bluuebunny/crossref_metadata_2025_split huggingface dataset:
An energy‐based approach to packing the 7‐helix bundle of bacteriorhodopsin
Authors: Chou Kuo‐Chen , Carlacci Louis , Maggiora Gerald M. , Parodi L. A. , Schulz M. W. 
Published: February 2009

AbstractBased on the heavy‐atom coordinates determined by the electron microscopy for the seven main helical regions of bacteriorhodopsin with the all‐trans retinal isomer, energy optimizations were carried out for helix bundles containing the all‐trans retinal and 13‐cis retinal chromophores, respectively. A combination of simulated annealing and energy minimization was utilized during the process of energy optimization. It was found that the 7‐helix bundle containing the all‐trans isomer is about 10 kcal/mol lower in conformational energy than that containing the 13‐cis isomer. An energetic analysis indicates that such a difference in energy is consistent with the observation that absorption of a 570‐nm proton is required for the conversion of a bacteriorhodopsin from its all‐trans to 13‐cis form.It was also found that the above conversion process is accompanied by a significant conformational perturbation around the chromophore, as reflected by the fact that the β‐ionone ring of retinal moves about 5.6 Å along the direction perpendicular to the membrane plane. This is consistent with the observation by Fodor et al. (Fodor, S.P.A., Ames, J.B., Gebhard, R., van der Berg, E.M.M., Stoeckenius, W., Lugtenburg, J., & Mathies, R.A., 1988, Biochemistry 27, 7097–7101). Furthermore, it is interesting to observe that although the retinal chromophore undergoes a significant change in its spatial position, the orientation of its transition dipole changes only slightly, in accord with experimental observations. In other words, even though orientation of the retinal transition dipole is very restricted, there is sufficient room, and degrees of freedom, for the retinal chromophore to readjust its position considerably. This finding provides new insight into the subtle change of the retinal microenvironment, which may be important for revealing the proton‐pumping mechanism of bacteriorhodopsin.The importance of electrostatic and nonbonded interactions in stabilizing the 7‐helix bundle structure has also been analyzed. Electrostatic interactions favor an antiparallel arrangement among adjacent helices. Nonbonded interactions, however, drive most of the closely packed helices into an arrangement in which the packing angles lie around – 160°, a value very near the – 154° value computed earlier as the most favorable packing arrangement of two poly(Ala) α‐helices (Chou, K.‐C., Némethy, G., & Scheraga, H.A., 1983, J. Phys. Chem. 87, 2869–2881).The structural features of the 7‐helix bundle and their relationship to those found in typical 4‐helix bundle proteins are also discussed. The knowledge thus obtained suggests that many 7‐helix membrane proteins might probably be built in a stepwise fashion from 4‐helix bundle groups.
Structural homology between rbs repressor and ribose binding protein implies functional similarity
Authors: Mauzy C.A. , Hermodson M.A. 
Published: February 2009

AbstractThe deduced amino acid sequence of the rbs repressor, RbsR, of Escherichia coli is homologous over its C‐terminal 272 residues to the entire sequence of the periplasmic ribose binding protein. RbsR is also homologous to a family of bacterial repressor proteins including LacI. This implies that the structure of the repressor consists of a two‐domain binding protein portion attached to a DNA‐binding domain having the four‐helix structure of the LacI headpiece. The implications of these relationships to the mechanism of this class of repressors are discussed.
Identification of the guanine binding domain peptide of the GTP‐binding site of glucagon
Authors: Shoemaker Mike , Lin Paul C. , Haley Boyd 
Published: February 2009

AbstractGlucagon, a peptide hormone synthesized and secreted by α islet cells, regulates glucose homeostasis by several mechanisms. Using [γ32P]8N3GTP, a proven photoaffinity probe for GTP, a specific nucleotide binding site on human glucagon was detected that showed preference for GTP. Half‐maximal saturation of photoinsertion into the polypeptide hormone was at 8–12 μM with either [α32P]8N3GTP or [γ32P]8N3GTP. GTP protected photolabeling with an apparent kd of 15 μM, whereas ATP was less effective as a protector, exhibiting an apparent kd of about 30 μM. Maximal protection by GTP and ATP was over 90%. UTP, CTP, GDP, ADP, GMP, AMP, guanosine, adenosine, guanine, and adenine were much less effective protectors, indicating that binding is specific for purine nucleoside triphosphates, particularly GTP. Mg2+ at 150 μM enhanced photoinsertion (twofold), whereas at 2–10 mM, it inhibited photoinsertion. Both Ca2+ and Zn2+ at 0.2 mM decreased photoinsertion about 45%. Purification of chymotryptic and tryptic digests of photolabeled glucagon by reverse‐phase high performance liquid chromatography (HPLC) revealed that the N‐terminal peptide, HSQGTF, was the only peptide region covalently photomodified by [32P]8N3GTP. GTP, if present during photolysis, greatly reduced both photoinsertion into glucagon and the amount of radiolabeled peptide recovered on HPLC. The specificity of binding to the N‐terminal region is suggestive of a physiological role for a glucagon‐GTP complex in the mechanism of action of this hormone.
Cis proline mutants of ribonuclease A. I. thermal stability
Authors: Schultz David A. , Baldwin Robert L. 
Published: February 2009

