bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dynamic NMR studies of ligand‐receptor interactions: Design and analysis of a rapidly exchanging complex of FKBP‐12/FK506 with a 24 kDa calcineurin fragment
Authors: Fejzo Jasna , Lepre Christopher A. , Peng Jeffrey W. , Su Michael S.‐S. , Thomson John A. , Moore Jonathan M. 
Published: February 2009

AbstractDynamic NMR methods, such as differential line broadening and transferred NOE spectroscopy, are normally reserved for the study of small molecule ligand interactions with large protein receptors. Using a combination of isotope labeling and isotope edited NMR, we have extended these techniques to characterize interactions of a much larger protein/drug complex, FKBP‐12/FK506 with its receptor protein, calcineurin. In order to examine this multicomponent system by dynamic NMR methods, the 93 kDa, tightly bound FKBP‐12/FK506/Cn complex was replaced with a lower affinity, rapidly exchanging system consisting of FKBP‐12/FK506 (13 kDa), recombinant calcineurin subunit B (CnB) (20 kDa), and a synthetic peptide (4 kDa) corresponding to the B binding domain (BBD) of calcineurin catalytic subunit A (CnA)S.Analysis of 1H‐13C HSQC data acquired for the FKBP‐12/13C‐FK506 and FKBP‐12/13C‐FK506/CnB/BBD complexes indicates that FKBP‐12/FK506 and CnB/BBD are in fast exchange in the quaternary complex. Comparison of proton line widths shows significant broadening of resonances along the macrocycle backbone at 13‐CH, 13‐OMe, 15‐OMe, 18‐CH2, 20‐CH, 21‐CH, and 25‐Me, as well as moderate broadening on the macrocycle backbone at 17‐Me, 24‐CH, and the pyranose 12‐CH2 protons. The tri‐substituted olefin and cyclohexyl groups also show moderate broadening at the 27‐Me, 28‐CH, and 30‐CH2 positions, respectively. Unexpectedly, little line broadening was observed for the allyl resonances of FK506 in the quaternary complex, although 13C longitudinal relaxation measurements suggest this group also makes contacts with calcineurin. In addition, intermolecular transfer NOE peaks were observed for the allyl 37‐CH2, 21‐CH, 30‐CH2, 13‐OMe, 15‐OMe, 17‐Me, 25‐Me, and 27‐Me groups, indicating that these are potential sites on the FK506 molecule that interact with calcineurin.
Purification and crystallization of cyclin‐dependent kinase inhibitor p21
Authors: Mayrose Dale R. , Ke Hengming , Xiong Yue , Nichols Michael A. 
Published: February 2009

Abstractp21, a universal inhibitor of mammalian cyclin‐dependent kinases (CDK), regulates cell cycle progression by forming various distinct protein complexes with cyclins, CDKs, and the proliferating cell nuclear antigen. We have overexpressed recombinant human p21 in E. coli and purified active p21 to near homogeneity on a large scale. Crystals of recombinant p21 have been grown in the space group P21 a = 157.4, b = 152.7, c = 90.6 Å, and β = 92.7°. The diffraction data of the recombinant p21 have been collected to 2.5 and 3.5 Å resolution for the native crystal and two heavy atom derivatives of mercury and iridium.
Members of the immunoglobulin superfamily in bacteria
Authors: Bateman Alex , Chothia Cyrus , Eddy Sean R. 
Published: February 2009

AbstractWe report a prediction that two prokaryotic proteins contain immunoglobulin superfamily domains. Immunoglobulin‐like folds have been identified previously in prokaryotic proteins, but these share no recognizable sequence similarity with eukaryotic immunoglobulin superfamily (IgSF) folds, and may be the result of the physics and chemistry of proteins favoring certain common folds. In contrast, the prokaryotic proteins identified have sequences whose match to the immunoglobulin superfamily can be detected by hidden Markov modeling, BLASTP matches, key residue analysis, and secondary structure predictions. We propose that these prokaryotic immunoglobulin‐like domains are almost certain to be related by divergence from a common ancestor to eukaryotic immunoglobulin superfamily domains.
Crystal structure of human interleukin‐10 at 1.6 Å resolution and a model of a complex with its soluble receptor
Authors: Zdanov Alexander , Schalk‐Hihi Céline , Wlodawer Alexander 
Published: February 2009

