bluuebunny/crossref_metadata_2025 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

Dynamic 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

p21, 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

We 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

The 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

Tricodon 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

The 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

A 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

The 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

We 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

The 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

Human 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

RNase 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

The 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

The 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

Mini 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

Various 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

Ecotin, 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.
Individual amino acids in the N‐terminal loop region determine the thermostability and unfolding characteristics of bacterial glucanases
Authors: Welfle Karin , Politz Oliver , Borriss Rainer , Misselwitz Rolf , Welfle Heinz
Published: February 2009

Thermostability and unfolding behavior of the wild‐type (1,3–1,4)‐β‐glucanases from Bacillus macerans (MAC) and Bacillus amyloliquefaciens (AMY) and of two hybrid enzymes H(A12‐M)ΔF14 and H(A12‐M)ΔY13F14A were studied by spectroscopic and microcalorimetric measurements. H(A12‐M)ΔF14 is constructed by the fusion of 12 N‐terminal amino acids of AMY with amino acids 13–214 of MAC, and by deletion of F14. In H(A12‐M)ΔY13F14A, the N‐terminal region of MAC is exchanged against the AMY sequence, Y13 is deleted, and Phe 14 is exchanged against Ala. The sequence of the N‐terminal loop region from Pro 9 to amino acid 16 (or 17) is very important for the properties of the enzymes and influences the effects of Ca2+ ions on the thermostability and unfolding behavior of the enzymes. The half transition temperatures Tm are higher in the presence of Ca2+ than in Ca2+ free buffer. Furthermore, the unfolding mechanism is influenced by Ca2+. In Ca2+‐free buffer, MAC, H(A12‐M)ΔF14 and H(A12‐M)ΔY13F14A unfold in a single cooperative transition from the folded state to the unfolded state, whereas for AMY, a two‐step unfolding was found. In the presence of Ca2+, the two‐step unfolding of AMY is strengthened. Furthermore, for H(A12‐M)ΔF14, a two‐step unfolding is induced by Ca2+. These data indicate a two‐domain structure of AMY and H(A12‐M)ΔF14 in the presence of Ca2+. Thus, point mutations in a peripheral loop region are decisive for thermal stabilities and unfolding mechanisms of the studied glucanases in the presence of Ca2+.
Characterization of the S1 binding site of the glutamic acid‐specific protease from Streptomyces griseus
Authors: Stennicke Henning R. , Breddam Klaus , Birktoft Jens J.
Published: February 2009

The glutamic acid‐specific protease from Streptomyces griseus (SGPE) is an 18.4‐kDa serine protease with a distinct preference for Glu in the P1 position. Other enzymes characterized by a strong preference for negatively charged residues in the P1 position, e.g., interleukin‐1β converting enzyme (ICE), use Arg or Lys residues as counterions within the S1 binding site. However, in SGPE, this function is contributed by a His residue (His 213) and two Ser residues (Ser 192 and S216). It is demonstrated that proSGPE is activated autocatalytically and dependent on the presence of a Glu residue in the — 1 position. Based on this observation, the importance of the individual S1 residues is evaluated considering that enzymes unable to recognize a Glu in the P1 position will not be activated. Among the residues constituting the S1 binding site, it is demonstrated that His 213 and Ser 192 are essential for recognition of Glu in the P1 position, whereas Ser 216 is less important for catalysis but has an influence on stabilization of the ground state. From the three‐dimensional structure, it appears that His 213 is linked to two other His residues (His 199 and His 228), forming a His triad extending from the S1 binding site to the back of the enzyme. This hypothesis has been tested by substitution of His 199 and His 228 with other amino acid residues. The catalytic parameters obtained with the mutant enzymes, as well as the pH dependence, do not support this theory; rather, it appears that His 199 is responsible for orienting His 213 and that His 228 has no function associated with the recognition of Glu in P1.
Identification and application of the concepts important for accurate and reliable protein secondary structure prediction
Authors: King Ross D. , Sternberg Michael J.E.
Published: February 2009

