bluuebunny/crossref_metadata_2025_split huggingface dataset:
Calretinin and calbindin D28k have different domain organizations
Authors: Palczewska Małgorzata , Groves Patrick , Batta Gyula , Heise Bert , Kuźnicki Jacek 
Published: August 2003

AbstractThe domain organization of calretinin (CR) was predicted to involve all six EF‐hand motifs (labeled I to VI) condensed into a single domain, as characterized for calbindin D28k (Calb), the closest homolog of calretinin. Unperturbed1H,15N HSQC NMR spectra of a15N‐labeled calretinin fragment (CR III–VI, residues 100–271) in the presence of the unlabeled complimentary fragment (CR I–II, residues 1–100) show that these fragments do not interact. Size exclusion chromatography and affinity chromatography data support this conclusion. The HSQC spectrum of15N‐labeled CR is similar to the overlaid spectra of individual15N‐labeled CR fragments (CR I–II and CR III–VI), also suggesting that these regions do not interact within intact CR. In contrast to these observations, but in accordance with the Calb studies, we observed interactions between other CR fragments: CR I (1–60) with CR II–VI (61–271), and CR I–III (1–142) with CR IV–VI (145–271). We conclude that CR is formed from at least two independent domains consisting of CR I–II and CR III–VI. The differences in domain organization of Calb and CR may explain the specific target interaction of Calb with caspase‐3. Most importantly, the comparison of CR and Calb domain organizations questions the value of homologous modeling of EF‐hand proteins, and perhaps of other protein families.
Screening for functional expression and overexpression of a family of diiron‐containing interfacial membrane proteins using the univector recombination system
Authors: Berthold Deborah A. , Stenmark Pål , Nordlund Pär 
Published: August 2003

AbstractThe large number of uncharacterized genes emerging from genome sequencing projects has resulted in a need for quick and reliable screening methods for protein expression parameters. We have utilized the univector plasmid recombination system (as previously reported) to develop a series of vectors for rapid screening for expression in Escherichia coli. A high level of recombinant protein expression is a requirement for purification of protein for structural determination and other purposes. In other applications, successful complementation of a missing enzyme activity in E. coli, as well as directed evolution studies and metabolic engineering, often require a much lower level of protein expression. In this report we describe the construction of a number of new pHOST vectors that can be screened for both low‐ and high‐level expression. We isolated a mutant vector for MBP fusions that exhibited a more optimal level of expression for complementation of aerobic respiration in hemA− E. coli, our functional assay for the alternative oxidase. We then demonstrated the use of our system to rapidly screen for both optimal functional expression and optimal overexpression of the alternative oxidase as well as two other members of a family of membrane‐bound diiron carboxylate proteins, the plastid terminal oxidase and 5‐demethoxyquinone hydroxylase.
Position‐dependent interactions between cysteine residues and the helix dipole
Authors: Miranda JJ L. 
Published: August 2003

AbstractA protein model was developed for studying the interaction between cysteine residues and the helix dipole. Site‐directed mutagenesis was used to introduce cysteine residues at the N‐terminus of helix H in recombinant sperm whale myoglobin. Based on the difference in thiol pKa between folded proteins and an unfolded peptide, the energy of interaction between the thiolate and the helix dipole was determined. Thiolates at the N1 and N2 positions of the helix were stabilized by 0.3 kcal/mole and 0.7 kcal/mole, respectively. A thiolate at the Ncap position was stabilized by 2.8 kcal/mole, and may involve a hydrogen bond. In context with other studies, an experimentally observed helix dipole effect may be defined in terms of two distinct components. A charge‐dipole component involves electrostatic interactions with peptide bond dipoles in the first two turns of the helix and affects residues at all positions of the terminus; a hydrogen bond component involves one or more backbone amide groups and is only possible at the capping position due to conformational restraints elsewhere. The nature and magnitude of the helix dipole effect is, therefore, position‐dependent. Results from this model system were used to interpret cysteine reactivity in rodent hemoglobins and the thioredoxin family.
NMR detection of multiple transitions to low‐populated states in azurin
Authors: Korzhnev Dmitry M. , Karlsson B. Göran , Orekhov Vladislav Yu. , Billeter Martin 
Published: August 2003

