bluuebunny/crossref_metadata_2025_split huggingface dataset:
Changes in structure and dynamics of the Fv fragment of a catalytic antibody upon binding of inhibitor
Authors: Kroon Gerard J.A. , Mo Huaping , Martinez‐Yamout Maria A. , Dyson H. Jane , Wright Peter E. 
Published: June 2003

AbstractBinding of the product inhibitor p‐nitrophenol to the monoclonal esterolytic antibody NPN43C9 has been investigated by performing NMR spectroscopy of the heterodimeric variable‐domain fragment (Fv) of the antibody in the presence and absence of inhibitor. Structural information from changes in chemical shift upon binding has been related to the changes in local dynamics in the active site of the catalytic antibody using NMR relaxation measurements. Significant changes in the chemical shifts of the backbone resonances upon binding extend beyond the immediate vicinity of the antigen binding site into the interface between the two associated polypeptides that form the Fv heterodimer, a possible indication that the binding of ligand causes a change in the relative orientations of the component light (VL) and heavy (VH) chain polypeptides. Significant differences in backbone dynamics were observed between the free Fv and the complex with p‐nitrophenol. A number of resonances, including almost all of the third hypervariable loop of the light chain (L3), were greatly broadened in the free form of the protein. Other residues in the antigen‐binding site showed less broadening of resonances, but still required exchange terms (Rex) in the model‐free dynamics analysis, consistent with motion on a slow timescale in the active site region of the free Fv. Binding of p‐nitrophenol caused these resonances to sharpen, but some Rex terms are still required in the analysis of the backbone dynamics. We conclude that the slow timescale motions in the antigen‐binding site are very different in the bound and free forms of the Fv, presumably due to the damping of large‐amplitude motions by the bound inhibitor.
Crystal structures of fusion proteins with large‐affinity tags
Authors: Smyth Douglas R. , Mrozkiewicz Marek K. , McGrath William J. , Listwan Pawel , Kobe Bostjan 
Published: June 2003

AbstractThe fusion of a protein of interest to a large‐affinity tag, such as the maltose‐binding protein (MBP), thioredoxin (TRX), or glutathione‐S‐transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large‐affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three‐ to five‐amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest.
A solution NMR study of the binding kinetics and the internal dynamics of an HIV‐1 protease‐substrate complex
Authors: Katoh Etsuko , Louis John M. , Yamazaki Toshimasa , Gronenborn Angela M. , Torchia Dennis A. , Ishima Rieko 
Published: June 2003

AbstractNMR studies of the binding of a substrate to an inactive HIV‐1 protease construct, containing an active site mutation PRD25N, are reported. Substrate titration measurements monitored by HSQC spectra and a 15N‐edited NOESY experiment show that the chromogenic substrate analog of the capsid/p2 cleavage site binds to PRD25N with an equilibrium dissociation constant, KD, of 0.27 ± 0.05 mM, and upper limits of the association and dissociation rate constants, 2×104 M−1s−1 and 10 s−1, respectively, at 20°C, pH 5.8. This association rate constant is not in the diffusion limit, suggesting that association is controlled by a rare event, such as opening of the protease flaps. Analysis of 15N relaxation experiments reveals a slight reduction of S2 values in the flap region, indicating a small increase in the amplitude of internal motion on the sub‐nsec timescale. In addition, several residues in the flap region are mobile on the conformational exchange timescale, msec–μsec. Flap dynamics of the protease‐substrate complex are compared with those of protease‐inhibitor complexes, and the implications of these results for substrate‐binding models are discussed.
Recognition of cysteine‐containing peptides through prompt fragmentation of the 4‐dimethylaminophenylazophenyl‐4′‐maleimide derivative during analysis by MALDI‐MS
Authors: Borges Chad R. , Watson J. Throck 
Published: June 2003

