bluuebunny/crossref_metadata_2025_split huggingface dataset:
Carbamate kinase: New structural machinery for making carbamoyl phosphate, the common precursor of pyrimidines and arginine
Authors: Marina Alberto , Alzari Pedro M. , Bravo Jerónimo , Uriarte Matxalen , Barcelona Belén , Fita Ignacio , Rubio Vicente 
Published: October 2010

AbstractThe enzymes carbamoyl phosphate synthetase (CPS) and carbamate kinase (CK) make carbamoyl phosphate in the same way: by ATP‐phosphorylation of carbamate. The carbamate used by CK is made chemically, whereas CPS itself synthesizes its own carbamate in a process involving the phosphorylation of bicarbonate. Bicarbonate and carbamate are analogs and the phosphorylations are carried out by homologous 40 kDa regions of the 120 kDa CPS polypeptide. CK can also phosphorylate bicarbonate and is a homodimer of a 33 kDa subunit that was believed to resemble the 40 kDa regions of CPS. Such belief is disproven now by the CK structure reported here. The structure does not conform to the biotin carboxylase fold found in the 40 kDa regions of CPS, and presents a new type of fold possibly shared by homologous acylphosphate‐making enzymes. A molecular 16‐stranded open β‐sheet surrounded by α‐helices is the hallmark of the CK dimer. Each subunit also contains two smaller sheets and a large crevice found at the location expected for the active center. Intersubunit interactions are very large and involve a central hydrophobic patch and more hydrophilic peripheral contacts. The crevice holds a sulfate that may occupy the site of an ATP phosphate, and is lined by conserved residues. Site‐directed mutations tested at two of these residues inactivate the enzyme. These findings support active site location in the crevice. The orientation of the crevices in the dimer precludes their physical cooperation in the catalytic process. Such cooperation is not needed in the CK reaction but is a requirement of the mechanism of CPSs.
Folding and binding cascades: Dynamic landscapes and population shifts
Authors: Kumar Sandeep , Ma Buyong , Tsai Chung‐Jung , Sinha Neeti , Nussinov Ruth 
Published: October 2010

AbstractWhereas previously we have successfully utilized the folding funnels concept to rationalize binding mechanisms (Ma B, Kumar S, Tsai CJ, Nussinov R, 1999, Protein Eng 12:713–720) and to describe binding (Tsai CJ, Kumar S, Ma B, Nussinov R, 1999, Protein Sci 8:1181–1190), here we further extend the concept of folding funnels, illustrating its utility in explaining enzyme pathways, multimolecular associations, and allostery. This extension is based on the recognition that funnels are not stationary; rather, they are dynamic, depending on the physical or binding conditions (Tsai CJ, Ma B, Nussinov R, 1999, Proc Natl Acad Sci USA 96:9970–9972). Different binding states change the surrounding environment of proteins. The changed environment is in turn expressed in shifted energy landscapes, with different shapes and distributions of populations of conformers. Hence, the function of a protein and its properties are not only decided by the static folded three‐dimensional structure; they are determined by the distribution of its conformational substates, and in particular, by the redistributions of the populations under different environments. That is, protein function derives from its dynamic energy landscape, caused by changes in its surroundings.
Antibody‐detected folding: Kinetics of surface epitope formation are distinct from other folding phases
Authors: Raman C.S. , Nall Barry T. , Jemmerson Ronald 
Published: October 2010

AbstractThe rate of macromolecular surface formation in yeast iso‐2 cytochrome c and its site‐specific mutant, N52I iso‐2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39. The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early. The antibody‐detected (ad) kinetic phase accompanying epitope formation has Kad = 0.2 s−1 and is ∼40‐fold slower than the fastest detectable event in the folding of yeast iso‐2 cytochrome c (k2f ∼ 8 s−1), but occurs prior to the absorbance‐ and fluorescence‐detected slow folding steps (K1a ∼ 0.06 s−1; k1b ∼ 0.09 s‐1). N52I iso‐2 cytochrome c exhibits similar kinetic behavior with respect to epitope formation. A detailed dissection of the mechanistic differences between the folding pathways of horse and yeast cytochromes c identifies possible reasons for the slow surface formation in the latter. Our results suggest that non‐native ligation involving H33 or H39 during refolding may slow down the formation of the tertiary epitope in iso‐2 cytochrome c. This study illustrates that surface formation can be coupled to early events in protein folding. Thus, the rate of macromolecular surface formation is fine tuned by the residues that make up the surface and the interactions they entertain during refolding.
Kinetic evidence of an on‐pathway intermediate in the folding of lysozyme
Authors: Bai Yawen 
Published: October 2010

