bluuebunny/crossref_metadata_2025_split huggingface dataset:
Three‐dimensional structures of three engineered cellulose‐binding domains of cellobiohydrolase I from Trichoderma reesei
Authors: Mattinen Maija‐Liisa , Kontteli Maarit , Kerovuo Janne , Drakenberg Torbjörn , Annila Arto , Linder Markus , Reinikainen Tapani , Lindeberg Gunnar 
Published: July 2010

AbstractThree‐dimensional solution structures for three engineered, synthetic CBDs (Y5A, Y31A, and Y32A) of cellobiohydrolase I (CBHI) from Trichoderma reesei were studied with nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. According to CD measurements the antiparallel β‐sheet structure of the CBD fold was preserved in all engineered peptides. The three‐dimensional NMR‐based structures of Y31A and Y32A revealed only small local changes due to mutations in the flat face of CBD, which is expected to bind to crystalline cellulose. Therefore, the structural roles of Y31 and Y32 are minor, but their functional importance is obvious because these mutants do not bind strongly to cellulose. In the case of Y5A, the disruption of the structural framework at the N‐terminus and the complete loss of binding affinity implies that Y5 has both structural and functional significance. The number of aromatic residues and their precise spatial arrangement in the flat face of the type I CBD fold appears to be critical for specific binding. A model for the CBD binding in which the three aligned aromatic rings stack onto every other glucose ring of the cellulose polymer is discussed.
The NMR side‐chain assignments and solution structure of enzyme HBcellobiose of the phosphoenolpyruvate‐dependent phosphotransferase system of Escherichia coli
Authors: Ab Eiso , Schuurman‐Wolters Gea , Dijkstra Klaas , Scheek Ruud M. , Robillard George T. , Reizer Jonathan , Saier Milton H. 
Published: July 2010

AbstractThe assignment of the side‐chain NMR resonances and the determination of the three‐dimensional solution structure of the C10S mutant of enzyme IIBcellobiose (IIBcel) of the phosphoenolpyruvate‐dependent phosphotransferase system of Escherichia coli are presented. The side‐chain resonances were assigned nearly completely using a variety of mostly heteronuclear NMR experiments, including HCCH‐TOCSY, HCCH‐COSY, and COCCH‐TOCSY experiments as well as CBCACOHA, CBCA(CO)NH, and HBHA(CBCA)(CO)NH experiments.In order to obtain the three‐dimensional structure, NOE data were collected from l5N‐NOESY‐HSQC, 13C‐HSQC‐NOESY, and 2D NOE experiments. The distance restraints derived from these NOE data were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In an iterative procedure, additional NOE assignments were derived from the calculated structures and new structures were calculated. The final set of structures, calculated with approximately 2000 unambiguous and ambiguous distance restraints, has an rms deviation of 1.1 Å on Cα atoms. IIBce consists of a four stranded parallel β‐sheet, in the order 2134. The sheet is flanked with two and three α‐helices on either side. Residue 10, a cysteine in the wild‐type enzyme, which is phosphorylated during the catalytic cycle, is located at the end of the first β‐strand. A loop that is proposed to be involved in the binding of the phosphoryl‐group follows the cysteine. The loop appears to be disordered in the unphosphorylated state.
Analysis of protein structure in intact cells: Crosslinking in vivo between introduced cysteines in the transmembrane domain of a bacterial chemoreceptor
Authors: Hughson Andrew G. , Hazelbauer Gerald L. , Lee Geoffrey F. 
Published: July 2010

AbstractOxidative crosslinking of cysteines introduced by site‐specific mutagenesis is a powerful tool for structural analysis of proteins, but the approach has been limited to studies in vitro. We recently reported that intact cells of Escherichia coli could be treated with Cu(II)‐(o‐phenanthroline)3 or molecular iodine in a way that left unperturbed flagellar function or general chemotactic response, yet crosslinks were quantitatively formed between select cysteines in adjoining trans‐membrane helices of chemoreceptor Trg. This suggested that oxidative crosslinking might be utilized for structural analysis in vivo. Thus, we used our comprehensive collection of Trg derivatives, each containing a single cysteine at one of the 54 positions in the two transmembrane segments of the receptor monomer to characterize patterns of crosslinking in vivo and in vitro for this homodimeric protein. We found that in vivo crosslinking compared favorably as a technique for structural analysis with the more conventional in vitro approach. Patterns of crosslinking generated by oxidation treatments of intact cells indicated extensive interaction of transmembrane segment 1 (TM1) with its homologous partner (TM1′) in the other subunit and a more distant placement of TM2 and TM2′, the same relationships identified by crosslinking in isolated membranes. In addition, the same helical faces for TM1‐TM1′ interaction and TM2‐TM2′ orientation were identified in vivo and in vitro. The correspondence of the patterns also indicates that structural features identified by analysis of in vitro crosslinking are relevant to the organization of the chemoreceptor in its native environment, the intact, functional cell. It appears that the different features of the two functionally benign treatments used for in vivo oxidations can provide insights into protein dynamics.
A novel methodology for assignment of disulfide bond pairings in proteins
Authors: Wu Jiang , Watson J. Throck 
Published: July 2010