AbstractUnder specified UV‐MALDI conditions, the 4‐dimethylaminophenylazophenyl‐4'‐maleimide (DABMI) derivative of cysteine‐containing peptides undergoes prompt fragmentation to produce a characteristic mass spectral pattern. As reported previously by others, derivatization with chromophoric DABMI allows cysteine‐containing peptides and proteins to be tracked during HPLC by absorbance at upper UV and visible wavelengths. Here, we describe methodology by which these same peptide derivatives can be recognized by their distinctive MALDI mass spectral fragmentation pattern, then mass mapped, allowing for easy, rapid identification of cysteine‐containing peptides.
Grafting a new metal ligand in the cocatalytic site of B. cereus metallo‐β‐lactamase: Structural flexibility without loss of activity
Authors: Rasia Rodolfo M. , Ceolín Marcelo , Vila Alejandro J. 
Published: June 2003

AbstractMetallo‐β‐lactamases are zinc enzymes able to hydrolyze the four‐membered ring of β‐lactam antibiotics, representing one of the latest generations of β‐lactamases. These enzymes belong to the zinc metallo‐hydrolase family of the β‐lactamase fold. Enzymes belonging to this family have a bimetallic active site whose structure varies among different members by point substitutions of the metal ligands. In this work, we have grafted new metal ligands into the metal binding site of BcII from Bacillus cereus that mimic the ligands present in other members of this superfamily. We have characterized spectroscopically and modeled the structure of the redesigned sites, which differ substantially from the wild‐type enzyme. Despite the changes introduced in the active site, the mutant enzymes retain almost full activity. These results shed some light on the possible evolutionary origin of these metalloenzymes.
Three monophyletic superfamilies account for the majority of the known glycosyltransferases
Authors: Liu Jing , Mushegian Arcady 
Published: June 2003

AbstractSixty‐five families of glycosyltransferases (EC 2.4.x.y) have been recognized on the basis of high‐sequence similarity to a founding member with experimentally demonstrated enzymatic activity. Although distant sequence relationships between some of these families have been reported, the natural history of glycosyltransferases is poorly understood. We used iterative searches of sequence databases, motif extraction, structural comparison, and analysis of completely sequenced genomes to track the origins of modern‐type glycosyltransferases. We show that >75% of recognized glycosyltransferase families belong to one of only three monophyletic superfamilies of proteins, namely, (1) a recently described GPGTF/GT‐B superfamily; (2) a nucleoside‐diphosphosugar transferase (GT‐A) superfamily, which is characterized by a DxD sequence signature and also includes nucleotidyltransferases; and (3) a GT‐C superfamily of integral membrane glycosyltransferases with a modified DxD signature in the first extracellular loop. Several developmental regulators in Metazoans, including Fringe and Egghead homologs, belong to the second superfamily. Interestingly, Tout‐velu/Exostosin family of developmental proteins found in all multicellular eukaryotes, contains separate domains belonging to the first and the second superfamilies, explaining multiple glycosyltransferase activities in one protein.
Structural studies of the interaction of S‐adenosylmethionine with the [4Fe‐4S] clusters in biotin synthase and pyruvate formate‐lyase activating enzyme
Authors: Cosper Michele M. , Cosper Nathaniel J. , Hong Wei , Shokes Jacob E. , Broderick William E. , Broderick Joan B. , Johnson Michael K. , Scott Robert A. 
Published: June 2003

AbstractThe diverse reactions catalyzed by the radical‐SAM superfamily of enzymes are thought to proceed via a set of common mechanistic steps, key among which is the reductive cleavage of S‐adenosyl‐L‐methionine (SAM) by a reduced [4Fe‐4S] cluster to generate an intermediate deoxyadenosyl radical. A number of spectroscopic studies have provided evidence that SAM interacts directly with the [4Fe‐4S] clusters in several of the radical‐SAM enzymes; however, the molecular mechanism for the reductive cleavage has yet to be elucidated. Selenium X‐ray absorption spectroscopy (Se‐XAS) was used previously to provide evidence for a close interaction between the Se atom of selenomethionine (a cleavage product of Se‐SAM) and an Fe atom of the [4Fe‐4S] cluster of lysine‐2,3‐aminomutase (KAM). Here, we utilize the same approach to investigate the possibility of a similar interaction in pyruvate formate‐lyase activating enzyme (PFL‐AE) and biotin synthase (BioB), two additional members of the radical‐SAM superfamily. The results show that the latter two enzymes do not exhibit the same Fe‐Se interaction as was observed in KAM, indicating that the methionine product of reductive cleavage of SAM does not occupy a well‐defined site close to the cluster in PFL‐AE and BioB. These results are interpreted in terms of the differences among these enzymes in their use of SAM as either a cofactor or a substrate.
Solution structure and thermal stability of ribosomal protein L30e from hyperthermophilic archaeonThermococcus celer
Authors: Wong Kam‐Bo , Lee Chi‐Fung , Chan Siu‐Hong , Leung Tak‐Yuen , Chen Yu Wai , Bycroft Mark 
Published: June 2003