AbstractBy means of a kinetic test, it was demonstrated that one of the folding intermediates (Iα) of hen lysozyme with α‐domain folded and β‐domain unfolded is on the folding pathway under the classical definition. lα folds to the native (N) state directly (unfolded (U) ⟺ Iα ⟺ N) without having to unfold to U and then refold to N through alternative folding pathways as in Iα ⟺ U ⟺ N.
NMR analysis of cleaved Escherichia coli thioredoxin (1–73/74–108) and its P76A variant: Cis/trans peptide isomerization
Authors: Yu Wen‐Feng , Tasayco María Luisa , Tung Chang‐Shung , Wang Hsin 
Published: October 2010

AbstractInspection of high resolution three‐dimensional (3D) structures from the protein database reveals an increasing number of cis‐Xaa‐Pro and cis‐Xaa‐Yaa peptide bonds. However, we are still far from being able to predict whether these bonds will remain cis upon single‐site substitution of Pro or Yaa and/or cleavage of a peptide bond close to it in the sequence. We have chosen oxidized Escherichia coli thioredoxin (Trx), a member of the Trx superfamily with a single α/β domain and cis P76 to determine the effect of single‐site substitution and/or cleavage on this isomer. Standard two‐dimensional (2D) NMR analysis were performed on cleaved Trx (1–73074‐108) and its P76A variant. Analysis of the NOE connectivities indicates remarkable similarity between the secondary and supersecondary structure of the noncovalent complexes and Trx. Analysis of the 2D version of the HCCH‐TOCSY and HMQC‐NOESY‐HMQC and 13C‐filtered HMQC‐NOESY spectra of cleaved Trx with uniformly 13C‐labeled 175 and P76 shows surprising conservation of both cis P76 and packing of 175 against W31. A similar NMR analysis of its P76A variant provides no evidence for cis A76 and shows only subtle local changes in both the packing of 175 and the interstrand connectivities between its most protected hydrophobic strands (β2 and β4). Indeed, a molecular simulation model for the trans P76A variant of Trx shows only subtle local changes around the substitution site. In conclusion, cleavage of R73 is insufficient to provoke cis/trans isomerization of P76, but cleavage and single‐site substitution (P76A) favors the trans isomer.
The crystal structures of human α‐thrombin complexed with active site‐directed diamino benzo[b] thiophene derivatives: A binding mode for a structurally novel class of inhibitors
Authors: Chirgadze N.Y. , Sall D.J. , Briggs S.L. , Clawson D.K. , Zhang M. , Smith G.F. , Schevitz R.W. 
Published: October 2010

AbstractThe crystal structures of four active site‐directed thrombin inhibitors, 1–4, in a complex with human α‐thrombin have been determined and refined at up to 2.0 Å resolution using X‐ray crystallography. These compounds belong to a structurally novel family of inhibitors based on a 2,3‐disubstituted benzo[b]thiophene structure. Compared to traditional active‐site directed inhibitors, the X‐ray crystal structures of these complexes reveal a novel binding mode. Unexpectedly, the lipophilic benzo[b]thiophene nucleus of the inhibitor appears to bind in the S1 specificity pocket. At the same time, the basic amine of the C‐3 side chain of the inhibitor interacts with the mostly hydrophobic proximal, S2, and distal, S3, binding sites. The second, basic amine side chain at C‐2 was found to point away from the active site, occupying a location between the S1 and S'1 sites. Together, the aromatic rings of the C‐2 and C‐3 side chains sandwich the indole ring of Trp60D contained in the thrombin S2 insertion loop defined by the sequence “Tyr‐Pro‐Pro‐Trp.” [The thrombin residue numbering used in this study is equivalent to that reported for chymotrypsinogen (Hartley BS, Shotton DM, 1971, The enzymes, vol. 3. New York: Academic Press. pp 323–373).] In contrast to the binding mode of more classical thrombin inhibitors (D‐Phe‐Pro‐Arg‐H, NAPAP, Argatroban), this novel class of benzo[b]thiophene derivatives does not engage in hydrogen bond formation with Gly216 of the thrombin active site. A detailed analysis of the three‐dimensional structures not only provides a clearer understanding of the interaction of these agents with thrombin, but forms a foundation for rational structure‐based drug design. The use of the data from this study has led to the design of derivatives that are up to 2,900‐fold more potent than the screening hit 1.
Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase
Authors: Knapp James E. , Carroll Donald , Lawson Janet E. , Ernst Stephen R. , Reed Lester J. , Hackert Marvin L. 
Published: October 2010