AbstractA novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2‐carboxyethyl)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by l‐cyano‐4‐dimethylamino‐pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed‐phase HPLC, are subjected to cleavage of the peptide bonds on the N‐terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI‐MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, β‐elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions.
Cooperative α‐helix formation of β‐lactoglobulin and melittin induced by hexafluoroisopropanol
Authors: Hirota Nami , Goto Yuji , Mizuno Kazuko 
Published: July 2010

AbstractAlcohols denature the native state of proteins, and also stabilize the α‐helical conformation in unfolded proteins and peptides. Among various alcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) are often used because of their high potential to induce such effects. However, the reason why TFE and HFIP are more effective than other alcohols is unknown. Using CD, we studied the effects of TFE and HFIP as well as reference alcohols, i.e., methanol, ethanol, and isopropanol, on the conformation of bovine β‐lactoglobulin and the bee venom melittin at pH 2. Upon addition of alcohols, β‐lactoglobulin exhibited a transformation from the native state, consisting of β‐sheets, to the α‐helical state, whereas melittin folded from the unfolded state to the α‐helical state. In both cases, the order of effectiveness of alcohols was shown to be: HFIP > TFE > isopropanol > ethanol > methanol. The alcohol‐induced transitions were analyzed assuming a two‐state mechanism to obtain the m value, a measure of the dependence of the free energy change on alcohol concentration. Comparison of the m values indicates that the high potential of TFE can be explained by the additive contribution of constituent groups, i.e., F atoms and alkyl group. On the other hand, the high potential of HFIP is more than that expected from the additive effects, suggesting that the cooperative formation of micelle‐like clusters of HFIP is important.
The role of helix VIII in the lactose permease of Escherichia coli: II. Site‐directed sulfhydryl modification
Authors: Frillingos Stathis , Kaback H. Ronald 
Published: July 2010

AbstractCys‐scanning mutagenesis of putative transmembrane helix VIII in the lactose permease of Escherichia coli (Frillingos S, Ujwal ML, Sun J, Kaback HR, 1997, Protein Sci 6:431–437) indicates that, although helix VIII contains only one irreplaceable residue (Glu 269), one face is important for active lactose transport. In this study, the rate of inactivation of each N‐ethylmaleimide (NEM)‐sensitive mutant is examined in the absence or presence of β,D‐galactopyranosyl l‐thio‐β,D‐galactopyranoside (TDG). Remarkably, the analogue affords protection against inactivation with mutants Val 264 → Cys, Gly 268 → Cys, and Asn 272 → Cys, and alkylation of these single‐Cys mutants in right‐side‐out membrane vesicles with [l4C]NEM is attenuated by TDG. In contrast, alkylation of Thr 265 → Cys, which borders the three residues that are protected by TDG, is enhanced markedly by the analogue. Furthermore, NEM‐labeling in the presence of the impermeant thiol reagent methanethiosulfonate ethylsulfonate demonstrates that ligand enhances the accessibility of position 265 to solvent. Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262 → Cys, Glu 269 → Cys, Ala 273 → Cys, Met 276 → Cys, Phe 277 → Cys, or Ala 279 → Cys. The findings indicate that a portion of one face of helix VIII (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand‐induced conformational change.
Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: A model for studying phosphagen kinases
Authors: Zhou Genfa , Parthasarathy Gopalakrishnan , Somasundaram Thayumanasamy , Ables Andrea , Roy Lance , Chapman Michael S. , Strong Scott J. , Ellington W. Ross 
Published: July 2010

AbstractPhosphagen kinases catalyze the reversible transfer of a phosphoryl group between guanidino phosphate compounds and ADP, thereby regenerating ATP during bursts of cellular activity. Large quantities of highly pure arginine kinase (EC 2.7.3.3), the phosphagen kinase present in arthropods, have been isolated fromE. coli, into which the cDNA for the horseshoe crab enzyme had been cloned. Purification involves size exclusion and anion exchange chromatographies applied in the denatured and refolded states. The recombinant enzyme has been crystallized as a transition state analog complex. Near complete native diffraction data have been collected to 1.86 Å resolution. Substitution of a recombinant source for a natural one, improvement in the purification, and data collection atcryotemperatures have all yielded significant improvements in diffraction.
Thermoanaerobacter brockii alcohol dehydrogenase: Characterization of the active site metal and its ligand amino acids
Authors: Bogin Oren , Peretz Moshe , Burstein Yigal 
Published: July 2010

