , Fütterer Klaus , Grucza Richard A. , Munoz Consuelo M. , Frazier William A. , Waksman Gabriel 
Published: July 2010

AbstractThe Src homologous and collagen‐like (SHC) protein plays an essential role in signal transduction pathways in that it participates in the chain of events that leads to the activation of the protein Ras. The crystal structure of the SH2 domain of SHC has been determined using the method of multiple isomorphous replacement at a resolution of 2.5 Å. The SH2 domain of SHC is similar in fold to other SH2 domains. The peptide‐binding surfaces resemble that of the SH2 domain of Src in that a deep pocket is formed where the third amino acid C‐terminal to the phosphotyrosine can insert. A novel feature of this structure is the observation of a disulfide bond and an extensive dimer interface between two symmetry‐related molecules. Solution studies under reducing conditions using analytical centrifugation and PAGE suggest that the SH2 domain of SHC dimerizes in a pH‐dependent manner where low pH conditions (∼pH 4.5) are conducive to dimer formation. Dimerization of SHC may have important biological implications in that it may promote the assembly of large heteromultimeric signaling complexes.
A novel, multilayer structure of a helical peptide
Authors: Taylor K. S. , Lou M.‐Z. , Chin T.‐M. , Yang N. C. , Garavito R. M. 
Published: July 2010

AbstractX‐ray diffraction analysis at 1.5 A resolution has confirmed the helical conformation of a de novo designed 18‐residue peptide. However, the crystal structure reveals the formation of continuous molecular layers of parallel‐packed amphiphilic helices as a result of much more extensive helix‐helix interactions than predicted. The crystal packing arrangement, by virtue of distinct antiparallel packing interactions, segregates the polar and apolar surfaces of the helices into discrete and well‐defined interfacial regions. An extensive “ridges‐into‐grooves” interdigitation characterizes the hydrophobic interface, whereas an extensive network of salt bridges and hydrogen bonds dominates the corresponding hydrophilic interface.
The structure of bovine mitochondrial adenylate kinase: Comparison with isoenzymes in other compartments
Authors: Schlauderer G. J. , Schulz G. E. 
Published: July 2010

AbstractIn vertebrates, there are different adenylate kinases in the compartments cytosol, mitochondrial intermembrane space, and mitochondrial matrix. Here, we report the spatial structure of the intermembrane species established in two crystal forms by X‐ray diffraction analyses at 1.92 and 2.1 Å resolution. In both structures, the enzyme is unligated, and thus in an “open” conformation. The enzyme was prepared from bovine liver, containing at least five variants arisen from posttranscriptional and posttranslational modifications. It could only be crystallized after removing some of these variants. A comparison with the known structures of the adenylate kinases from cytosol and mitochondrial matrix reveals structural differences that should play a role in protein targeting because none of these enzymes contains a cleavable signal peptide. A further comparison with adenylate kinases from Gram‐positive bacteria showed that the structural Zn2+ ion of these species is replaced by a strictly conserved assembly of hydrogen bonded residues.
Electrospray mass spectrometric investigation of the chaperone SecB
Authors: Smith Virginia F. , Schwartz Brenda L. , Randall Linda L. , Smith Richard D. 
Published: July 2010

AbstractElectrospray ionization mass spectrometry was used to investigate the structure of the Escherichia coli chaperone protein SecB. It was determined that the N‐terminal methionine of SecB has been removed and that more than half of all SecB monomers are additionally modified, most likely by acetylation of the N‐terminus or a lysine. The use of gentle mass spectrometer interface conditions showed that the predominant, oligomeric form of SecB is a tetramer that is stable over a range of solution pH conditions and mass spectrometer interface heating (i.e., inlet capillary temperatures). At very high pH, SecB dimers are observed. SecB contains a region that is hypersensitive to cleavage by proteinase K and is thought to be involved in conformational changes that are crucial to the function of SecB. We identified the primary site of cleavage to be between Leu 141 and Gln 142. Fourteen amino acids are removed, but the truncated form remains a tetramer with stability similar to that of the intact form.
Three‐dimensional solution structure of the HIV‐1 protease complexed with DMP323, a novel cyclic urea‐type inhibitor, determined by nuclear magnetic resonance spectroscopy
Authors: Yamazaki Toshimasa , Hinck Andrew P. , Wang Yun‐Xing , Nicholson Linda K. , Torchia Dennis A. , Wingfield Paul , Stahl Stephen J. , Kaufman Joshua D. , Chang Chong‐Hwan , Domaille Peter J. , Lam Patrick Y.S. 
Published: July 2010