AbstractTransitions to conformational states with very low populations were detected for the reduced blue copper protein azurin from Pseudomonas aeruginosa by applying constant relaxation time CPMG measurements to the backbone 15N nuclei at three magnetic fields (11.7, 14.1, and 18.8 T) and three temperatures (25.7, 35.4, and 44.8°C). Two exchange processes with different rate constants could be discriminated despite populations of the excited states below 1% and spatial neighborhood of the two processes. The group of 15N nuclei involved in the faster process exhibits at 44.8°C a forward rate constant of 11.7±2.4 s−1 and a population of the exited state of 0.39±0.07%. They surround the aromatic ring of histidine 35 whose protonation state is coupled to the flipping of a neighboring peptide plane. For the slower process, the forward rate constant and population of the exited state at 44.8°C are 4.1±0.1 s−1 and 0.45±0.02%, respectively. The residues involved cluster nearby the copper ion, which is separated from the protonation site of histidine 35 by about 8 Å, indicating conformational rearrangements involving the copper coordinating loops. The dependence of the equilibrium constant on the temperature is consistent with an enthalpy‐dominated transition around the copper, but an entropy‐controlled transition near histidine 35. The detection by nuclear magnetic resonance of millisecond to second conformational transitions near the copper ion suggests a low energy‐cost rearrangement of the copper‐binding site that may be necessary for efficient electron transfer.
Leucine 145 of the ribotoxin α‐sarcin plays a key role for determining the specificity of the ribosome‐inactivating activity of the protein
Authors: Masip Manuel , García‐Ortega Lucía , Olmo Nieves , García‐Mayoral Ma Flor , Pérez‐Cañadillas José Manuel , Bruix Marta , Oñaderra Mercedes , Martínez del Pozo Álvaro , Gavilanes José G. 
Published: August 2003

AbstractSecreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as α‐sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of α‐sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild‐type α‐sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of α‐sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of α‐sarcin on ribosomes.
Structural genomics: Computational methods for structure analysis
Authors: Goldsmith‐Fischman Sharon , Honig Barry 
Published: August 2003

AbstractThe success of structural genomics initiatives requires the development and application of tools for structure analysis, prediction, and annotation. In this paper we review recent developments in these areas; specifically structure alignment, the detection of remote homologs and analogs, homology modeling and the use of structures to predict function. We also discuss various rationales for structural genomics initiatives. These include the structure‐based clustering of sequence space and genome‐wide function assignment. It is also argued that structural genomics can be integrated into more traditional biological research if specific biological questions are included in target selection strategies.
Contact order revisited: Influence of protein size on the folding rate
Authors: Ivankov Dmitry N. , Garbuzynskiy Sergiy O. , Alm Eric , Plaxco Kevin W. , Baker David , Finkelstein Alexei V. 
Published: August 2003

AbstractGuided by the recent success of empirical model predicting the folding rates of small two‐state folding proteins from the relative contact order (CO) of their native structures, by a theoretical model of protein folding that predicts that logarithm of the folding rate decreases with the protein chain length L as L2/3, and by the finding that the folding rates of multistate folding proteins strongly correlate with their sizes and have very bad correlation with CO, we reexamined the dependence of folding rate on CO and L in attempt to find a structural parameter that determines folding rates for the totality of proteins. We show that the Abs_CO = CO × L, is able to predict rather accurately folding rates for both two‐state and multistate folding proteins, as well as short peptides, and that this Abs_CO scales with the protein chain length as L0.70 ± 0.07 for the totality of studied single‐domain proteins and peptides.
Structural signatures of the complex formed between 3‐nitro‐4‐hydroxybenzoate and the Zn(II)‐substituted R6 insulin hexamer
Authors: Olsen Helle Birk , Leuenberger‐Fisher Melissa R. , Kadima Webe , Borchardt Dan , Kaarsholm Niels C. , Dunn Michael F. 
Published: August 2003

