bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nucleotide binding to the G12V‐mutant of Cdc42 investigated by X‐ray diffraction and fluorescence spectroscopy: Two different nucleotide states in one crystal
Authors: Rudolph Markus Georg , Wittinghofer Alfred , Vetter Ingrid Renate 
Published: October 2010

AbstractThe 2.5 Å crystal structure of the full length human placental isoform of the Gly12 to Val mutant Cdc42 protein (Cdc42(G12V)) bound to both GDP/Mg2+ and GDPNH2 (guanosine‐5′‐diphospho‐β‐amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg2+, while the other has bound GDPNH2 without a Mg2+ ion. Crystallization of the protein was induced via hydrolysis of the Cdc42 · GppNHp complex by the presence of contaminating alkaline phosphatase activity in combination with the crystallization conditions. This prompted us to compare the binding characteristics of GDPNH2 vs. GDP. The amino group of GDPNH2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg2+ ion, and apparently also increases the conformational flexibility of the protein as seen in the crystal. Both the switch I and switch II regions are visible in the electron density of the GDP‐bound molecule, but not in the molecule bound to GDPNH2. The C‐terminus containing the CaaX‐motif is partly ordered in both molecules due to an intramolecular disulfide bond formed between Cys105/Cys188 and Cys305/Cys388, respectively.
Nucleolar protein B23 has molecular chaperone activities
Authors: Szebeni Attila , Olson Mark O.J. 
Published: October 2010

AbstractProtein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone‐like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature‐dependent and ‐independent aggregation of the HIV‐1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine‐HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis.
Computational studies on mutant protein stability: The correlation between surface thermal expansion and protein stability
Authors: Palma Rocio , Curmi Paul M.G. 
Published: October 2010

AbstractThermal stability of mutant proteins has been investigated using temperature dependent molecular dynamics (MD) simulations in vacuo. The numerical modeling was aimed at mimicking protein expansion upon heating. After the conditions for an expanding protein accessible surface area were established for T4 lysozyme and barnase wild‐type proteins, MD simulations were carried out under the same conditions using the crystal structures of several mutant proteins. The computed thermal expansion of the accessible surface area of mutant proteins was found to be strongly correlated with their experimentally measured stabilities. A similar, albeit weaker, correlation was observed for model mutant proteins. This opens the possibility of obtaining stability information directly from protein structure.
Disulfide engineering at the dimer interface of lactobacillus casei thymidylate synthase: Crystal structure of the T155C/E188C/C244T mutant
Authors: Velanker S.S. , Gokhale R.S. , Ray Soumya S. , Gopal B. , Parthasarathy S. , Santi D.V. , Balaram P. , Murthy M.R.N. 
Published: October 2010

AbstractThe crystal structure of a covalently cross‐linked Lactobacillus casei thymidylate synthase has been determined at 2.8 å resolution. The sites for mutation to achieve the bis‐disulfide linked dimer were identified using the disulfide modeling program MODIP. The mutant so obtained was found to be remarkably thermostable. This increase in stability has been reasoned to be entirely a consequence of the covalent gluing between the two subunits.
Substrate‐assisted catalysis: Molecular basis and biological significance
Authors: Dall'Acqua William , Carter Paul 
Published: October 2010

AbstractSubstrate‐assisted catalysis (SAC) is the process by which a functional group in a substrate contributes to catalysis by an enzyme. SAC has been demonstrated for representatives of three major enzyme classes: serine proteases, GTPases, and type II restriction endonucleases, as well as lysozyme and hexose‐1‐phosphate uridylyltransferase. Moreover, structure‐based predictions of SAC have been made for many additional enzymes. Examples of SAC include both naturally occurring enzymes such as type II restriction endonucleases as well as engineered enzymes including serine proteases. In the latter case, a functional group from a substrate can substitute for a catalytic residue replaced by site‐directed mutagenesis. From a protein engineering perspective, SAC provides a strategy for drastically changing enzyme substrate specificity or even the reaction catalyzed. From a biological viewpoint, SAC contributes significantly to the activity of some enzymes and may represent a functional intermediate in the evolution of catalysis. This review focuses on advances in engineering enzyme specificity and activity by SAC, together with the biological significance of this phenomenon.
Affinity cleavage at the divalent metal site of porcine NAD‐specific isocitrate dehydrogenase
Authors: Huang Yu‐Chu , Soundar Sambanthamurthy , Colman Roberta F. 
Published: October 2010