AbstractThe dihydrolipoamide succinyltransferase (E2o) component of the α‐ketoglutarate dehydrogenase complex catalyzes the transfer of a succinyl group from the S‐succinyldihydrolipoyl moiety to coenzyme A. E2o is normally a 24‐mer, but is found as a trimer when E2o is expressed with a C‐terminal [His]6 tag. The crystal structure of the trimeric form of the catalytic domain (CD) of the Escherichia coli E2o has been solved to 3.0 Å resolution using the Molecular Replacement method. The refined model contains an intact trimer in the asymmetric unit and has an R‐factor of 0.257 (Rfree = 0.286) for 18,699 reflections between 10.0 and 3.0 Å resolution. The core of tE2oCD (residues 187–396) superimposes onto that of the cubic E2oCD with an RMS difference of 0.4 Å for all main‐chain atoms. The C‐terminal end of tE2oCD (residues 397–404) rotates by an average of 378 compared to cubic E2oCD, disrupting the normal twofold interface. Despite the alteration of quaternary structure, the active site of tE2oCD shows no significant differences from that of the cubic E2oCD, although several side chains in the active site are more ordered in the trimeric form of E2oCD. Analysis of the available sequence data suggests that the majority of E2 components have active sites that resemble that of E. coli E2oCD. The remaining E2 components can be divided into three groups based on active‐site sequence similarity. Analysis of the surface properties of both crystal forms of E. coli E2oCD suggests key residues that may be involved in the protein‐protein contacts that occur between the catalytic and lipoyl domains of E2o.
The use of nucleotide analogs to evaluate the mechanism of the heterotropic response of Escherichia coli aspartate transcarbamoylase
Authors: Sakash Jessica B. , Tsen Anita , Kantrowitz Evan R. 
Published: October 2010

AbstractAs an alternative method to study the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase, a series of nucleotide analogs were used. These nucleotide analogs have the advantage over site‐specific mutagenesis experiments in that interactions between the backbone of the protein and the nucleotide could be evaluated in terms of their importance for function. The ATP analogs purine 5′‐triphosphate (PTP), 6‐chloropurine 5′‐triphosphate (Cl‐PTP), 6‐mercaptopurine 5′‐triphosphate (SH‐PTP), 6‐methylpurine 5′‐triphosphate (Me‐PTP), and 1‐methyladenosine 5′‐triphosphate (Me‐ATP) were partially synthesized from their corresponding nucleosides. Kinetic analysis was performed on the wild‐type enzyme in the presence of these ATP analogs along with GTP, ITP, and XTP. PTP, Cl‐PTP, and SH‐PTP each activate the enzyme at subsaturating concentrations of L‐aspartate and saturating concentrations of carbamoyl phosphate, but not to the same extent as does ATP. These experiments suggest that the interaction between N6‐amino group of ATP and the backbone of the regulatory chain is important for orienting the nucleotide and inducing the displacements of the regulatory chain backbone necessary for initiation of the regulatory response. Me‐PTP and Me‐ATP also activate the enzyme, but in a more complex fashion, which suggests differential binding at the two sites within each regulatory dimer. The purine nucleotides GTP, ITP, and XTP each inhibit the enzyme but to a lesser extent than CTP. The influence of deoxy and dideoxynucleotides on the activity of the enzyme was also investigated. These experiments suggest that the 2′ and 3′ ribose hydroxyl groups are not of significant importance for binding and orientation of the nucleotide in the regulatory binding site. 2′‐dCTP inhibits the enzyme to the same extent as CTP, indicating that the interactions of the enzyme to the O2‐carbonyl of CTP are critical for CTP binding, inhibition, and the ability of the enzyme to discriminate between ATP and CTP. Examination of the electrostatic surface potential of the nucleotides and the regulatory chain suggest that the complimentary electrostatic interactions between the nucleotides and the regulatory chain are important for binding and orientation of the nucleotide necessary to induce the local conformational changes that propagate the heterotropic effect.
Deleterious effects of β‐branched residues in the S1specificity pocket ofStreptomyces griseusproteinase B (SGPB): Crystal structures of the turkey ovomucoid third domain variants Ile18I, Val18I, Thr18I, and Ser18I in complex with SGPB
Authors: Bateman Katherine S. , James Michael N.G. , Anderson Stephen , Lu Wuyuan , Qasim M.A. , Laskowski Michael 
Published: October 2010