AbstractA chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60–89). The expressed protein, which contains an additional N‐terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site‐directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give ΔTm ≅ 10 °C and ΔΔG0 (apparent) = 2–3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis ⟺ trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild‐type conformation, that allows the substituted amino acid to form a trans peptide bond.
83‐Kilodalton heat shock proteins of trypanosomes are potent peptide‐stimulated ATPases
Authors: Nadeau Kari , Walsh Christopher T. , Bradley Mark , Sullivan Mary Anne , Engman David M. 
Published: February 2009

AbstractA Crithidia fasciculata 83‐kDa protein purified during a separate study of C. fasciculata trypanothione synthetase was shown to have ATPase activity and to belong to the hsp90 family of stress proteins. Because no ATPase activity has previously been reported for the hsp90 class, ATP utilization by C. fasciculata hsp83 was characterized: this hsp83 has an ATPase kcat of 150 min−1 and a Km of 60 μM, whereas the homologous mammalian hsp90 binds ATP but has no ATPase activity. Crithidia fasciculata hsp83 undergoes autophosphorylation on serine and threonine at a rate constant of 3.3 × 10−3 min−1. Similar analysis was performed on recombinant Trypanosoma cruzi hsp83, and comparable ATPase parameters were obtained (kcat = 100 min−1, Km = 80 μM, kautophosphorylation = 6.3 × 10−3 min−1). The phosphoenzyme is neither on the ATPase hydrolytic pathway nor does it affect ATPase catalytic efficiency. Both C. fasciculata and T. cruzi hsp83 show up to fivefold stimulation of ATPase activity by peptides of 6–24 amino acids.
Chimeric prion protein expression in cultured cells and transgenic mice
Authors: Scott Michael R. , Köhler Ruth , Foster Dallas , Prusiner Stanley B. 
Published: February 2009

AbstractThe efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558–1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847–857; Prusiner, S.B., et al., 1990, Cell 63, 673–686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease‐resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa‐specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.
Catalytically competent human and bovine ζ‐thrombin and chimeras generated from unfolded polypeptide chains
Authors: Lewis Sidney D. , Brezniak Diane V. , Shafer Jules A. , Fenton John W. 
Published: February 2009

AbstractHuman and bovine α‐thrombin cleaved at the B‐chain by chymotrypsin generates catalytically competent ζ‐thrombins, which are comprised of two noncovalently linked fragments: a 36‐ (human) or 49‐ (bovine) residue A‐chain linked by a disulfide to B‐chain residues B1–148 (ζ1‐thrombin) and B‐chain residues B149–259 (ζ2‐thrombin). Human and bovine d‐Phe‐Pro‐Arg‐CH2‐ζ‐ and PhMeSO2‐ζ‐thrombins were prepared by reaction of the active‐site histidine (H‐B43) and serine (S‐B205) with PPACK and PMSF, respectively. Unfolding and dissociation of the noncovalently linked polypeptide chains of either human or bovine d‐Phe‐Pro‐Arg‐CH2‐ζ‐ and PhMeSO2‐ζ‐thrombins in 4.5 M guanidine‐HCl and refolding upon 30‐fold dilution in 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 0.1% PEG resulted in biphasic generation of catalytic activity. The slow phase was eliminated in the presence of the competitive inhibitor benzamidine‐HCl. Unfolding and refolding mixtures of the appropriate inactive precursors generated the active chimeric thrombins bovine ζ1‐thrombin:human ζ2‐thrombin and human ζ1‐thrombin:bovine ζ2‐thrombin. Human ζ1‐thrombin and ζ2‐thrombin were isolated, and, upon recom‐bining, the isolated fragments refolded to generate catalytically competent ζ‐thrombin with an active‐site content, specific activity toward Chromozym‐TH, and a specificity constant (kcat/Km) for FPA release from fibrinogen that were all within 60% of those of native α‐thrombin. Both α‐ and ζ‐thrombin refolded via first‐order processes (k = 0.011–0.014 s−1), which in the case of ζ‐thrombin were independent of whether ζ1‐thrombin and/or ζ2‐thrombin was incubated in refolding buffer prior to mixing. This observation, together with others, is consistent with the view that generation of catalytically competent enzyme proceeds via a kinetic pathway wherein ζ1‐thrombin and ζ2‐thrombin independently partially refold and form a noncovalent complex that undergoes a rate‐determining rearrangement to active thrombin.
Crystal structure of a complex of HIV‐1 protease with a dihydroxyethylene‐containing inhibitor: Comparisons with molecular modeling
Authors: Thanki Narmada , Rao J.K. Mohana , Foundling Stephen I. , Wlodawer Alexander , Howe W. Jeffrey , Moon Joseph B. , Hui John O. , Tomasselli Alfredo G. , Heinrikson Robert L. , Thaisrivongs Suvit 
Published: February 2009