AbstractTo understand the structural basis of thermostability, we have determined the solution structure of a thermophilic ribosomal protein L30e fromThermococcus celerby NMR spectroscopy. The conformational stability ofT. celerL30e was measured by guanidine and thermal‐induced denaturation, and compared with that obtained for yeast L30e, a mesophilic homolog. The melting temperature ofT. celerL30e was 94°C, whereas the yeast protein denatured irreversibly at temperatures >45°C. The two homologous proteins also differ greatly in their stability at 25°C: the free energy of unfolding was 45 kJ/mole forT. celerL30e and 14 kJ/mole for the yeast homolog. The solution structure ofT. celerL30e was compared with that of the yeast homolog. Although the two homologous proteins do not differ significantly in their number of hydrogen bonds and the amount of solvent accessible surface area buried with folding, the thermophilicT. celerL30e was found to have more long‐range ion pairs, more proline residues in loops, and better helix capping residues in helix‐1 and helix‐4. A K9A variant ofT. celerL30e was created by site‐directed mutagenesis to examine the role of electrostatic interactions on protein stability. Although the melting temperatures of the K9A variant is ∼8°C lower than that of the wild‐type L30e, their difference in Tmis narrowed to ∼4.2°C at 0.5 M NaCl. This salt‐dependency of melting temperatures strongly suggests that electrostatic interactions contribute to the thermostability ofT. celerL30e.
Folding specificity induced by loop stiffness
Authors: Spagnolo Laura , Ventura Salvador , Serrano Luis 
Published: June 2003

AbstractTo test the importance of loop stiffness in restricting the heterogeneity of transition state ensemble, we relaxed the distal loop of 10 unstable redesigned hydrophobic core mutants of α‐spectrin SH3 domain. This was achieved by replacing Asp48 by Gly at the tip of the distal hairpin. Although the change was local in nature, the effect on stabilization was not uniform across the core mutants tested. There is an inverse rough correlation between the stabilization and the increase in buried hydrophobic volume, with respect to the wild type. Interestingly enough, proteins that although unstable are properly folded become molten globule‐like after relaxation of the distal loop. These results highlight the importance of stiffness in restricting the conformational heterogeneity of a protein during the folding reaction. An interplay between unspecific hydrophobic interactions and constraint induced by polar interactions, or in this case local stiffness, is essential to achieve a well‐ordered folded structure.
Crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima
Authors: Shin Dong Hae , Roberts Anne , Jancarik Jaru , Yokota Hisao , Kim Rosalind , Wemmer David E. , Kim Sung‐Hou 
Published: June 2003

AbstractWe have determined the crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima, TM0651 (gi 4981173), at 2.2 Å resolution by selenomethionine single‐wavelength anomalous diffraction (SAD) techniques. TM0651 is a member of the haloacid dehalogenase (HAD) superfamily, with sequence homology to trehalose‐6‐phosphate phosphatase and sucrose‐6F‐phosphate phosphohydrolase. Selenomethionine labeled TM0651 crystallized in space group C2 with three monomers per asymmetric unit. Each monomer has approximate dimensions of 65 × 40 × 35 Å3, and contains two domains: a domain of known hydrolase fold characteristic of the HAD family, and a domain with a new tertiary fold consisting of a six‐stranded β‐sheet surrounded by four α‐helices. There is one disulfide bond between residues Cys35 and Cys265 in each monomer. One magnesium ion and one sulfate ion are bound in the active site. The superposition of active site residues with other HAD family members indicates that TM0651 is very likely a phosphatase that acts through the formation of a phosphoaspartate intermediate, which is supported by both NMR titration data and a biochemical assay. Structural and functional database searches and the presence of many aromatic residues in the interface of the two domains suggest the substrate of TM0651 is a carbohydrate molecule. From the crystal structure and NMR data, the protein likely undergoes a conformational change upon substrate binding.
The inactivating factor of glutamine synthetase, IF7, is a “natively unfolded” protein
Authors: Muro‐Pastor M. Isabel , Barrera Francisco N. , Reyes José C. , Florencio Francisco J. , Neira José L. 
Published: June 2003

