bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determinants of protein hydrogen exchange studied in equine cytochrome c
Authors: Milne J.S. , Mayne L. , Roder H. , Wand A.J. , Englander S. W. 
Published: July 2010

AbstractThe exchange of a large number of amide hydrogens in oxidized equine cytochrome c was measured by NMR and compared with structural parameters. Hydrogens known to exchange through local structural fluctuations and through larger unfolding reactions were separately considered. All hydrogens protected from exchange by factors greater than 103 are in defined H‐bonds, and almost all H‐bonded hydrogens including those at the protein surface were measured to exchange slowly. H‐exchange rates do not correlate with H‐bond strength (length) or crystallographic B factors. It appears that the transient structural fluctuation necessary to bring an exchangeable hydrogen into H‐bonding contact with the H‐exchange catalyst (OH−‐ion) involves a fairly large separation of the H‐bond donor and acceptor, several angstroms at least, and therefore depends on the relative resistance to distortion of immediately neighboring structure. Accordingly, H‐exchange by way of local fluctuational pathways tends to be very slow for hydrogens that are neighbored by tightly anchored structure and for hydrogens that are well buried. The slowing of buried hydrogens may also reflect the need for additional motions that allow solvent access once the protecting H‐bond is separated, although it is noteworthy that burial in a protein like cytochrome c does not exceed 4 Å. When local fluctuational pathways are very slow, exchange can become dominated by a different category of larger, cooperative, segmental unfolding reactions reaching up to global unfolding.
Structural and functional characterization of recombinant human cellular retinaldehyde‐binding protein
Authors: Crabb John W. , Chen Yang , Goldflam Steve , Intres Richard , West Karen A. , Hulmes Jeffery D. , Kapron James T. , Luck Linda A. , Carlson Anne , Bok Dean , Horwitz Joseph 
Published: July 2010

AbstractCellular retinaldehyde‐binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Miiller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11‐cis‐retinal and/or 11‐cis‐retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde‐binding protein (rCRALBP) has been over expressed as non‐fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15‐20% of the soluble bacterial lysate protein and after purification, yield about 3‐8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV‐visible absorption spectroscopy, and solution state 19F‐NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid‐binding properties. Ligand‐dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9‐cis‐retinal. These recombinant preparations provide valid models for human CRALBP structure‐function studies.
Mass spectrometric analysis of integral membrane proteins: Application to complete mapping of bacteriorhodopsins and rhodopsin
Authors: Wang Jianyao , Ball Lauren E. , Oatis John E. , Dharmasiri Kuruppu , Busman Mark , Cowden Lloraine B. , Galijatovic Alema , Knapp Daniel R. , Chen Ning , Crouch Rosalie K. 
Published: July 2010

AbstractIntegral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC‐electrospray ionization mass spectrometry (ESI‐MS) and ‐tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G‐protein coupled receptor, rhodopsin (Rh).In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)‐MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins.
The N‐terminal segment of antithrombin acts as a steric gate for the binding of heparin
Authors: Fitton Hazel L. , Skinner Richard , Dafforn Timothy R. , Jin Lei , Pike Robert N. 
Published: July 2010

AbstractThe binding of heparin causes a conformational change in antithrombin to give an increased heparin binding affinity and activate the inhibition of thrombin and factor Xa. The areas of antithrombin involved in binding heparin and stabilizing the interaction in the high‐affinity form have been partially resolved through the study of both recombinant and natural variants. The role of a section of the N‐terminal segment of antithrombin, residues 22‐46 (segment 22‐46), in heparin binding was investigated using rapid kinetic analysis of the protein cleaved at residues 29‐30 by limited proteolysis with thermolysin. The cleaved antithrombin had 5.5‐fold lowered affinity for heparin pentasaccharide and 1.8‐fold for full‐length, high‐affinity heparin. It was shown that, although the initial binding of heparin is slightly enhanced by the cleavage, it dissociates much faster from the cleaved form, giving rise to the overall decrease in heparin affinity. This implies that the segment constituting residues 22‐46 in the N terminus of antithrombin hinders access to the binding site for heparin, hence the increased initial binding for the cleaved form, whereas, when heparin is bound, segment 22‐46 is involved in the stabilization of the binding interaction, as indicated by the increased dissociation constant. When the heparin pentasaccharide is bound to antithrombin prior to incubation with thermolysin, it protects the N‐terminal cleavage site, implying that segment 22‐46 moves to interact with heparin in the conformational change and thus stabilizes the complex.
Crystal structure of recombinant human tissue kallikrein at 2.0 Å resolution
Authors: Katz Bradley A. , Liu Beishan , Barnes Micael , Springman Eric B. 
Published: July 2010