AbstractThe active‐site metal ion and the associated ligand amino acids in the NADP‐linked, tetrameric enzyme Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized by atomic absorption spectroscopy analysis and site‐directed mutagenesis. Our preliminary results indicating the presence of a catalytic zinc and the absence of a structural metal ion in TBADH (Peretz & Burstein. 1989. Biochemistry 25:6549–6555) were verified. To determine the role of the putative active‐site zinc, we investigated whether exchanging the zinc for other metal ions would affect the structural and/or the enzymatic properties of the enzyme. Substituting various metal ions for zinc either enhanced or diminished enzymatic activity, as follows: Mn2+ (240%); Co2+ (130%); Cd2+ (20%); Cu2+ or V3+ (<5%). Site‐directed mutagenesis to replace any one of the three putative zinc ligands of TBADH, Cys 37, His 59, or Asp 150, with the non‐chelating residue, alanine, abolished not only the metal‐binding capacity of the enzyme but also its catalytic activity, without affecting the overall secondary structure of the enzyme. Replacing the three putative catalytic zinc ligands of TBADH with the respective chelating residues serine, glutamine, or cysteine damaged the zinc‐binding capacity of the mutated enzyme and resulted in a loss of catalytic activity that was partially restored by adding excess zinc to the reaction. The results imply that the zinc atom in TBADH is catalytic rather than structural and verify the involvement of Cys 37, His 59, and Asp 150 of TBADH in zinc coordination.
Evidence for PDZ domains in bacteria, yeast, and plants
Authors: Ponting Christopher P. 
Published: July 2010

AbstractSeveral dozen signaling proteins are now known to contain 80‐100 residue repeats, called PDZ (or DHR or GLGF) domains, several of which interact with the C‐terminal tetrapeptide motifs X‐Ser/Thr‐X‐Val‐COO− of ion channels and/or receptors. PDZ domains have previously been noted only in mammals, flies, and worms, suggesting that the primordial PDZ domain arose relatively late in eukaryotic evolution. Here, techniques of sequence analysis—including local alignment, profile, and motif database searches—indicate that PDZ domain homologues are present in yeast, plants, and bacteria. It is suggested that two PDZ domains occur in bacterial high‐temperature requirement A (htrA) and one in tail‐specific protease (tsp) homologues, and that a yeast htrA homologue contains four PDZ domains. Sequence comparisons suggest that the spread of PDZ domains in these diverse organisms may have occurred via horizontal gene transfer. The known affinity of Escherichia coli tsp for C‐terminal polypeptides is proposed to be mediated by its PDZ‐like domain, in a similar manner to the binding of C‐terminal polypeptides by animal PDZ domains.
Remodeling domain interfaces to enhance heterodimer formation
Authors: Zhu Zhenping , Presta Leonard G. , Zapata Gerardo , Carter Paul 
Published: July 2010

AbstractAn anti‐p185HER2/anti‐CD3 humanized bispecific diabody was previously constructed from two cross‐over single‐chain Fv in which VH and VL domains of the parent antibodies are present on different polypeptides. Here this diabody is used to evaluate domain interface engineering strategies for enhancing the formation of functional heterodimers over inactive homodimers. A disulfide‐stabilized diabody was obtained by introducing two cysteine mutations, VL L46C and VH D101C, at the anti‐p185HER2 VL/VH interface. The fraction of recovered diabody that was functional following expression in Escherichia coli was improved for the disulfide‐stabilized compared to the parent diabody (>96% versus 72%), whereas the overall yield was >60‐fold lower. Eleven “knob‐into‐hole” diabodies were designed by molecular modeling of sterically complementary mutations at the two VL/VH interfaces. Replacements at either interface are sufficient to improve the fraction of functional heterodimer, while maintaining overall recoverable yields and affinity for both antigens close to that of the parent diabody. For example, diabody variant v5 containing the mutations VL Y87A:F98M and VH V37F:L45W at the anti‐p185HER2 VL/VH interface was recovered as 92% functional heterodimer while maintaining overall recovered yield within twofold of the parent diabody. The binding affinity of v5 for p185HER2 extracellular domain and T cells is eightfold weaker and twofold stronger than for the parent diabody, respectively. Domain interface remodeling based upon either sterically complementary mutations or interchain disulfide bonds can facilitate the production of a functional diabody heterodimer. This study expands the scope of domain interface engineering by demonstrating the enhanced assembly of proteins interacting via two domain interfaces.
The kinetic basis for the stabilization of staphylococcal nuclease by xylose
Authors: Frye Kelly J. , Royer Catherine A. 
Published: July 2010