AbstractGlutamine synthetase (GS) is the key enzyme responsible for the primary assimilation of ammonium in all living organisms, and it catalyses the synthesis of glutamine from glutamic acid, ATP, and ammonium. One of the recently discovered mechanisms of GS regulation involves protein‐protein interactions with a small 65‐residue‐long protein named IF7. Here, we study the structure and stability of IF7 and its binding properties to GS, by using several biophysical techniques (fluorescence, circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopies, and gel filtration chromatography) which provide complementary structural information. The findings show that IF7 has a small amount of residual secondary structure, but lacks a well defined tertiary structure, and is not compact. Thus, all of the studies indicate that IF7 is a “natively unfolded” protein. The binding of IF7 to GS, its natural binding partner, occurs with an apparent dissociation constant ofKD= 0.3 ± 0.1 μM, as measured by fluorescence. We discuss the implications for the GS regulation mechanisms of IF7 being unfolded.
Interaction of the 18.5‐kD isoform of myelin basic protein with Ca2+‐calmodulin: Effects of deimination assessed by intrinsic Trp fluorescence spectroscopy, dynamic light scattering, and circular dichroism
Authors: Libich David S. , Hill Christopher M.D. , Bates Ian R. , Hallett F. Ross , Armstrong Souzan , Siemiarczuk Aleksander , Harauz George 
Published: June 2003

AbstractThe effects of deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) on its binding to calmodulin (CaM) have been examined. Four species of MBP were investigated: unmodified recombinant murine MBP (rmMBP‐Cit0), an engineered protein with six quasi‐citrullinyl (i.e., glutaminyl) residues per molecule (rmMBP‐qCit6), human component C1 (hMBP‐Cit0), and human component C8 (hMBP‐Cit6), both obtained from a patient with multiple sclerosis (MS). Both rmMBP‐Cit0 and hMBP‐Cit0 bound CaM in a Ca2+‐dependent manner and primarily in a 1:1 stoichiometry, which was verified by dynamic light scattering. Circular dichroic spectroscopy was unable to detect any changes in secondary structure in MBP upon CaM‐binding. Inherent Trp fluorescence spectroscopy and a single‐site binding model were used to determine the dissociation constants: Kd = 144 ± 76 nM for rmMBP‐Cit0, and Kd = 42 ± 15 nM for hMBP‐Cit0. For rmMBP‐qCit6 and hMBP‐Cit6, the changes in fluorescence were suggestive of a two‐site interaction, although the dissociation constants could not be accurately determined. These results can be explained by a local conformational change induced in MBP by deimination, exposing a second binding site with a weaker association with CaM, or by the existence of several conformers of deiminated MBP. Titration with the collisional quencher acrylamide, and steady‐state and lifetime measurements of the fluorescence at 340 nm, showed both dynamic and static components to the quenching, and differences between the unmodified and deiminated proteins that were also consistent with a local conformational change due to deimination.
Analysis of accessible surface of residues in proteins
Authors: Lins Laurence , Thomas Annick , Brasseur Robert 
Published: June 2003