AbstractHuman tissue kallikrein, a trypsin‐like serine protease involved in blood pressure regulation and inflammation processes, was expressed in a deglycosylated form at high levels inPichia pastoris, purified, and crystallized. The crystal structure at 2.0 Å resolution is described and compared with that of porcine kallikrein and of other trypsin‐like proteases. The active and S1 sites (nomenclature of Schechter I, Berger A, 1967,Biochem Biophys Res Commun 27:157‐162) are similar to those of porcine kallikrein. Compared to trypsin, the S1 site is enlarged owing to the insertion of an additional residue,cis‐Pro 219. The replacement Tyr 228 → Ala further enlarges the S1 pocket. However, the replacement of Gly 226 in trypsin with Ser in human tissue kallikrein restricts accessibility of substrates and inhibitors to Asp 189 at the base of the S1 pocket; there is a hydrogen bond between Oδ1Asp189and Oγser226. These changes in the architecture of the S1 site perturb the binding of inhibitors or substrates from the modes determined or inferred for trypsin. The crystal structure gives insight into the structural differences responsible for changes in specificity in human tissue kallikrein compared with other trypsin‐like proteases, and into the structural basis for the unusual specificity of human tissue kallikrein in cleaving both an Arg‐Ser and a Met‐Lys peptide bond in its natural protein substrate, kininogen. A Zn+2‐dependent, small‐molecule competitive inhibitor of kallikrein (Ki= 3.3 μM) has been identified and the bound structure modeled to guide drug design.
The BPI/LBP family of proteins: A structural analysis of conserved regions
Authors: Beamer Lesa J. , Carroll Stephen F. , Eisenberg David 
Published: July 2010

AbstractTwo related mammalian proteins, bactericidal/permeability‐increasing protein (BPI) and lipopolysaccharide‐binding protein (LBP), share high‐affinity binding to lipopolysaccharide (LPS), a glycolipid found in the outer membrane of Gram‐negative bacteria. The recently determined crystal structure of human BPI permits a structure/function analysis, presented here, of the conserved regions of these two proteins sequences. In the seven known sequences of BPI and LBP, 102 residues are completely conserved and may be classified in terms of location, side‐chain chemistry, and interactions with other residues. We find that the most highly conserved regions lie at the interfaces between the tertiary structural elements that help create two apolar lipid‐binding pockets. Most of the conserved polar and charged residues appear to be involved in inter‐residue interactions such as H‐bonding. However, in both BPI and LBP a subset of conserved residues with positive charge (lysines 42, 48, 92, 95, and 99 of BPI) have no apparent structural role. These residues cluster at the tip of the NH2‐terminal domain, and several coincide with residues known to affect LPS binding; thus, it seems likely that these residues make electrostatic interactions with negatively charged groups of LPS. Overall differences in charge and electrostatic potential between BPI and LBP suggest that BPI's bactericidal activity is related to the high positive charge of its NH2‐terminal domain. A model of human LBP derived from the BPI structure provides a rational basis for future experiments, such as site‐directed mutagenesis and inhibitor design.
NMR determination of the global structure of the 113Cd derivative of desulforedoxin: Investigation of the hydrogen bonding pattern at the metal center
Authors: Goodfellow B.J. , Moura I. , Moura J.J.G. , Rusnak F. , Domke T. 
Published: July 2010

AbstractDesulforedoxin (Dx) is a simple homodimeric protein isolated from Desulfovibrio gigas (Dg) containing a distorted rubredoxin‐like center with one iron coordinated by four cysteinyl residues (7.9 kDa with 36 amino acids per monomer). In order to probe the geometry and the H‐bonding at the active site of Dx, the protein was reconstituted with 113Cd and the solution structure determined using 2D NMR methods. The structure of this derivative was initially compared with the NMR solution structure of the Zn form (Goodfellow BJ et al., 1996, J Biol Inorg Chem 1:341‐353). Backbone amide protons for G4, D5, G13, L11 NH, and the Q14 NH side‐chain protons, H‐bonded in the X‐ray structure, were readily exchanged with solvent. Chemical shift differences observed for amide protons near the metal center confirm the H‐bonding pattern seen in the X‐ray model (Archer M et al., 1995, J Mol Biol 251:690‐702) and also suggest that H‐bond lengths may vary between the Fe, Zn, and 113Cd forms. The H‐bonding pattern was further probed using a heteronuclear spin echo difference (HSED) experiment; the results confirm the presence of NH‐S H‐bonds inferred from D2O exchange data and observed in the NMR family of structures. The presence of “H‐bond mediated” coupling in Dx indicates that the NH‐S H‐bonds at the metal center have significant covalent character. The HSED experiment also identified an intermonomer “through space” coupling for one of the L26 methyl groups, indicating its proximity to the 113Cd center in the opposing monomer. This is the first example of an intermonomer “through space” coupling. Initial structure calculations produced subsets of NMR families with the S of C28 pointing away from or toward the L26 methyl: only the subset with the C28 sulfur pointing toward the L26 methyl could result in a “through space” coupling. The HSED result was therefore included in the structure calculations. Comparison of the Fe, Zn, and 113Cd forms of Dx suggests that the geometry of the metal center and the global fold of the protein does not vary to any great extent, although the H‐bond network varies slightly when Cd is introduced. The similarity between the H‐bonding pattern seen at the metal center in Dx, Rd (including H‐bonded and through space‐mediated coupling), and many zinc‐finger proteins suggests that these H‐bonds are structurally vital for stabilization of the metal centers in these proteins.
Flexible ligand docking using conformational ensembles
Authors: Lorber David M. , Shoichet Brian K. 
Published: July 2010