AbstractTurkey ovomucoid third domain (OMTKY3) is a canonical inhibitor of serine proteinases. Upon complex formation, the inhibitors fully exposed P1residue becomes fully buried in the preformed cavity of the enzyme. All 20 P1variants of OMTKY3 have been obtained by recombinant DNA technology and their equilibrium association constants have been measured with six serine proteinases. To rationalize the trends observed in this data set, high resolution crystal structures have been determined for OMTKY3 P1variants in complex with the bacterial serine proteinase,Streptomyces griseusproteinase B (SGPB). Four high resolution complex structures are being reported in this paper; the three β‐branched variants, Ile18I, Val18I, and Thr18I, determined to 2.1, 1.6, and 1.7 Å resolution, respectively, and the structure of the Ser18I variant complex, determined to 1.9 Å resolution. Models of the Cys18I, Hse18I, and Ape18I variant complexes are also discussed. The β‐branched side chains are not complementary to the shape of the S1binding pocket in SGPB, in contrast to that of the wild‐type γ‐branched P1residue for OMTKY3, Leu18I.X1angles of approximately 40° are imposed on the side chains of Ile18I, Val18I, and Thr18I within the S1pocket. Dihedral angles of +60°, ‐60°, or 180° are more commonly observed but 40° is not unfavorable for the β‐branched side chains. Thr18I Oγ1also forms a hydrogen bond with Ser195 Oγin this orientation. The Ser18I side chain adopts two alternate conformations within the S1pocket of SGPB, suggesting that the side chain is not stable in either conformation.
Dynamics of the Hck‐SH3 domain: Comparison of experiment with multiple molecular dynamics simulations
Authors: Horita David A. , Byrd R. Andrew , Zhang Weixing , Smithgall Thomas E. , Gmeiner William H. 
Published: October 2010

AbstractMolecular dynamics calculations provide a method by which the dynamic properties of molecules can be explored over timescales and at a level of detail that cannot be obtained experimentally from NMR or X‐ray analyses. Recent work (Philippopoulos M, Mandel AM, Palmer AG III, Lim C, 1997, Proteins 28:481–493) has indicated that the accuracy of these simulations is high, as measured by the correspondence of parameters extracted from these calculations to those determined through experimental means. Here, we investigate the dynamic behavior of the Src homology 3 (SH3) domain of hematopoietic cell kinase (Hck) via 15N backbone relaxation NMR studies and a set of four independent 4 ns solvated molecular dynamics calculations. We also find that molecular dynamics simulations accurately reproduce fast motion dynamics as estimated from generalized order parameter (S 2) analysis for regions of the protein that have experimentally well‐defined coordinates (i.e., stable secondary structural elements). However, for regions where the coordinates are not well defined, as indicated by high local root‐mean‐square deviations among NMR‐determined structural family members or high B‐factors/low electron density in X‐ray crystallography determined structures, the parameters calculated from a short to moderate length (less than 5–10 ns) molecular dynamics trajectory are dependent on the particular coordinates chosen as a starting point for the simulation.
A novel method of affinity‐purifying proteins using a bis‐arsenical fluorescein
Authors: Thorn Kurt S. , Naber Nartman , Matuska Marija , Cooke Roger , Vale Ronald D. 
Published: October 2010

AbstractGenetically‐encoded affinity tags constitute an important strategy for purifying proteins. Here, we have designed a novel affinity matrix based on the bis‐arsenical fluorescein dye FlAsH, which specifically recognizes short α‐helical peptides containing the sequence CCXXCC (Griffin BA, Adams SR, Tsien RY, 1998, Science 281:269–212). We find that kinesin tagged with this cysteine‐containing helix binds specifically to FlAsH resin and can be eluted in a fully active form. This affinity tag has several advantages over polyhistidine, the only small affinity tag in common use. The protein obtained with this single chromatographic step from crude Escherichia coli lysates is purer than that obtained with nickel affinity chromatography of 6xHis tagged kinesin. Moreover, unlike nickel affinity chromatography, which requires high concentrations of imidazole or pH changes for elution, protein bound to the FlAsH column can be completely eluted by dithiothreitol. Because of these mild elution conditions, FlAsH affinity chromatography is ideal for recovering fully active protein and for the purification of intact protein complexes.
Protein global fold determination using site‐directed spin and isotope labeling
Authors: Gaponenko Vadim , Howarth Jack W. , Gasmi‐Seabrook Genevieve , Yuan Jie , Columbus Linda , Hubbell Wayne L. , Rosevear Paul R. 
Published: October 2010