AbstractThe crystal structure of human interleukin‐10 (IL‐10) was refined at 1.6 Å resolution against X‐ray diffraction data collected at 100 K with the use of synchrotron radiation. Although similar to the IL‐10 structure determined previously at room temperature, this low‐temperature IL‐10 structure contains, in addition, four N‐terminal residues, three sulfate anions, and 175 extra water molecules. Whereas the main‐chain conformation is preserved, about 30% of the side chains, most of them on the protein surface, assume different conformations. A computer model of a complex of IL‐10 with its two soluble receptors was generated based on the topological similarity of IL‐10 to interferon‐γ. The contact region between the cytokine and each receptor shows excellent complementarity of polar and hydrophobic interactions, suggesting that the model is generally correct and should be useful in guiding mutagenesis experiments.
Protein secondary structural types are differentially coded on messenger RNA
Authors: Thanaraj T. A. , Argos Patrick 
Published: February 2009

AbstractTricodon regions on messenger RNAs corresponding to a set of proteins from Escherichia coli were scrutinized for their translation speed. The fractional frequency values of the individual codons as they occur in mRNAs of highly expressed genes from Escherichia coli were taken as an indicative measure of the translation speed. The tricodons were classified by the sum of the frequency values of the constituent codons. Examination of the conformation of the encoded amino acid residues in the corresponding protein tertiary structures revealed a correlation between codon usage in mRNA and topological features of the encoded proteins. Alpha helices on proteins tend to be preferentially coded by translationally fast mRNA regions while the slow segments often code for beta strands and coil regions. Fast regions correspondingly avoid coding for beta strands and coil regions while the slow regions similarly move away from encoding alpha helices. Structural and mechanistic aspects of the ribosome peptide channel support the relevance of sequence fragment translation and subsequent conformation. A discussion is presented relating the observation to the reported kinetic data on the formation and stabilization of protein secondary structural types during protein folding. The observed absence of such strong positive selection for codons in non‐highly expressed genes is compatible with existing theories that mutation pressure may well dominate codon selection in non‐highly expressed genes.
The 1.8‐Å X‐ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding
Authors: Sugiyama Shigeru , Matsuo Yo , Vassylyev Dmitry G. , Matsushima Masaaki , Morikawa Kosuke , Maenaka Katsumi , Kashiwagi Keiko , Igarashi Kazuei 
Published: February 2009

AbstractThe PotD protein from Escherichia coli is one of the components of the polyamine transport system present in the periplasm. This component specifically binds either spermidine or putrescine. The crystal structure of the E. coli PotD protein complexed with spermidine was solved at 1.8 Å resolution and revealed the detailed substrate‐binding mechanism. The structure provided the detailed conformation of the bound spermidine. Furthermore, a water molecule was clearly identified in the binding site lying between the amino‐terminal domain and carboxyl‐terminal domain. Through this water molecule, the bound spermidine molecule forms two hydrogen bonds with Thr 35 and Ser 211. Another periplasmic component of polyamine transport, the PotF protein, exhibits 35% sequence identity with the PotD protein, and it binds only putrescine, not spermidine. To understand these different substrate specificities, model building of the PotF protein was performed on the basis of the PotD crystal structure. The hypothetical structure suggests that the side chain of Lys 349 in PotF inhibits spermidine binding because of the repulsive forces between its positive charge and spermidine. On the other hand, putrescine could be accommodated into the binding site without any steric hindrance because its molecular size is much smaller than that of spermidine, and the positively charged amino group is relatively distant from Lys 349.
Construction and analysis of a profile library characterizing groups of structurally known proteins
Authors: Ogiwara Atsushi , Uchiyama Ikuo , Takagi Toshihisa , Kanehisa Minoru 
Published: February 2009

AbstractA new sequence motif library StrProf was constructed characterizing the groups of related proteins in the PDB three‐dimensional structure database. For a representative member of each protein family, which was identified by cross‐referencing the PDB with the PIR superfamily classification, a group of related sequences was collected by the BLAST search against the nonredundant protein sequence database. For every group, the motifs were identified automatically according to the criteria of conservation and uniqueness of pentapeptide patterns and with a dual dynamic programming algorithm. In the StrProf library, motifs are represented by profile matrices rather than consensus patterns to allow more flexible search capabilities. Another dynamic programming algorithm was then developed to search this motif library. When the computationally derived StrProf was compared with PROSITE, which is a manually derived motif library in the best consensus pattern representation, the numbers of identified patterns were comparable. StrProf missed about one third of the PROSITE motifs, but there were also new motifs lacking in PROSITE. The new library was incorporated in SMART (Sequence Motif Analysis and Retrieval Tool), a computer tool designed to help search and annotate biologically important sites in an unknown protein sequence. The client program is available free of charge through the Internet.
Phosphoribosyl anthranilate isomerase from Thermotoga maritima is an extremely stable and active homodimer
Authors: Sterner Reinhard , Kleemann Gerd R. , Szadkowski Halina , Lustig Ariel , Kirschner Kasper , Hennig Michael 
Published: February 2009