AbstractA divalent metal ion, such as Mn2+, is required for the catalytic reaction and allosteric regulation of pig heart NAD‐dependent isocitrate dehydrogenase. The enzyme is irreversibly inactivated and cleaved by Fe2+ in the presence of O2 and ascorbate at pH 7.0. Mn2+ prevents both inactivation and cleavage. Nucleotide ligands, such as NAD, NADPH, and ADP, neither prevent nor promote inactivation or cleavage of the enzyme by Fe2+. The NAD‐specific isocitrate dehydrogenase is composed of three distinct subunits in the ratio 2α:1 β:1 γ. The results indicate that the oxidative inactivation and cleavage are specific and involve the 40 kDa α subunit of the enzyme. A pair of major peptides is generated during Fe2+ inactivation: 29.5 + 10.5 kDa, as determined by SDS‐PAGE. Amino‐terminal sequencing reveals that these peptides arise by cleavage of the Val262‐His263 bond of the α subunit. No fragments are produced when enzyme is incubated with Fe2+ and ascorbate under denaturing conditions in the presence of 6 M urea, indicating that the native structure is required for the specific cleavage. These results suggest that His263 of the α subunit may be a ligand of the divalent metal ion needed for the reaction catalyzed by isocitrate dehydrogenase. Isocitrate enhances the inactivation of enzyme caused by Fe2+ in the presence of oxygen, but prevents the cleavage, suggesting that inactivation occurs by a different mechanism when metal ion is bound to the enzyme in the presence of isocitrate: oxidation of cysteine may be responsible for the rapid inactivation in this case. Affinity cleavage caused by Fe2+ implicates α as the catalytic subunit of the multisubunit porcine NAD‐dependent isocitrate dehydrogenase.
Apoflavodoxin (un) folding followed at the residue level by NMR
Authors: Van Mierlo Carlo P.M. , Van Den Oever Jos M.P. , Steensma Elles 
Published: October 2010

AbstractThe denaturant‐induced (un)folding of apoflavodoxin from Azotobacter vinelandii has been followed at the residue level by NMR spectroscopy. NH groups of 21 residues of the protein could be followed in a series of 1H‐15N heteronuclear single‐quantum coherence spectra recorded at increasing concentrations of guanidinium hydrochloride despite the formation of protein aggregate. These NH groups are distributed throughout the whole apoflavodoxin structure. The midpoints of unfolding determined by NMR coincide with the one obtained by fluorescence emission spectroscopy. Both techniques give rise to unfolding curves with transition zones at significantly lower denaturant concentrations than the one obtained by circular dichroism spectroscopy. The NMR (un)folding data support a mechanism for apoflavodoxin folding in which a relatively stable intermediate is involved. Native apoflavodoxin is shown to cooperatively unfold to a molten globule‐like state with extremely broadened NMR resonances. This initial unfolding step is slow on the NMR chemical shift timescale. The subsequent unfolding of the molten globule is faster on the NMR chemical shift timescale and the limited appearance of 1H‐15N HSQC cross peaks of unfolded apoflavodoxin in the denaturant range studied indicates that it is noncooperative.
Amide proton hydrogen exchange rates for sperm whale myoglobin obtained from 15N‐1H NMR spectra
Authors: Cavagnero Silvia , Thériault Yves , Narula Surinder S. , Dyson H. Jane , Wright Peter E. 
Published: October 2010

AbstractThe hydrogen exchange behavior of exchangeable protons in proteins can provide important information for understanding the principles of protein structure and function. The positions and exchange rates of the slowly‐exchanging amide protons in sperm whale myoglobin have been mapped using 15N‐1H NMR spectroscopy. The slowest‐exchanging amide protons are those that are hydrogen bonded in the longest helices, including members of the B, E, and H helices. Significant protection factors were observed also in the A, C, and G helices, and for a few residues in the D and F helices. Knowledge of the identity of slowly‐exchanging amide protons forms the basis for the extensive quench‐flow kinetic folding experiments that have been performed for myoglobin, and gives insights into the tertiary interactions and dynamics in the protein.
X‐ray crystal structure and molecular dynamics simulations of silver hake parvalbumin (Isoform B)
Authors: Richardson Robert C. , King Nancy M. , Sun Hongmao , Nelson Donald J. , Harrington Daniel J. , Royer William E. 
Published: October 2010