Abstract3‐Nitro‐4‐hydroxybenzoate (3N4H) is a probe of the structure and dynamics of the metal‐centered His B10 assembly sites of the insulin hexamer. Each His B10 site consists of a ∼12 Å‐long cavity situated on the threefold symmetry axis. These sites play an important role in the storage and release of insulin in vivo. The allosteric behavior of the insulin hexamer is modulated by ligand binding to the His B10 zinc sites and to the phenolic pockets. Binding to these sites drives transitions among three allosteric states, designated T6, T3R3, and R6. Although a wide variety of mono anions bind to the His B10 zinc sites of R3, X‐ray structures of ligands complexed to this site exist only for H2O, Cl–, and SCN–. This work combines one‐ and two‐dimensional 1H NMR and UV‐Vis absorbance studies of the structure and dynamics of the 3N4H complex, which establish the following: (1) relative to the NMR time scale, 3N4H exchange between free and bound states is slow, while flipping among three equivalent orientations about the site threefold axis is fast; (2) binding of 3N4H perturbs resonances within the His B10 zinc site and generates NOEs between ligand resonances and the insulin C‐α and side chain resonances of ValB2, AsnB3, LeuB6, and CysB7; and (3) 3N4H exchange for other ligands is limited by a protein conformational transition. These results are consistent with coordination of the 3N4H carboxylate to the His B10 zinc ion and van der Waals interactions with Val B2, Asn B3, Leu B6, and Cys A7.
Structure–activity relationships in HIV‐1 reverse transcriptase revealed by radiation target analysis
Authors: Sluis‐Cremer Nicolas , Kempner Ellis , Parniak Michael A. 
Published: August 2003

AbstractRadiation target analysis is a powerful technique that can be used to determine both the structural and functional sizes of macromolecules. We have used this technique to probe the structure–function relationships of the recombinant forms of HIV‐1 reverse transcriptase (RT). For the p66/p51 and p66/p66 dimeric forms of HIV‐1 RT, both the structural and functional target sizes corresponded to that of the dimeric protein, indicating that a primary ionization in one subunit of the HIV‐1 RT enzyme results in the concomitant polymer scission of both subunits. In contrast to p66/p51 and p66/p66 RT, the individually isolated p51 subunit of HIV‐1 RT inactivated as a monomer. However, in the presence of a DNA template/primer substrate, the radiation inactivation analyses of p51 yielded a structural target size corresponding to that of a dimeric protein. This would indicate that the DNA substrate acted as a scaffold or template for p51 RT homodimer formation. In light of this observation, radiation inactivation studies can readily be applied to other DNA polymerase enzymes, such as the murine leukemia virus RT, for which the functional form of the enzyme has yet to be determined.
Site2 binding energetics of the regulatory step of growth hormone–induced receptor homodimerization
Authors: Walsh Scott T.R. , Jevitts Liz M. , Sylvester Juliesta E. , Kossiakoff Anthony A. 
Published: August 2003

AbstractReceptor signaling in the growth hormone (GH)–growth hormone receptor (GHR) system is controlled through a sequential two‐step hormone‐induced dimerization of two copies of the extracellular domain (ECD) of the receptor. The regulatory step of this process is the binding of the second ECD (ECD2) to the stable preassociated 1 : 1 GH/ECD1 complex on the cell surface. To determine the energetics that governs this step, the binding kinetics of 38 single‐ and double‐alanine mutants in the hGH Site2 contact with ECD2 were measured by using trimolecular surface plasmon resonance (TM‐SPR). We find that the Site2 interface of hGH does not have a distinct binding hot‐spot region, and the most important residues are not spatially clustered, but rather are distributed over the whole binding surface. In addition, it was determined through analysis of a set of pairwise double alanine mutations that there is a significant degree of negative cooperativity among Site2 residues. Residues that show little effect or even improved binding on substitution with alanine, when paired with D116A‐hGH, display significant negative cooperativity. Because most of these pairwise mutated residues are spatially separated by ≥10 Å, this indicates that the Site2 binding interface of the hGH–hGHR ternary complex displays both structural and energetic malleability.
Crystal structure of mycothiol synthase (Rv0819) from Mycobacterium tuberculosis shows structural homology to the GNAT family of N‐acetyltransferases
Authors: Vetting Matthew W. , Roderick Steven L. , Yu Michael , Blanchard John S. 
Published: August 2003