AbstractMolecular docking algorithms suggest possible structures for molecular complexes. They are used to model biological function and to discover potential ligands. A present challenge for docking algorithms is the treatment of molecular flexibility. Here, the rigid body program, DOCK, is modified to allow it to rapidly fit multiple conformations of ligands. Conformations of a given molecule are pre‐calculated in the same frame of reference, so that each conformer shares a common rigid fragment with all other conformations. The ligand conformers are then docked together, as an ensemble, into a receptor binding site. This takes advantage of the redundancy present in differing conformers of the same molecule. The algorithm was tested using three organic ligand protein systems and two protein‐protein systems. Both the bound and unbound conformations of the receptors were used. The ligand ensemble method found conformations that resembled those determined in X‐ray crystal structures (RMS values typically less than 1.5 Å). To test the method's usefulness for inhibitor discovery, multi‐compound and multi‐conformer databases were screened for compounds known to bind to dihydrofolate reductase and compounds known to bind to thymidylate synthase. In both cases, known inhibitors and substrates were identified in conformations resembling those observed experimentally. The ligand ensemble method was 100‐fold faster than docking a single conformation at a time and was able to screen a database of over 34 million conformations from 117,000 molecules in one to four CPU days on a workstation.
A monomeric mutant of Clostridium symbiosum glutamate dehydrogenase: Comparison with a structured monomeric intermediate obtained during refolding
Authors: Millevoi Stefania , Chiaraluce Roberta , Consalvi Valerio , Giangiacomo Laura , Pasquo Alessandra , Linda Britton K. , Stillman Timothy J. , Rice David W. , Engel Paul C. 
Published: July 2010

AbstractThe refolding ofClostridium symbiosumglutamate dehydrogenase (GDH) involves the formation of an inactive structured monomeric intermediate prior to its concentration‐dependent association. The structured monomer obtained after removal of guanidinium chloride was stable and competent for reconstitution into active hexamers. Site‐directed mutagenesis ofC.symbiosum gdhgene was performed to replace the residues Arg‐61 and Phe‐187 which are involved in subunit‐subunit interactions, as determined by three‐dimensional structure analysis. Heterologous over‐expression inEscherichia coliof the double mutant (R61E/F187D) led to the production of a soluble protein with a molecular mass consistent with the monomeric form of clostridial GDH. This protein is catalytically inactive but cross‐reacts with an anti‐wild‐type GDH antibody preparation. The double mutant R61E/F187D does not assemble into hexamers. The physical properties and the stability toward guanidinium chloride and urea of R61E/F187D were studied and compared to those of the structured monomeric intermediate.
The dimerization motif of the glycophorin A transmembrane segment in membranes: Importance of glycine residues
Authors: Brosig Bettina , Langosch Dieter 
Published: July 2010

AbstractThe glycophorin A transmembrane segment homodimerizes to a right‐handed pair of α‐helices. Here, we identified the amino acid motif mediating this interaction within a natural membrane environment. Critical residues were grafted onto two different hydrophobic host sequences in a stepwise manner and self‐assembly of the hybrid sequences was determined with the ToxR transcription activator system. Our results show that the motif LIxxGxxxGxxxT elicits a level of self‐association equivalent to that of the original glycophorin A transmembrane segment. This motif is very similar to the one previously established in detergent solution. Interestingly, the central GxxxG motif by itself already induced strong self‐assembly of host sequences and the three‐residue spacing between both glycines proved to be optimal for the interaction. The GxxxG element thus appears to be the most crucial part of the interaction motif.
Treatment response criteria for prostatic cancer
Authors: Schroeder F. H. 
Published: July 2010