AbstractWe describe a simple experimental approach for the rapid determination of protein global folds. This strategy utilizes site‐directed spin labeling (SDSL) in combination with isotope enrichment to determine long‐range distance restraints between amide protons and the unpaired electron of a nitroxide spin label using the paramagnetic effect on relaxation rates. The precision and accuracy of calculating a protein global fold from only paramagnetic effects have been demonstrated on barnase, a well‐characterized protein. Two monocysteine derivatives of barnase, (H102C) and (H102A/Q15C), were 15N enriched, and the paramagnetic nitroxide spin label, MTSSL, attached to the single Cys residue of each. Measurement of amide 1H longitudinal relaxation times, in both the oxidized and reduced states, allowed the determination of the paramagnetic contribution to the relaxation processes. Correlation times were obtained from the frequency dependence of these relaxation processes at 800, 600, and 500 MHz. Distances in the range of 8 to 35 Å were calculated from the magnitude of the paramagnetic contribution to the relaxation processes and individual amide 1H correlation times. Distance restraints from the nitroxide spin to amide protons were used as restraints in structure calculations. Using nitroxide to amide 1H distances as long‐range restraints and known secondary structure restraints, barnase global folds were calculated having backbone RMSDs <3 å from the crystal structure. this approach makes it possible to rapidly obtain the overall topology of a protein using a limited number of paramagnetic distance restraints.
Proline inhibits aggregation during protein refolding
Authors: Samuel Dharmaraj , Ganesh Gopal , Yang Pey‐Wen , Chang Mei‐Ming , Wang Sue‐Lein , Hwang Kuo‐Chu , Yu Chin , Jayaraman Gurunathan , Kumar Thallampuranam Krishnaswamy S. , Trivedi Vishwa Deo , Chang Ding‐Kwo 
Published: October 2010

AbstractThe in vitro refolding of hen egg‐white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate(s) and trapping the folding intermediate(s) into enzymatically inactive, “aggregation‐insensitive” state(s). However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations (>1.5 M), proline is shown to form loose, higher‐order molecular aggregate(s). The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher‐order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone.
Human RhoA/RhoGDI complex expressed in yeast: Gtp exchange is sufficient for translocation of RhoA to liposomes
Authors: Read Paul W. , Liu Xiaopu , Longenecker Kenton , Dipierro Charles G. , Walker Lori A. , Somlyo Avril V. , Somlyo Andrew P. , Nakamoto Robert K. 
Published: October 2010

AbstractThe human small GTPase, RhoA, expressed in Saccharomyces cerevisiae is post‐translationally processed and, when co‐expressed with its cytosolic inhibitory protein, RhoGDI, spontaneously forms a heterodimer in vivo. The RhoA/RhoGDI complex, purified to greater than 98% at high yield from the yeast cytosolic fraction, could be stoichiometrically ADP‐ribosylated by Clostridium botulinum C3 exoenzyme, contained stoichiometric GDP, and could be nucleotide exchanged fully with [3H]GDP or partially with GTP in the presence of submicromolar Mg2+. The GTP‐RhoA/RhoGDI complex hydrolyzed GTP with a rate constant of 4.5 × 10−5 s−1, considerably slower than free RhoA. Hydrolysis followed pseudo‐first‐order kinetics indicating that the RhoA hydrolyzing GTP was RhoGDI associated. The constitutively active G14V‐RhoA mutant expressed as a complex with RhoGDI and purified without added nucleotide also bound stoichiometric guanine nucleotide: 95% contained GDP and 5% GTP. Microinjection of the GTP‐bound G14V‐RhoA/RhoGDI complex (but not the GDP form) into serum‐starved Swiss 3T3 cells elicited formation of stress fibers and focal adhesions. In vitro, GTP‐bound‐RhoA spontaneously translocated from its complex with RhoGDI to liposomes, whereas GDP‐RhoA did not. These results show that GTP‐triggered translocation of RhoA from RhoGDI to a membrane, where it carries out its signaling function, is an intrinsic property of the RhoA/RhoGDI complex that does not require other protein factors or membrane receptors.