AbstractMycothiol is the predominant low‐molecular weight thiol produced by actinomycetes, including Mycobacterium tuberculosis. The last reaction in the biosynthetic pathway for mycothiol is catalyzed by mycothiol synthase (MshD), which acetylates the cysteinyl amine of cysteine–glucosamine–inositol (Cys–GlcN–Ins). The crystal structure of MshD was determined in the presence of coenzyme A and acetyl–CoA. MshD consists of two tandem‐repeated domains, each exhibiting the Gcn5‐related N‐acetyltransferase (GNAT) fold. These two domains superimpose with a root‐mean‐square deviation of 1.7 Å over 88 residues, and each was found to bind one molecule of coenzyme, although the binding sites are quite different. The C‐terminal domain has a similar active site to many GNAT members in which the acetyl group of the coenzyme is presented to an open active site slot. However, acetyl–CoA bound to the N‐terminal domain is buried, and is apparently not positioned to promote acetyl transfer. A modeled substrate complex indicates that Cys–GlcN–Ins would only fill a portion of a negatively charged channel located between the two domains. This is the first structure determined for an enzyme involved in the biosynthesis of mycothiol.
Dissecting interdomain communication within cAPK regulatory subunit type IIβ using enhanced amide hydrogen/deuterium exchange mass spectrometry (DXMS)
Authors: Zawadzki Kerri M. , Hamuro Yoshitomo , Kim Jack S. , Garrod Siv , Stranz David D. , Taylor Susan S. , Woods Virgil L. 
Published: August 2003

AbstractcAMP‐dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dimer bound to two catalytic (C) subunits and is involved in numerous cell signaling pathways. The C‐subunit is activated allosterically when two cAMP molecules bind sequentially to the cAMP‐binding domains, designated A and B (cAB‐A and cAB‐B, respectively). Each cAMP‐binding domain contains a conserved Arg residue that is critical for high‐affinity cAMP binding. Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP‐binding domains, and cAPK activation. To better understand the local and long‐range effects that the Arg‐to‐Lys mutation has on the dynamic properties of the R‐subunit, the amide hydrogen/deuterium exchange in the RIIβ subunit was probed by electrospray mass spectrometry. Mutant proteins containing the Arg‐to‐Lys substitution in either cAMP‐binding domain were deuterated for various times and then, prior to mass spectrometry analysis, subjected to pepsin digestion to localize the deuterium incorporation. Mutation of this Arg in cAB‐A (Arg230) causes an increase in amide hydrogen exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is indicative of the importance of this Arg in domain organization. Mutation of Arg359 (cAB‐B) leads to increased exchange in the adjacent cAB‐A domain, particularly in the cAB‐A domain C‐helix that lies on top of the cAB‐B domain and is believed to be functionally linked to the cAB‐B domain. This interdomain communication appears to be a unidirectional pathway, as mutation of Arg230 in cAB‐A does not effect dynamics of the cAB‐B domain.
Structure of the Escherichia coli malate synthase G:pyruvate:acetyl‐coenzyme A abortive ternary complex at 1.95 Å resolution
Authors: Anstrom David M. , Kallio Karen , Remington S. James 
Published: August 2003