AbstractResponse criteria for phase II and phase III trials of prostate carcinoma patients of the EORTC Genito Urinary‐Group are described. These criteria, initially closely related to National Prostatic Cancer Project criteria, have gone through a development into the direction of more stringency. Admission of patients to phase II trials is now restricted to those showing objectively measurable lesions, excluding bone metastases. World Health Organization criteria are applied to these patients. For phase III trials, progression to Metastatic TNM system status, time to progression, and duration of survival are recommended as end points. Measurable marker lesions, as for phase II trials and subjective and nonspecific response criteria, are accepted as parameters for progression. Response usually is not evaluated in these studies.Based on recent literature and personal experiences, the author suggests that serum acid phosphatase (SPAP) and volume changes of the primary tumor can be used as indicators for response under certain conditions.There is obviously a great need for further development of objective response criteria for prostatic cancer patients.
Cryotherapy
Authors: Loening Stefan A. , Bonney William W. , Fallon Bernard , Gerber Walter L. , Hawtrey Charles E. , Lubaroff David M. , Narayana Ambati S. , Culp David A. 
Published: July 2010

AbstractSince 1969 cryosurgical perineal destruction of the prostate has been developed at the University of Iowa for treatment of patients with prostatic cancer. The technique of this procedure has been the subject of previous reviews. This is a summary of our experience of patient survival and the effect on local prostatic cancer growth in those patients with various stages of the disease subjected to perineal cryosurgical destruction of their cancer. We also report briefly on our experience in the laboratory where the Dunning tumor model is used to study the effect of cryosurgery as well as combinations of immunological modifications.
Cytochrome P450 Inhibition Assay for Standardized Extract of Terminalia chebula Retz.
Authors: Ponnusankar S. , Pandit S. , Venkatesh M. , Bandyopadhyay A. , Mukherjee Pulok K. 
Published: July 2010

AbstractThe hydroalcoholic extract of fruit pulp of Terminalia chebula Retz. was standardized and evaluated for its safety through cytochrome P450 (CYP 450) inhibition assay. Standardization was performed through high performance thin layer chromatography (HPTLC) using gallic acid (GA) standard. Cytochrome P450‐CO complex microplate assay was performed using rat liver microsomes. The effect of standardized extract, its fraction and bioactive marker compound were comparatively evaluated for its effect on CYP P450 enzymes. The extract of fruit pulp was used for HPTLC, where the Rf value of the marker was found to be 0.43. The calibration plot was linear in the range of 2–14 µg of GA and correlation co‐efficient of 0.99965. The mean quantity of GA was found to be 2.5% w/w. The CYP P450 concentration of the rat liver microsome sample used in the study was found to be 0.417 nmol/mg protein. The in vitro effect of various concentrations of extracts and fractions showed a linear concentration‐dependent inhibition of cytochrome P450 up to 60 µL. The study showed more inhibition of fraction when compared to the extract and GA. Still, the inhibition showed by fraction is less when compared with standard Ketoconazole. Thus, this study indicated the in vitro cytochrome P450 inhibition potential of T. Chebula.
Anti‐allergic effects of total bakkenolides from Petasites tricholobus in ovalbumin‐sensitized rats
Authors: Zhang Fu‐Jin , Wang Qin , Wang Yan , Guo Mei‐Li 
Published: July 2010

AbstractThe anti‐allergic effect of total bakkenolides from the rhizome of Petasites tricholobus (BAPT) was evaluated in an ovalbumin‐induced allergic rhinitis model in male Wistar rats. The major components of the bakkenolide fraction are bakkenolide‐D, bakkenolide‐B, bakkenolide‐IIIa and bakkenolide‐IVa, which account for 60.04% of the total. The rats were treated with 40 mg/kg, 20 mg/kg, 10 mg/kg or 5 mg/kg BAPT, and 0.942 mg/kg loratadine and 0.5% gum tragacanth were used as positive and negative controls, respectively. The frequency of nose rubbing and sneezing was observed, the number of eosinophils infiltrating into the nasal tissue was counted, and serum levels of IL‐4 and histamine were determined by ELISA. The results showed that BAPT had a beneficial effect on allergic rhinitis in ovalbumin‐sensitized Wistar rats, which was evidenced by a significant decrease in the frequency of sneezing, the number of eosinophils infiltrating into the nasal tissue, and the serum levels of IL‐4 and histamine. BAPT may therefore be a potential antiallergic drug. Copyright © 2010 John Wiley & Sons, Ltd.