AbstractThe effect of xylose on the rates of folding and unfolding of staphylococcal nuclease (nuclease) have been investigated using fluorescence‐detected pressure‐jump relaxation kinetics in order to establish the kinetic basis for the observed stabilization of nuclease by this sugar (Frye KJ, Perman CS, Royer CA, 1996, Biochemistry 35:10234–10239). The activation volumes for both folding and unfolding and the equilibrium volume change for folding were all positive. Their values were within experimental error of those reported previously (Vidugiris GJA, Markley JL, Royer CA, 1995, Biochemistry 34:4909–4912) and were independent of xylose concentration. The major effect of xylose concentration was to increase significantly the rate of folding. The large positive activation volume for folding was interpreted previously as indicating that the rate‐limiting step in nuclease folding involves dehydration of a significant amount of surface area. A large effect of xylose on the rate constant for folding provides strong support for this interpretation, because xylose, an osmolyte, stabilizes the folded state of proteins through surface tension effects. These studies further characterize the transition state in nuclease folding as lying closer to the folded, rather than the unfolded state along the folding coordinate in terms of the degree of burial of surface area. The image of the transition state that emerges is consistent with a dry molten globule.
Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site
Authors: Weaver Todd , Lees Mason , Banaszak Leonard 
Published: July 2010

AbstractTwo mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C‐terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A‐ and B‐, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A‐site resulted in a large decrease in specific activity, while the H129N mutation at the B‐site had essentially no effect. From the results, we conclude that the A‐site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B‐site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation.
Monitoring calcium‐induced conformational changes in recoverin by electrospray mass spectrometry
Authors: Neubert Thomas A. , Walsh Kenneth A. , Hurley James B. , Johnson Richard S. 
Published: July 2010

AbstractRecoverin is a calcium‐binding protein that regulates the vertebrate photoresponse by inhibiting rhodopsin kinase in response to high calcium concentrations. It is heterogeneously N‐acylated by myristoyl and related fatty acyl residues that are thought to act as “calcium‐myristoyl switches,” whereby, in the presence of Ca2+, the N‐terminal acyl group is extended away from recoverin and, in the absence of calcium, it is more closely associated with the protein. Here we use electrospray ionization mass spectrometry (ESI/MS) to examine hydrogen isotopic exchange rates for specific regions of both acylated and nonacylated recoverin in the presence and absence of calcium. The deuterium exchange rates of three regions in the hydrophobic myristoyl binding pocket of acylated recoverin decreased in the absence of calcium. This effect is most likely due to the closer association of the acyl group with the protein under these conditions. In contrast, rates of deuterium incorporation increased in the absence of calcium for other regions, including the two functional calcium‐binding sites. In addition to supporting the calcium‐myristoyl switch hypothesis, a comparison of the behavior of acylated and unacylated recoverin revealed that the N‐acyl group (N‐lauroyl or N‐myristoyl) exerts a significant stabilizing influence on the dynamics of recoverin. We demonstrate that the new technique of monitoring hydrogen isotopic exchange by ESI/MS can be used to obtain useful information concerning protein structures in solution using smaller amounts of protein and under more physiologically relevant conditions than is typically possible with NMR or X‐ray crystallography.
An electrophile‐nucleophile interaction in metalloprotein structures
Authors: Chakrabarti Pinak , Pal Debnath 
Published: July 2010

AbstractA new attractive interaction in metalloprotein structures, between the thiolate anion of a metal‐bound cysteine (acting as a nucleophile) and a carbonyl carbon of a peptide group (an electrophile), has been identified. From 82 cases extracted from 23 metalloprotein structures, the interacting S and C atoms are found to be at a distance of 3.2 (±2) Å, such that the angle S···C — O is 109° (±15°). Usually, the interacting atoms are from the same Cys residue, and to allow the S to interact with the carbonyl group the side‐chain and the main‐chain torsion angles deviate from those found in cysteines not bound by metals. There is a good correlation between the S···C distance and the angular deviation of the S···C vector from the normal to the peptide plane. Various data points may be envisaged to represent “snapshots” along the reaction coordinate for the intra‐residue attack of Cys S on the CO group.