AbstractMalate synthase, an enzyme of the glyoxylate pathway, catalyzes the condensation and subsequent hydrolysis of acetyl‐coenzyme A (acetyl‐CoA) and glyoxylate to form malate and CoA. In the present study, we present the 1.95 Å–resolution crystal structure of Escherichia coli malate synthase isoform G in complex with magnesium, pyruvate, and acetyl‐CoA, and we compare it with previously determined structures of substrate and product complexes. The results reveal how the enzyme recognizes and activates the substrate acetyl‐CoA, as well as conformational changes associated with substrate binding, which may be important for catalysis. On the basis of these results and mutagenesis of active site residues, Asp 631 and Arg 338 are proposed to act in concert to form the enolate anion of acetyl‐CoA in the rate‐limiting step. The highly conserved Cys 617, which is immediately adjacent to the presumed catalytic base Asp 631, appears to be oxidized to cysteine‐sulfenic acid. This can explain earlier observations of the susceptibility of the enzyme to inactivation and aggregation upon X‐ray irradiation and indicates that cysteine oxidation may play a role in redox regulation of malate synthase activity in vivo. There is mounting evidence that enzymes of the glyoxylate pathway are virulence factors in several pathogenic organisms, notably Mycobacterium tuberculosis and Candida albicans. The results described in this study add insight into the mechanism of catalysis and may be useful for the design of inhibitory compounds as possible antimicrobial agents.
Solution structure of Pi4, a short four‐disulfide‐bridged scorpion toxin specific of potassium channels
Authors: Guijarro J. Iñaki , M'Barek Sarrah , Gómez‐Lagunas Froylan , Garnier Damien , Rochat Hervé , Sabatier Jean‐Marc , Possani Lourrival D. , Delepierre Muriel 
Published: August 2003

AbstractPi4 is a short toxin found at very low abundance in the venom of Pandinus imperator scorpions. It is a potent blocker of K+ channels. Like the other members of the α‐KTX6 subfamily to which it belongs, it is cross‐linked by four disulfide bonds. The synthetic analog (sPi4) and the natural toxin (nPi4) have been obtained by solid‐phase synthesis or from scorpion venom, respectively. Analysis of two‐dimensional 1H NMR spectra of nPi4 and sPi4 indicates that both peptides have the same structure. Moreover, electrophysiological recordings of the blocking of Shaker B K+ channels by sPi4 (KD = 8.5 nM) indicate that sPi4 has the same blocking activity of nPi4 (KD = 8.0 nM), previously described. The disulfide bonds have been independently determined by NMR and structure calculations, and by Edman‐degradation/mass‐spectrometry identification of peptides obtained by proteolysis of nPi4. Both approaches indicate that the pairing of the half‐cystines is 6C–27C, 12C–32C, 16C–34C, and 22C–37C. The structure of the toxin has been determined by using 705 constraints derived from NMR data on sPi4. The structure, which is well defined, shows the characteristic α/β scaffold of scorpion toxins. It is compared to the structure of the other α‐KTX6 subfamily members and, in particular, to the structure of maurotoxin, which shows a different pattern of disulfide bridges despite its high degree of sequence identity (76%) with Pi4. The structure of Pi4 and the high amounts of synthetic peptide available, will enable the detailed analysis of the interaction of Pi4 with K+ channels.
Phosphohydrolase and transphosphatidylation reactions of two Streptomyces phospholipase D enzymes: Covalent versus noncovalent catalysis
Authors: Yang Hongying , Roberts Mary F. 
Published: August 2003

AbstractA kinetic comparison of the hydrolase and transferase activities of two bacterial phospholipase D (PLD) enzymes with little sequence homology provides insights into mechanistic differences and also the more general role of Ca2+ in modulating PLD reactions. Although the two PLDs exhibit similar substrate specificity (phosphatidylcholine preferred), sensitivity to substrate aggregation or Ca2+, and pH optima are quite distinct. Streptomyces sp. PMF PLD, a member of the PLD superfamily, generates both hydrolase and transferase products in parallel, consistent with a mechanism that proceeds through a covalent phosphatidylhistidyl intermediate where the rate‐limiting step is formation of the covalent intermediate. For Streptomyces chromofuscus PLD, the two reactions exhibit different pH profiles, a result consistent with a mechanism likely to involve direct attack of water or an alcohol on the phosphorus. Ca2+, not required for monomer or micelle hydrolysis, can activate both PLDs for hydrolysis of PC unilamellar vesicles. In the case of Streptomyces sp. PMF PLD, Ca2+ relieves product inhibition by interactions with the phosphatidic acid (PA). A similar rate enhancement could occur with other HxKx4D‐motif PLDs as well. For S. chromofuscus PLD, Ca2+ is absolutely critical for binding of the enzyme to PC vesicles and for PA activation. That the Ca2+‐PA activation involves a discreet site on the protein is suggested by the observation that the identity of the C‐terminal residue in S. chromofuscus PLD can modulate the extent of product activation.