AbstractWe analyzed the total, hydrophobic, and hydrophilic accessible surfaces (ASAs) of residues from a nonredundant bank of 587 3D structure proteins. In an extended fold, residues are classified into three families with respect to their hydrophobicity balance. As expected, residues lose part of their solvent‐accessible surface with folding but the three groups remain. The decrease of accessibility is more pronounced for hydrophobic than hydrophilic residues. Amazingly, Lysine is the residue with the largest hydrophobic accessible surface in folded structures. Our analysis points out a clear difference between the mean (other studies) and median (this study) ASA values of hydrophobic residues, which should be taken into consideration for future investigations on a protein‐accessible surface, in order to improve predictions requiring ASA values. The different secondary structures correspond to different accessibility of residues. Random coils, turns, and β‐structures (outside β‐sheets) are the most accessible folds, with an average of 30% accessibility. The helical residues are about 20% accessible, and the difference between the hydrophobic and the hydrophilic residues illustrates the amphipathy of many helices. Residues from β‐sheets are the most inaccessible to solvent (10% accessible). Hence, β‐sheets are the most appropriate structures to shield the hydrophobic parts of residues from water. We also show that there is an equal balance between the hydrophobic and the hydrophilic accessible surfaces of the 3D protein surfaces irrespective of the protein size. This results in a patchwork surface of hydrophobic and hydrophilic areas, which could be important for protein interactions and/or activity.
Predicting the topology of transmembrane helical proteins using mean burial propensity and a hidden‐Markov‐model‐based method
Authors: Zhou Hongyi , Zhou Yaoqi 
Published: June 2003

AbstractHelices in membrane spanning regions are more tightly packed than the helices in soluble proteins. Thus, we introduce a method that uses a simple scale of burial propensity and a new algorithm to predict transmembrane helical (TMH) segments and a positive‐inside rule to predict amino‐terminal orientation. The method (the topology predictor of transmembrane helical proteins using mean burial propensity [THUMBUP]) correctly predicted the topology of 55 of 73 proteins (or 75%) with known three‐dimensional structures (the 3D helix database). This level of accuracy can be reached by MEMSAT 1.8 (a 200‐parameter model‐recognition method) and a new HMM‐based method (a 111‐parameter hidden Markov model, UMDHMMTMHP) if they were retrained with the 73‐protein database. Thus, a method based on a physiochemical property can provide topology prediction as accurate as those methods based on more complicated statistical models and learning algorithms for the proteins with accurately known structures. Commonly used HMM‐based methods and MEMSAT 1.8 were trained with a combination of the partial 3D helix database and a 1D helix database of TMH proteins in which topology information were obtained by gene fusion and other experimental techniques. These methods provide a significantly poorer prediction for the topology of TMH proteins in the 3D helix database. This suggests that the 1D helix database, because of its inaccuracy, should be avoided as either a training or testing database. A Web server of THUMBUP and UMDHMMTMHP is established for academic users at http://www.smbs.buffalo.edu/phys_bio/service.htm. The 3D helix database is also available from the same Web site.
Role of a solvent‐exposed tryptophan in the recognition and binding of antibiotic substrates for a metallo‐β‐lactamase
Authors: Huntley James J.A. , Fast Walter , Benkovic Stephen J. , Wright Peter E. , Dyson H. Jane 
Published: June 2003

AbstractNumerous X‐ray crystal structures of the metallo‐β‐lactamase from Bacteroides fragilis and related organisms show a β‐hairpin loop immediately adjacent to the active‐site zinc atom(s). Both crystallographic and NMR information show that the end of this β‐hairpin loop, which contains a solvent exposed tryptophan residue, Trp49, is highly flexible in the absence of substrates or other ligands, giving rise in some of the X‐ray structures to a lack of observable electron density in this region. We report an investigation of the role of this mobile, solvent‐exposed tryptophan using site‐directed mutagenesis, steady state kinetics measurements and characterization by NMR. Trp49 appears to have a role both in substrate binding and in promotion of catalysis. Substitution of this residue with a number of different side chains indicates that the binding interaction depends on the bulky hydrophobic and aromatic nature of the indole ring, which can provide relatively non‐specific interactions with a variety of antibiotic substrates. In this way, the tryptophan at this position provides a large degree of the breadth of substrate specificity for the metallo‐β‐lactamase. Previous studies established that the antibiotic binding site was sufficiently plastic that the derivatization of existing antibiotics is unlikely to result in the successful treatment of bacterial infections incorporating this resistance element. Rather, a more productive approach may be to design therapeutics directed towards this solvent‐exposed tryptophan residue.