AbstractParvalbumins constitute a class of calcium‐binding proteins characterized by the presence of several helix‐loop‐helix (EF‐hand) motifs. In a previous study (Revett SP, King G, Shabanowitz J, Hunt DF, Hartman KL, Laue TM, Nelson DJ, 1997, Protein Sci 7:2397–2408), we presented the sequence of the major parvalbumin isoform from the silver hake (Merluccius bilinearis) and presented spectroscopic and structural information on the excised “EF‐hand” portion of the protein. In this study, the X‐ray crystal structure of the silver hake major parvalbumin has been determined to high resolution, in the frozen state, using the molecular replacement method with the carp parvalbumin structure as a starting model. The crystals are orthorhombic, space group C2221, with a = 75.7 Å, b = 80.7 Å, and c = 42.1 Å. Data were collected from a single crystal grown in 15% glycerol, which served as a cryoprotectant for flash freezing at ‐188 °C. The structure refined to a conventional R‐value of 21% (free R 25%) for observed reflections in the range 8 to 1.65 Å [I > 2σ(I)]. The refined model includes an acetylated amino terminus, 108 residues (characteristic of a β parvalbumin lineage), 2 calcium ions, and 114 water molecules per protein molecule. The resulting structure was used in molecular dynamics (MD) simulations focused primarily on the dynamics of the ligands coordinating the Ca2+ ions in the CD and EF sites. MD simulations were performed on both the fully Ca2+ loaded protein and on a Ca2+ deficient variant, with Ca2+ only in the CD site. There was substantial agreement between the MD and X‐ray results in addressing the issue of mobility of key residues in the calcium‐binding sites, especially with regard to the side chain of Ser55 in the CD site and Asp92 in the EF site.
2.9 Å crystal structure of ligand‐free tryptophanyl‐tRNA synthetase: Domain movements fragment the adenine nucleotide binding site
Authors: Ilyin Valentin A. , Temple Brenda , Hu Mei , Carter Charles W. , Li Genpei , Yin Yuhui , Vachette Patrice 
Published: October 2010

AbstractThe crystal structure of ligand‐free tryptophanyl‐tRNA synthetase (TrpRS) was solved at 2.9 Å using a combination of molecular replacement and maximum‐entropy map/phase improvement. The dimeric structure (R = 23.7, Rfree = 26.2) is asymmetric, unlike that of the TrpRS tryptophanyl‐5′AMP complex (TAM; Doublie S, Bricogne G, Gilmore CJ, Carter CW Jr, 1995, Structure 3:17–31). In agreement with small‐angle solution X‐ray scattering experiments, unliganded TrpRS has a conformation in which both monomers open, leaving only the tryptophan‐binding regions of their active sites intact. The amino terminal αA‐helix, TIGN, and KMSKS signature sequences, and the distal helical domain rotate as a single rigid body away from the dinucleotide‐binding fold domain, opening the AMP binding site, seen in the TAM complex, into two halves. Comparison of side‐chain packing in ligand‐free TrpRS and the TAM complex, using identification of nonpolar nuclei (Ilyin VA, 1994, Protein Eng 7:1189–1195), shows that significant repacking occurs between three relatively stable core regions, one of which acts as a bearing between the other two. These domain rearrangements provide a new structural paradigm that is consistent in detail with the“induced‐fit” mechanism proposed for TyrRS by Fersht et al. (Fersht AR, Knill‐Jones JW, Beduelle H, Winter G, 1988, Biochemistry 27:1581–1587). Coupling of ATP binding determinants associated with the two catalytic signature sequences to the helical domain containing the presumptive anticodon‐binding site provides a mechanism to coordinate active‐site chemistry with relocation of the major tRNA binding determinants.
Characterization of the functional role of asp 141, asp 194, and asp464 residues in the Mn2+‐l‐malate binding of pigeon liver malic enzyme
Authors: Chou Wei‐Yuan , Chang Hwei‐Ping , Huang Chien‐Hsiun , Kuo Cheng‐Chin , Chang Gu‐Gang , Tong Liang 
Published: October 2010

AbstractPigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+‐ascorbate system in acidic environment. Site‐specific mutagenesis was performed at these putative metal‐binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild‐type malic enzyme (WT) were successfully cloned and expressed inEscherichia colicells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q‐Sepharose and adenosine‐2′,5′‐bisphosphate‐agarose columns. The mutants showed similar apparentKm,nadpvalues to that of the WT. TheKm, Mal value was increased in the D141N and D194N mutants. TheKm, Mnvalue, on the other hand, was increased only in the D141N mutant by 14‐fold, corresponding to ∼ 1.6 kcal/mol for the Asp141‐Mn2+binding energy. Substrate inhibition by L‐malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp 194, and Asp464 were analyzed by the double‐mutation cycles and triple‐mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [kcat/(Kd, MnKm, MalKm, NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10‐ to 100‐fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+‐L‐malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+‐L‐malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.
Structure‐based thermodynamic analysis of the dissociation of protein phosphatase‐1 catalytic subunit and microcystin‐LR docked complexes
Authors: Lavigne Pierre , Willard Leigh , Sykes Brian D. , Bagu John R. , Boyko Robert , Holmes Charles E. B. 
Published: October 2010