AbstractThe metabolism of hyperthermophilic microorganisms can function properly at temperatures close to 100 °C. It follows that they are equipped with both thermostable enzymes and mechanisms that handle labile metabolites. We wanted to understand how stable and active phosphoribosyl anthranilate isomerase (tPRAI) from the hyperthermophile Thermotoga maritima is at its optimum growth temperature of 80 °C, and how its thermolabile substrate, N‐(5′‐phosphoribosyl)‐anthranilate (PRA), is protected from rapid decomposition. To this end, the trpF gene of T. maritima was expressed heterologously in Escherichia coli and tPRAI was purified. In contrast to most PRAIs from mesophiles, which are monomers with the eightfold βα (or TIM) barrel fold, tPRAI is a homodimer. It is strongly resistant toward inactivation by temperatures up to 95 °C, by acidification to pH 3.2, and by proteases in the presence and absence of detergents. tPRAI is about 35‐fold more active at its physiologic temperature than is the enzyme from E. coli (ePRAI) at 37 °C. This high catalytic efficiency of tPRAI is likely to compete successfully with the rapid spontaneous hydrolysis of PRA at 80 °C. Thus, with respect to both stability and function, tPRAI appears well adapted to the extreme habitat of T. maritima. Single crystals of tPRAI have been obtained that are suitable for X‐ray analysis at high resolution.
A fast conformational search strategy for finding low energy structures of model proteins
Authors: Beutler Thomas C. , Dill Ken A. 
Published: February 2009

AbstractWe describe a new computer algorithm for finding low‐energy conformations of proteins. It is a chain‐growth method that uses a heuristic bias function to help assemble a hydrophobic core. We call it the Core‐directed chain Growth method (CG). We test the CG method on several well‐known literature examples of HP lattice model proteins [in which proteins are modeled as sequences of hydrophobic (H) and polar (P) monomers], ranging from 20–64 monomers in two dimensions, and up to 88‐mers in three dimensions. Previous nonexhaustive methods—Monte Carlo, a Genetic Algorithm, Hydrophobic Zippers, and Contact Interactions—have been tried on these same model sequences. CG is substantially better at finding the global optima, and avoiding local optima, and it does so in comparable or shorter times. CG finds the global minimum energy of the longest HP lattice model chain for which the global optimum is known, a 3D 88‐mer that has only been reachable before by the CHCC complete search method. CG has the potential advantage that it should have nonexponential scaling with chain length. We believe this is a promising method for conformational searching in protein folding algorithms.
Detection and characterization of an ovine placental lactogen stable intermediate in the urea‐induced unfolding process
Authors: Cymes Gisela D. , Delfino Jose M. , Wolfenstein‐Todel Carlota , Grosman Claudio 
Published: February 2009

AbstractThe urea‐induced equilibrium unfolding of ovine placental lactogen, purified from ovine placenta, was followed by size‐exclusion chromatography, far‐UV CD, and intrinsic tryptophan fluorescence. The data obtained by each of these methods showed a poor fit to a two‐state model involving only a native and an unfolded form. A satisfactory fit required, instead, a model that involved a stable, partially folded form in addition to the native and unfolded ones. The results obtained from the best‐fitting theoretical curves for the three‐state model indicated that this intermediate state, which is the predominant species in solution at 3.6 M of urea activity, is compact, largely α‐helical, and changes considerably the native‐like tertiary packing around its tryptophan residues. These findings suggest that this stable intermediate exhibits properties similar to those that characterize the molten globule state.
NMR characterization of structure, backbone dynamics, and glutathione binding of the human macrophage migration inhibitory factor (MIF)
Authors: Mühlhahn Peter , Czisch Michael , Georgescu Julia , Renner Christian , Ross Alfred , Holak Tad A. , Bernhagen Jürgen , Bucala Richard 
Published: February 2009