A protein secondary structure prediction method from multiply aligned homologous sequences is presented with an overall per residue three‐state accuracy of 70.1%. There are two aims: to obtain high accuracy by identification of a set of concepts important for prediction followed by use of linear statistics; and to provide insight into the folding process. The important concepts in secondary structure prediction are identified as; residue conformational propensities, sequence edge effects, moments of hydrophobicity, position of insertions and deletions in aligned homologous sequence, moments of conservation, auto‐correlation, residue ratios, secondary structure feedback effects, and filtering. Explicit use of edge effects, moments of conservation, and auto‐correlation are new to this paper. The relative importance of the concepts used in prediction was analyzed by stepwise addition of information and examination of weights in the discrimination function. The simple and explicit structure of the prediction allows the method to be reimplemented easily. The accuracy of a prediction is predictable a priori. This permits evaluation of the utility of the prediction: 10% of the chains predicted were identified correctly as having a mean accuracy of >80%. Existing high‐accuracy prediction methods are “black‐box” predictors based on complex nonlinear statistics (e.g., neural networks in P.HD: Rost & Sander, 1993a). For medium‐ to short‐length chains (≥90 residues and <170 residues), the prediction method is significantly more accurate (P < 0.01) than the PHD algorithm (probably the most commonly used algorithm). In combination with the PHD, an algorithm is formed that is significantly more accurate than either method, with an estimated overall three‐state accuracy of 72.4%, the highest accuracy reported for any prediction method.
Characterization of a buried neutral histidine residue inBacillus circulansxylanase: Nmr assignments, pH titration, and hydrogen exchange
Authors: Plesniak Leigh A. , Connelly Gregory P. , Mcintosh Lawrence P. , Wakarchuk Warren W.
Published: February 2009

Bacillus circulansxylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic‐aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the1H,13C, and15N resonances and tautomeric forms of the imidazole rings were obtained from two‐dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded1Hϕ2of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 × 10−5s−1at pH*7.04 and 30°C) is retarded by > 106‐fold relative to an exposed histidine. The pKaof His 156 is unperturbed at ˜6.5, as measured from the pH dependence of the15N‐ and1H‐NMR spectra of BCX. In contrast, His 149 has a pKa< 2.3, existing in the neutral Nϕ2H tautomeric state under all conditions examined. BCX unfolds at low pH and 30°C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.
A novel organic solvent‐based coupling method for the preparation of covalently immobilized proteins on gold
Authors: Parker Marie‐Claire , Patel Nikin , Davies Martyn C. , Roberts Clive J. , Tendler Saul J.B. , Williams Philip M.
Published: February 2009

A novel organic solvent‐based coupling method has been developed for the covalent immobilization of biological material to gold surfaces. The method employs the polar organic solvent anhydrous 2,2,2‐trifluoroethanol as the reaction medium and involves dissolution of the protein (catalase) in the solvent allowing protein coupling to proceed under basic conditions in a dry organic environment. The advantage of this method is that protein attachment is favored over hydrolysis of the coupling reagent. We have shown qualitatively and quantitatively that following attachment to the gold surface a significant proportion of the enzyme catalase remains catalytically active (at least 20–31%).
Comparison of the NMR and X‐ray structures of the HIV‐1 matrix protein: Evidence for conformational changes during viral assembly
Authors: Massiah Michael A. , Worthylake David , Christensen Allyson M. , Sundquist Wesley I. , Hill Christopher P. , Summers Michael F.
Published: February 2009

The three‐dimensional solution‐ and solid‐state structures of the human immunodeficiency virus type‐1 (HIV‐1) matrix protein have been determined recently in our laboratories by NMR and X‐ray crystallographic methods (Massiah et al. 1994. J Mol Biol 244:198–223; Hill et al. 1996. Proc Natl Acad Sci USA 93:3099–3104). The matrix protein exists as a monomer in solution at low millimolar protein concentrations, but forms trimers in three different crystal lattices. Although the NMR and X‐ray structures are similar, detailed comparisons have revealed an approximately 6 Å displacement of a short 310 helix (Pro 66∼Gly 71) located at the trimer interface. High quality electron density and nuclear Overhauser effect (NOE) data support the integrity of the X‐ray and NMR models, respectively. Because matrix apparently associates with the viral membrane as a trimer, displacement of the 310 helix may reflect a physiologically relevant conformational change that occurs during virion assembly and disassembly. These findings further suggest that Pro 66 and Gly 71, which bracket the 310 helix, serve as “hinges” that allow the 310 helix to undergo this structural reorientation.
Activator anion binding site in pyridoxal phosphorylase b: The binding of phosphite, phosphate, and fluorophosphate in the crystal
Authors: Oikonomakos Nikos G. , Zographos Spyros E. , Tsitsanou Katerina E. , Johnson Louise N. , Acharya K. Ravi
Published: February 2009