AbstractThe three‐dimensional solution structure of the HIV‐1 protease homodimer, MW 22.2 kDa, complexed to a potent, cyclic urea‐based inhibitor, DMP323, is reported. This is the first solution structure of an HIV protease/inhibitor complex that has been elucidated. Multidimensional heteronuclear NMR spectra were used to assemble more than 4,200 distance and angle constraints. Using the constraints, together with a hybrid distance geometry/simulated annealing protocol, an ensemble of 28 NMR structures was calculated having no distance or angle violations greater than 0.3 Å or 5°, respectively. Neglecting residues in disordered loops, the RMS deviation (RMSD) for backbone atoms in the family of structures was 0.60 Å relative to the average structure. The individual NMR structures had excellent covalent geometry and stereochemistry, as did the restrained minimized average structure. The latter structure is similar to the 1.8‐Å X‐ray structure of the protease/DMP323 complex (Chang CH et al., 1995, Protein Science, submitted); the pairwise backbone RMSD calculated for the two structures is 1.22 Å. As expected, the mismatch between the structures is greatest in the loops that are disordered and/or flexible. The flexibility of residues 37–42 and 50–51 may be important in facilitating substrate binding and product release, because these residues make up the respective hinges and tips of the protease flaps. Flexibility of residues 4–8 may play a role in protease regulation by facilitating autolysis.
Control of aggregation in protein refolding: The temperature‐leap tactic
Authors: Xie Yansheng , Wetlaufer Donald B. 
Published: July 2010

AbstractThe kinetics of renaturation of bovine carbonic anhydrase II (CAII) were studied from 4° to 36°, at the relatively high [CAII] of 4 mg/mL. Following dilution to 1 M guanidinium chloride, aggregate formation is very rapid and reduces the formation of active enzyme. The CAII activity yield at 150 min, 20° (∼60%), is greater than that at either 4° or 36°. However, if refolding is conducted at 4°, aggregation is reduced dramatically and 37% yield is obtained at 120 min. If the solution is then rapidly warmed to 36°, the yield rises rapidly to 95% at 150 min. This is an example of the “temperature leap” tactic. These results can be understood on the basis of two slow‐folding intermediates whose kinetics have been studied. Only the first of these forms aggregates. Kinetic simulations show that, at 4°, the first intermediate is depleted after 120 min, and the second intermediate rapidly isomerizes to active enzyme on warming. A series of experiments was conducted where the initial (120 min) folding temperature was systematically varied, followed by a “leap” to 36° for 30 additional minutes. With initial incubations from 4° to 12°, the final yield is >90%, drops rapidly from 12° to 20°, and decreases more gradually to ∼45% at 36°. The overall results qualitatively fit the simple idea of ordinary temperature‐accelerated reactions in competition with hydrophobic aggregation, which is strongly suppressed in the cold. Qualifications are discussed for the temperature‐leap approach to find application in refolding other proteins.
Mass spectrometric‐based revision of the structure of a cysteine‐rich peptide toxin with γ‐carboxyglutamic acid, TxVIIA, from the sea snail, Conus textile
Authors: Nakamura Takemichi , Yu Zhonghua , Fainzilber Michael , Burlingame Alma L. 
Published: July 2010

AbstractA mollusk‐specific toxin, TxVIIA, having potent paralytic activity was isolated from the venom of sea snail Conus textile (Fainzilber M et al., 1991, Eur J Biochem 202:589–595). The structure reported above was based upon amino acid analysis and the Edman degradation. We have recently reinvestigated this toxin employing some of the most novel techniques in mass spectrometry. We now report a revised structure based primarily on high‐energy collision‐induced dissociation analysis of the two Asp17‐N peptides of the reduced, pyridinylethyl derivative representing the entire sequence using matrix‐assisted laser desorption ionization (MALDI) as CGGYSTYCγVDSγ CCSDNCVRSYCTLF‐NH2 (γ, γ‐carboxyglutamic acid or Gla).The N‐terminus of the previous sequence was incorrect, apparently due to a side reaction of reduction and alkylation, which led to the erroneous assignment of Trp for the N‐terminal residue. In addition, the last two C‐terminal amino acids and the C‐terminal amidation had not been detected. Also, a combination of electrospray ionization mass spectrometry and positive and negative ion MALDI mass spectrometry provided information on the molecular weights of the native and derivatized toxin and presence of two Gla residues. Thus, TxVIIA does not have an “unusual” sequence as previously reported, but in fact belongs to the conserved Cys framework for ω‐ and δ‐conotoxins. However, the four net negative charges with the cysteine‐rich structure of this revised sequence is highly unusual for conopeptides.
Classification of mutations in the human CD40 ligand, gp39, that are associated with X‐linked hyper IgM syndrome
Authors: Bajorath Jürgen , Seyama Kuniaki , Nonoyama Shigeaki , Ochs Hans D. , Aruffo Alejandro 
Published: July 2010