AbstractHuman macrophage migration inhibitory factor is a 114 amino acid protein that belongs to the family of immunologic cytokines. Assignments of 1H, l5N, and 13C resonances have enabled the determination of the secondary structure of the protein, which consists of two α‐helices (residues 18–31 and 89–72) and a central four‐stranded β‐sheet. In the β ‐sheet, two parallel β ‐sheets are connected in an antiparallel sense. From the total of three cysteines present in the primary structure of MIF, none was found to form disulfide bridges. 1H‐15N heteronuclear T1, T2, and steady‐state NOE measurements indicate that the backbone of MIF exists in a rigid structure of limited conformational flexibility (on the nanosecond to picosecond time scale). Several residues located in the loop regions and at the N termini of two helices exhibit internal motions on the 1–3 ns time scale. The capacity to bind glutathione was investigated by titration of a uniform l5N‐labeled sample and led us to conclude that MIF has, at best, very low affinity for glutathione.
Dynamics of ribonuclease A and ribonuclease S: Computational and experimental studies
Authors: Nadig Gautham , Ratnaparkhi Girish S. , Varadarajan Raghavan , Vishveshwara Saraswathi 
Published: February 2009

AbstractRNase S is a complex consisting of two proteolytic fragments of RNase A: the S peptide (residues 1–20) and S protein (residues 21–124). RNase S and RNase A have very similar X‐ray structures and enzymatic activities. Previous experiments have shown increased rates of hydrogen exchange and greater sensitivity to tryptic cleavage for RNase S relative to RNase A. It has therefore been asserted that the RNase S complex is considerably more dynamically flexible than RNase A. In the present study we examine the differences in the dynamics of RNase S and RNase A computationally, by MD simulations, and experimentally, using trypsin cleavage as a probe of dynamics. The fluctuations around the average solution structure during the simulation were analyzed by measuring the RMS deviation in coordinates. No significant differences between RNase S and RNase A dynamics were observed in the simulations. We were able to account for the apparent discrepancy between simulation and experiment by a simple model. According to this model, the experimentally observed differences in dynamics can be quantitatively explained by the small amounts of free S peptide and S protein that are present in equilibrium with the RNase S complex. Thus, folded RNase A and the RNase S complex have identical dynamic behavior, despite the presence of a break in polypeptide chain between residues 20 and 21 in the latter molecule. This is in contrast to what has been widely believed for over 30 years about this important fragment complementation system.
Participation of water in hin recombinase‐DNA recognition
Authors: Robinson Clifford R. , Sugar Stephen G. 
Published: February 2009

AbstractThe participation of water molecules in the interaction between the Hin recombinase and its operator DNA has been detected by analysis of the dissociation constant in the presence of varying concentrations of neutral solutes and cosolvents. The dissociation constant as measured by gel mobility shift assays increased as the concentration of dimethyl sulfoxide, glycerol, sucrose, or polyethylene glycol was increased. Osmotic pressure is the only property that correlates with the change in the dissociation constant for all compounds. This data indicates that binding of a small population of water molecules accompanies formation of the Hin‐DNA complex, and points to a novel role for solvent molecules in assisting site specific interaction between DNA‐binding proteins and their cognate DNA sequence.
1H NMR assignments of apo calcyclin and comparative structural analysis with calbindin D9k and S100β
Authors: Potts Barbara C.M. , Carlström Göran , Okazaki Katsuo , Hidaka Hiroyoshi , Chazin Walter J. 
Published: February 2009