It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5′‐phosphate (Chang YC, McCalmont T, Graves DJ. 1983. Biochemistry 22:4987–4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X‐ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R‐state‐like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T‐state inhibitor glucose. Co‐crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P43212, with native‐like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5–19.2%, for data between 8 and 2.4 Å resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5′‐phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 Å). The structural results show that the structures of the pyridoxal phosphorylase b‐anion‐glucose‐IMP complexes are overall similar to the glucose complex of native T‐state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis.
Crystal structure of dUTP pyrophosphatase from feline immunodeficiency virus
Authors: Prasad G. Sridhar , Stura E.A. , Mcree D.E. , Laco G.S. , Hasselkus‐Light C. , Elder J.H. , Stout C.D.
Published: February 2009

We have determined the crystal structure of dUTP pyrophosphatase (dUTPase) from feline immunodeficiency virus (FIV) at 1.9 Å resolution. The structure has been solved by the multiple isomorphous replacement (MIR) method using a P63crystal form. The results show that the enzyme is a trimer of 14.3 kDa subunits with marked structural similarity toE. colidUTPase. In both enzymes the C‐terminal strand of an anti‐parallel β‐barrel participates in the β‐sheet of an adjacent subunit to form an interdigitated, biologically functional trimer. In the P63crystal form one trimer packs on the 63 screw‐axis and another on the threefold axis so that there are two independent monomers per asymmetric unit. A Mg2+ion is coordinated by three asparate residues on the threefold axis of each trimer. Alignment of 17 viral, prokaryotic, and eukaryotic dUTPase sequences reveals five conserved motifs. Four of these map onto the interface between pairs of subunits, defining a putative active site region; the fifth resides in the C‐terminal 16 residues, which is disordered in the crystals. Conserved motifs from all three subunits are required to create a given active site. With respect to viral protein expression, it is particularly interesting that the gene for dUTPase (DU) resides in the middle of the Pol gene, the enzyme cassette of the retroviral genome. Other enzymes encoded in the Pol polyprotein, including protease (PR), reverse transcriptase (RT), and most likely integrase (IN), are dimeric enzymes, which implies that the stoichiometry of expression of active trimeric dUTPase is distinct from the other Pol‐encoded enzymes. Additionally, due to structural constraints, it is unlikely that dUTPase can attain an active form prior to cleavage from the polyprotein.
Significant hydrogen exchange protection in GroEL‐bound DHFR is maintained during iterative rounds of substrate cycling
Authors: Groß Michael , Robinson Carol V. , Mayhew Mark , Hartl F. Ulrich , Radford Sheena E.
Published: February 2009

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP‐driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATR Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL‐assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.
The ubiquitous cofactor NADH protects against substrate‐induced inhibition of a pyridoxal enzyme
Authors: Jones Wanda M. , Van Ophem Peter W. , Pospischil Maria A. , Ringe Dagmar , Petsko Gregory , Soda Kenji , Manning James M.
Published: February 2009

In the usual reaction catalyzed by D‐amino acid transaminase, cleavage of the alpha‐H bond is followed by the reversible transfer of the alpha‐NH2 to a keto acid cosubstrate in a two‐step reaction mediated by the two vitamin B6 forms pyridoxal 5′‐phosphate (PLP) and pyridoxamine 5′‐phosphate (PMP). We report here a reaction not on the main pathway, i.e., β‐decarboxylation of D‐aspartate to D‐alanine, which occurs at 0.01% the rate of the major transaminase reaction. In this reaction, β‐C‐C bond cleavage of the single substrate D‐aspartate occurs rather than the usual α‐bond cleavage in the transaminase reaction. The D‐alanine produced from D‐aspartate slowly inhibits both transaminase and decarboxylase activities, but NADH or NADPH instantaneously prevent D‐aspartate turnover and D‐alanine formation, thereby protecting the enzyme against inhibition. NADH has no effect on the enzyme spectrum itself in the absence of substrates, but it acts on the enzymeD‐aspartate complex with an apparent dissociation constant of 16 μM. Equivalent concentrations of NAD or thiols have no such effect. The suppression of β‐decarboxylase activity by NADH occurs concomitant with a reduction in the 415‐nm absorbance due to the PLP form of the enzyme and an increase at 330 nm due to the PMP form of the enzyme. α‐Ketoglutarate reverses the spectral changes caused by NADH and regenerates the active PLP form of the enzyme from the PMP form with an equilibrium constant of 10 μM. In addition to its known role in shuttling electrons in oxidation‐reduction reactions, the niacin derivative NADH may also function by preventing aberrant damaging reactions for some enzyme‐substrate intermediates. The D‐aspartate‐induced effect of NADH may indicate a slow transition between protein conformational studies if the reaction catalyzed is also slow.
Kinetic and crystallographic studies of Escherichia coli UDP‐N‐acetylmuramate:L‐alanine ligase
Authors: Emanuele John J. , Jin Haiyong , Jacobson Bruce L. , Chang Chiehying Y. , Einspahr Howard M. , Villafranca Joseph J.
Published: February 2009