AbstractThe interaction between the T cell activation antigen gp39 and CD40, its receptor CD40 on B cells, plays a critical role in the regulation of humoral immune responses. Using a detailed three‐dimensional model of the gp39 extracellular region, we have analyzed 20 mutations in gp39 that were, with one exception, isolated from patients with X‐linked hyper IgM (XHIM) syndrome. On the basis of this analysis, the mutations were classified according to their predicted locations and effects. Twelve mutations are thought to compromise the gp39 structure by affecting interactions in hydrophobic core regions or at monomer interfaces, whereas seven others map closely to gp39 residues important for interaction with CD40. The latter mutations may thus, directly or indirectly, interfere with CD40 binding. One naturally occurring mutant whose carrier displays normal immune responses maps to a solvent‐exposed position in a loop region of the molecule.
Simulated annealing with restrained molecular dynamics using CONGEN: Energy refinement of the NMR solution structures of epidermal and type‐α transforming growth factors
Authors: Tejero Roberto , Bassolino‐Klimas Donna , Bruccoleri Robert E. , Montelione Gaetano T. 
Published: July 2010

AbstractThe new functionality of the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137–168; Bassolino‐Klimas D et al., 1996, Protein Sci 5:593–603) has been applied for energy refinement of two previously determined solution NMR structures, murine epidermal growth factor (mEGF) and human type‐α transforming growth factor (hTGFα). A summary of considerations used in converting experimental NMR data into distance constraints for CONGEN is presented. A general protocol for simulated annealing with restrained molecular dynamics is applied to generate NMR solution structures using CONGEN together with real experimental NMR data. A total of 730 NMR‐derived constraints for mEGF and 424 NMR‐derived constraints for hTGFα were used in these energy‐refinement calculations. Different weighting schemes and starting conformations were studied to check and/or improve the sampling of the low‐energy conformational space that is consistent with all constraints. The results demonstrate that loosened (i.e., “relaxed”) sets of the EGF and hTGFα internuclear distance constraints allow molecules to overcome local minima in the search for a global minimum with respect to both distance restraints and conformational energy. The resulting energy‐refined structures of mEGF and hTGFα are compared with structures determined previously and with structures of homologous proteins determined by NMR and X‐ray crystallography.
Redox‐dependent dynamics of putidaredoxin characterized by amide proton exchange
Authors: Lyons Teresa A. , Ratnaswamy Gayathri , Pochapsky Thomas C. 
Published: July 2010

AbstractMultidimensional NMR methods were used to obtain 1H‐15N correlations and 15N resonance assignments for amide and side‐chain nitrogens of oxidized and reduced putidaredoxin (Pdx), the Fe2S2 ferredoxin, which acts as the physiological reductant of cytochrome P‐450cam (CYP101). A model for the solution structure of oxidized Pdx has been determined recently using NMR methods (Pochapsky TC, Ye XM, Ratnaswamy G, Lyons TA, 1994, Biochemistry 53:6424–6432) and redox‐dependent 1H NMR spectral features have been described (Pochapsky TC, Ratnaswamy G, Patera A, 1994, Biochemistry 55:6433–6441). 15N assignments were made with NOESY‐(1H/15N) HMQC and TOCSY‐(1H/15N) HSQC spectra obtained using samples of Pdx uniformly labeled with 15N. Local dynamics in both oxidation states of Pdx were then characterized by comparison of residue‐specific amide proton exchange rates, which were measured by a combination of saturation transfer and H2O/D2O exchange methods at pH 6.4 and 7.4 (uncorrected for isotope effects). In general, where exchange rates for a given site exhibit significant oxidation‐state dependence, the oxidized protein exchanges more rapidly than the reduced protein. The largest dependence of exchange rate upon oxidation state is found for residues near the metal center and in a region of compact structure that includes the loop‐turn Val 74‐Ser 82 and the C‐terminal residues (Pro 102‐Trp 106). The significance of these findings is discussed in light of the considerable dependence of the binding interaction between Pdx and CYP101 upon the oxidation state of Pdx.
Structure of a secreted aspartic protease from C. albicans complexed with a potent inhibitor: Implications for the design of antifungal agents
Authors: Abad‐Zapatero Cele , Goldman Robert , Muchmore Steven W. , Hutchins Charles , Stewart Kent , Navaza Jorge , Payne Candia D. , Ray Thomas L. 
Published: July 2010

AbstractThe three‐dimensional structure of a secreted aspartic protease from Candida albicans complexed with a potent inhibitor reveals variations on the classical aspartic protease theme that dramatically alter the specificity of this class of enzymes. The structure presents: (1) an 8‐residue insertion near the first disulfide (Cys 45‐Cys 50, pepsin numbering) that results in a broad flap extending toward the active site; (2) a 7‐residue deletion replacing helix hN2 (Ser 110‐Tyr 114), which enlarges the S3 pocket; (3) a short polar connection between the two rigid body domains that alters their relative orientation and provides certain specificity; and (4) an ordered 11‐residue addition at the carboxy terminus. The inhibitor binds in an extended conformation and presents a branched structure at the P3 position. The implications of these findings for the design of potent antifungal agents are discussed.