AbstractThe relationship between the structure of a free ligand in solution and the structure of its bound form in a complex is of great importance to the understanding of the energetics and mechanism of molecular recognition and complex formation. In this study, we use a structure‐based thermodynamic approach to study the dissociation of the complex between the toxin microcystin‐LR (MLR) and the catalytic domain of protein phosphatase‐1 (PP‐lc) for which the crystal structure of the complex is known. We have calculated the thermodynamic parameters (enthalpy, entropy, heat capacity, and free energy) for the dissociation of the complex from its X‐ray structure and found the calculated dissociation constant (4.0 × 10−11) to be in excellent agreement with the reported inhibitory constant (3.9 × 10−11). We have also calculated the thermodynamic parameters for the dissociation of 47 PP‐1c:MLR complexes generated by docking an ensemble of NMR solution structures of MLR onto the crystal structure of PP‐1c. In general, we observe that the lower the root‐mean‐square deviation (RMSD) of the docked complex (compared to the X‐ray complex) the closer its free energy of dissociation (δG°d) is to that calculated from the X‐ray complex. On the other hand, we note a significant scatter between the δG°d and the RMSD of the docked complexes. We have identified a group of seven docked complexes with δG°d values very close to the one calculated from the X‐ray complex but with significantly dissimilar structures. The analysis of the corresponding enthalpy and entropy of dissociation shows a compensation effect suggesting that MLR molecules with significant structural variability can bind PP‐1c and that substantial conformational flexibility in the PP‐1c:MLR complex may exist in solution.
Substrate‐ and pH‐dependent contribution of oxyanion binding site to the catalysis of prolyl oligopeptidase, a paradigm of the serine oligopeptidase family
Authors: Szeltner Zoltán , Renner Veronika , Polgár László 
Published: October 2010

AbstractProlyl oligopeptidase, an enzyme implicated in memory disorders, is a member of a new serine peptidase family. Crystallographic studies (Fülöp et al., 1998) revealed a novel oxyanion binding site containing a tyrosine residue, Tyr473. To study the importance of Tyr473 OH, we have produced prolyl oligopeptidase and its Tyr473Phe variant in Escherichia coli. The specificity rate constant, kcat/Km, for the modified enzyme decreased by a factor of 8–40 with highly specific substrates, Z‐Gly‐Pro‐Nap, and a fluorogenic octapeptide. With these compounds, the decline in kcat was partly compensated for by reduction in Km, a difference from the extensively studied subtilisin. With the less specific suc‐Gly‐Pro‐Nap, the Km value, which approximates Ks, was not significantly changed, resulting in greater diminution (∼500‐fold) in kcat/Km. The second‐order rate constant for the reaction with Z‐Pro‐prolinal, a slow tight‐binding transition‐state analogue inhibitor, and the Kj values for a slow substrate and two product‐like inhibitors were not significantly affected by the Tyr473 OH group. The mechanism of transition‐state stabilization was markedly dependent upon the nature of substrate and varied with pH as the enzyme interconverted between its two catalytically competent forms.
Contribution of proton linkage to the thermodynamic stability of the major cold‐shock protein of Escherichia coli CspA
Authors: Petrosian Susan A. , Makhatadze George I. 
Published: October 2010

AbstractThe stability of protein is defined not only by the hydrogen bonding, hydrophobic effect, van der Waals interactions, and salt bridges. Additional, much more subtle contributions to protein stability can arise from surface residues that change their properties upon unfolding. The recombinant major cold shock protein of Escherichia coli CspA an all‐β protein unfolds reversible in a two‐state manner, and behaves in all other respects as typical globular protein. However, the enthalpy of CspA unfolding strongly depends on the pH and buffer composition. Detailed analysis of the unfolding enthalpies as a function of pH and buffers with different heats of ionization shows that CspA unfolding in the pH range 5.5–9.0 is linked to protonation of an amino group. This amino group appears to be the N‐terminal α‐amino group of the CspA molecule. It undergoes a 1.6 U shift in pKa values between native and unfolded states. Although this shift in pKa is expected to contribute ∼5 kJ/mol to CspA stabilization energy the experimentally observed stabilization is only ∼1 kJ/mol. This discrepancy is related to a strong enthalpy—entropy compensation due, most likely, to the differences in hydration of the protonated and deprotonated forms of the α‐amino group.
Entropic barriers, transition states, funnels, and exponential protein folding kinetics: A simple model
Authors: Bicout D. J. , Szabo Attila 
Published: October 2010

AbstractThis paper presents an analytically tractable model that captures the most elementary aspect of the protein folding problem, namely that both the energy and the entropy decrease as a protein folds. In this model, the system diffuses within a sphere in the presence of an attractive spherically symmetric potential. The native state is represented by a small sphere in the center, and the remaining space is identified with unfolded states. The folding temperature, the time‐dependence of the populations, and the relaxation rate are calculated, and the folding dynamics is analyzed for both golf‐course and funnel‐like energy landscapes. This simple model allows us to illustrate a surprising number of concepts including entropic barriers, transition states, funnels, and the origin of single exponential relaxation kinetics.