Uridine diphosphate‐N‐acetylmuramate:L‐alanine ligase (EC 6.3.2.8, UNAM:L‐Ala ligase or MurC gene product) catalyzes the ATP‐dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram‐positive and gram‐negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady‐state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studied included: cation specificity, ionic strength, buffer composition and pH. At 37 °C and pH 8.0, kcat was equal to 980 40 min−1, while Km values for ATP, UNAM, and L‐alanine were, 130 10, 44 3, and 48 6 μM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of Vmax/Km for the three substrates and of Vmax were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X‐ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 Å, β = 105°, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L‐alanine. This L‐alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kcat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L‐alanine. Numerous L‐alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L‐alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L‐alanine analogs tested as inhibitors were competitive versus L‐alanine.
Recombinant human pigment epithelium‐derived factor (PEDF): Characterization of PEDF overexpressed and secreted by eukaryotic cells
Authors: Stratikos Efstratios , Alberdi Elena , Gettins Peter G.W. , Becerra S. Patricia
Published: February 2009

Pigment epithelium‐derived factor (PEDF) is a serpin found in the interphotoreceptor matrix of the eye, which, although not a proteinase inhibitor, possesses a number of important biological properties, including promotion of neurite outgrowth and differential expression in quiescent versus senescent states of certain cell types. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. We describe here the expression and secretion of full‐length glycosylated human recombinant PEDF at high levels (>20 μg/mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and fluorescence spectroscopies and neurite outgrowth assay. By these assays, the recombinant protein behaves as expected for a correctly folded full‐length human PEDF. The availability of milligram amounts of PEDF has permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards thermal and guanidine hydrochloride denaturation.
Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance
Authors: Conejero‐Lara Francisco , Parrado Juan , Azuaga Ana I. , Smith Richard A.G. , Ponting Christopher P. , Dobson Christopher M.
Published: February 2009

Streptococcus equisimilis streptokinase (SK) is a single‐chain protein of 414 residues that is used extensively in the clinical treatment of acute myocardial infarction due to its ability to activate human plasminogen (Plg). The mechanism by which this occurs is poorly understood due to the lack of structural details concerning both molecules and their complex. We reported recently (Parrado J et al., 1996, Protein Sci 5:693–704) that SK is composed of three structural domains (A, B, and C) with a C‐terminal tail that is relatively unstructured. Here, we report thermal unfolding experiments, monitored by CD and NMR, using samples of intact SK, five isolated SK fragments, and two two‐chain noncovalent complexes between complementary fragments of the protein. These experiments have allowed the unfolding processes of specific domains of the protein to be monitored and their relative stabilities and interdomain interactions to be characterized. Results demonstrate that SK can exist in a number of partially unfolded states, in which individual domains of the protein behave as single cooperative units. Domain B unfolds cooperatively in the first thermal transition at approximately 46 °C and its stability is largely independent of the presence of the other domains. The high‐temperature transition in intact SK (at approximately 63 °C) corresponds to the unfolding of both domains A and C. Thermal stability of domain C is significantly increased by its isolation from the rest of the chain. By contrast, cleavage of the Phe 63—Ala 64 peptide bond within domain A causes thermal destabilization of this domain. The two resulting domain portions (A1 and A2) adopt unstructured conformations when separated. A1 binds with high affinity to all fragments that contain the A2 portion, with a concomitant restoration of the native‐like fold of domain A. This result demonstrates that the mechanism whereby A1 stimulates the plasminogen activator activities of complementary SK fragments is the reconstitution of the native‐like structure of domain A.
Proteolytic mapping of heat shock transcription factor domains
Authors: Zhong Min , Wu Carl
Published: February 2009

Heat shock transcription factors (HSFs) of higher eukaryotes respond to physical and cellular stress signals by tri‐merizing, binding to a specific site on DNA, and transactivating genes encoding the heat shock proteins. In this work, limited proteolysis was used as a biochemical probe of the domain organization of Drosophila HSF. Both unshocked monomelic and heat‐shocked trimeric HSF possess an internal protease‐sensitive region located between the aminoterminal and carboxyl‐terminal hydrophobic heptad repeats, suggesting that this is a less structured region compared to those defined for DNA‐binding, trimerization, and transactivation. For a few cleavage sites, the heat‐shocked form of HSF is more accessible to proteases than the unshocked form, providing an additional diagnostic marker for inducible changes in conformation or modification between the latent and activated forms of HSF.