AbstractThe homodimeric S100 protein calcyclin has been studied in the apo state by two‐dimensional 1H NMR spectroscopy. Using a combination of scalar correlation and NOE experiments, sequence‐specific 1H NMR assignments were obtained for all but one backbone and >90% of the side‐chain resonances. To our knowledge, the 2 × 90 residue (20 kDa) calcyclin dimer is the largest protein system for which such complete assignments have been made by purely homo‐nuclear methods. Sequential and medium‐range NOEs and slowly exchanging backbone amide protons identified directly the four helices and the short antiparallel β‐type interaction between the two binding loops that comprise each subunit of the dimer. Further analysis of NOEs enabled the unambiguous assignment of 556 intrasubunit distance constraints, 24 intrasubunit hydrogen bonding constraints, and 2 × 26 intersubunit distance constraints. The conformation of the monomer subunit was refined by distance geometry and restrained molecular dynamics calculations using the intrasubunit constraints only. Calculation of the dimer structure starting from this conformational ensemble has been reported elsewhere. The extent of structural homology among the apo calcyclin subunit, the monomer subunit of apo S100β, and monomeric apo calbindin D9k has been examined in detail by comparing 1H NMR chemical shifts and secondary structures. This analysis was extended to a comprehensive comparison of the three‐dimensional structures of the calcyclin monomer subunit and calbindin D9k, which revealed greater similarity in the packing of their hydrophobic cores than was anticipated previously. Together, these results support the hypothesis that all members of the S100 family have similar core structures and similar modes of dimerization. Analysis of the amphiphilicity of Helix IV is used to explain why calbindin D9k is monomeric, but full‐length S100 proteins form homodimers.
Solution structure of a mini IGF‐1
Authors: Wolf Elke De , Gill Raj , Geddes Stella , Pitts Jim , Wollmer Axel , Grötzinger Joachim 
Published: February 2009

AbstractMini insulin‐like growth factor 1, an inactive insulin‐like growth factor 1 mutant lacking the C region, was studied by 2D NMR spectroscopy. Resonances were assigned for almost all protons of the 57 amino acid residues. The 3D structure of the protein was determined by distance geometry methods. Three helical segments; Ala 8‐Cys 18, Gly 42‐Phe 49, and Leu 54‐Cys 61, were identified, corresponding to those present in wild‐type insulin‐like growth factor 1 and in single‐chain insulin. Their relative orientation, however, was found to be changed. This change is connected with a displacement of the Phe 23‐Tyr 24‐Phe 25‐Asn 26 β‐strand‐like segment, i.e., of aromatic side chains known to be important for receptor binding. Thus, deletion of the C region of IGF‐1 results in a substantial tertiary structural rearrangement that accounts for the loss of receptor affinity.
Serpin α1proteinase inhibitor probed by intrinsic tryptophan fluorescence spectroscopy
Authors: Koloczek Henryk , Banbula Agnieszka , Potempa Jan , Salvesen Guy S. 
Published: February 2009

AbstractVarious conformational forms of the archetypal serpin human α1proteinase inhibitor (α1PI), including ordered polymers, active and inactive monomers, and heterogeneous aggregates, have been produced by refolding from mild denaturing conditions. These forms presumably originate by different folding pathways during renaturation, under the influence of the A and C sheets of the molecule. Because α1PI contains only two Trp residues, at positions 194 and 238, it is amenable to fluorescence quenching resolved spectra and red‐edge excitation measurements of the Trp environment. Thus, it is possible to define the conformation of the various forms based on the observed fluorescent properties of each of the Trp residues measured under a range of conditions. We show that denaturation in GuHCl, or thermal denaturation in Tris, followed by renaturation, leads to the formation of polymers that contain solvent‐exposed Trp 238, which we interpret as ordered head‐to‐tail polymers (A‐sheet polymers). However, thermal denaturation in citrate leads to shorter polymers where some of the Trp 238 residues are not solvent accessible, which we interpret as polymers capped by head‐to‐head interactions via the C sheet. The latter treatment also generates monomers thought to represent a latent form, but in which the environment of Trp 238 is occluded by ionized groups. These data indicate that the folding pathway of α1PI, and presumably other serpins, is sensitive to solvent composition that affects the affinity of the reactive site loop for the A sheet or the C sheet.
Crystal structure analyses of uncomplexed ecotin in two crystal forms: Implications for its function and stability
Authors: Shin Dong Hae , Song Hyun Kyu , Suh Se Won , Seong Ihn Sik , Lee Cheol Soon , Chung Chin Ha 
Published: February 2009

AbstractEcotin, a homodimeric protein composed of 142 residue subunits, is a novel serine protease inhibitor present in Escherichia coli. Its thermostability and acid stability, as well as broad specificity toward proteases, make it an interesting protein for structural characterization. Its structure in the uncomplexed state, determined for two different crystalline environments, allows a structural comparison of the free inhibitor with that in complex with trypsin. Although there is no gross structural rearrangement of ecotin when binding trypsin, the loops involved in binding trypsin show relatively large shifts in atomic positions. The inherent flexibility of the loops and the highly nonglobular shape are the two features essential for its inhibitory function. An insight into the understanding of the structural basis of thermostability and acid stability of ecotin is also provided by the present structure.