AbstractThe structure of a crystal complex of recombinant human immunodeficiency virus type 1 (HIV‐1) protease with a peptide‐mimetic inhibitor containing a dihydroxyethylene isostere insert replacing the scissile bond has been determined. The inhibitor is Noa‐His‐Hch[CH(OH)CH(OH)]Vam‐Ile‐Amp (U‐75875), and its Ki for inhibition of the HIV‐1 protease is <1.0 nM (Noa = 1‐naphthoxyacetyl, Hch = a hydroxy‐modified form of cyclohexylalanine, Vam = a hydroxy‐modified form of valine, Amp = 2‐pyridylmethylamine). The structure of the complex has been refined to a crystallographic R factor of 0.169 at 2.0 Å resolution by using restrained least‐squares procedures. Root mean square deviations from ideality are 0.02 Å and 2.4°, for bond lengths and angles, respectively. The bound inhibitor diastereomer has the R configurations at both of the hydroxyl chiral carbon atoms. One of the diol hydroxyl groups is positioned such that it forms hydrogen bonds with both the active site aspartates, whereas the other interacts with only one of them. Comparison of this X‐ray structure with a model‐built structure of the inhibitor, published earlier, reveals similar positioning of the backbone atoms and of the side‐chain atoms in the P2‐P2′ region, where the interaction with the protein is strongest. However, the X‐ray structure and the model differ considerably in the location of the P3 and P3′ end groups, and also in the positioning of the second of the two central hydroxyl groups. Reconstruction of the central portion of the model revealed the source of the hydroxyl discrepancy, which, when corrected, provided a P1‐P1′ geometry very close to that seen in the X‐ray structure.
Crystallization of proton channel peptides
Authors: Lovejoy Brett , Åkerfeldt Karin S. , Degrado William F. , Eisenberg David 
Published: February 2009

AbstractCrystals have been grown of two similar peptides that form ion‐conducting channels in diphytanoyl phosphatidylcholine bilayers. These crystals were grown by slow evaporation of the organic solvent, 2,2,2‐trifluoroethanol. Crystals of one of the peptides have been characterized by X‐ray diffraction, and X‐ray data have been measured to 2.3 Å resolution. Earlier it was proposed that the ion‐conducting channels formed by these peptides consist of four peptides associated as a parallel α‐helical tetramer. On the basis of the space group and unit cell dimensions of the crystals, a packing scheme for the peptide is proposed that is consistent with a tetrameric channel.
Control of phosphorylase b conformation by a modified cofactor: Crystallographic studies on R‐state glycogen phosphorylase reconstituted with pyridoxal 5′‐diphosphate
Authors: Leonidas D. D. , Oikonomakos N.G. , Papageorgiou A. C. , Acharya K. R. , Barford D. , Johnson L. N. 
Published: February 2009

AbstractPrevious crystallographic studies on glycogen phosphorylase have described the different conformational states of the protein (T and R) that represent the allosteric transition and have shown how the properties of the 5′‐phosphate group of the cofactor pyridoxal phosphate are influenced by these conformational states. The present work reports a study on glycogen phosphorylase b (GPb) complexed with a modified cofactor, pyridoxal 5′‐diphosphate (PLPP), in place of the natural cofactor. Solution studies (Withers, S.G., Madsen, N.B., & Sykes, B.D., 1982, Biochemistry 21, 6716–6722) have shown that PLPP promotes R‐state properties of the enzyme indicating that the cofactor can influence the conformational state of the protein. GPb complexed with pyridoxal 5′‐diphosphate (PLPP) has been crystallized in the presence of IMP and ammonium sulfate in the monoclinic R‐state crystal form and the structure refined from X‐ray data to 2.8 Å resolution to a crystallographic R value of 0.21. The global tertiary and quaternary structure in the vicinity of the Ser 14 and the IMP sites are nearly identical to those observed for the R‐state GPb‐AMP complex. At the catalytic site the second phosphate of PLPP is accommodated with essentially no change in structure from the R‐state structure and is involved in interactions with the side chains of two lysine residues (Lys 568 and Lys 574) and the main chain nitrogen of Arg 569. Superposition of the T‐state structure shows that were the PLPP to be incorporated into the T‐state structure there would be a close contact with the 280s loop (residues 282–285) that would encourage the T to R allosteric transition. The second phosphate of the PLPP occupies a site that is distinct from other dianionic binding sites that have been observed for glucose‐1‐phosphate and sulfate (in the R state) and for heptulose‐2‐phosphate (in the T state). The results indicate mobility in the dianion recognition site, and the precise position is dependent on other linkages to the dianion. In the modified cofactor the second phosphate site is constrained by the covalent link to the first phosphate of PLPP. The observed position in the crystal suggests that it is too far from the substrate site to represent a site for catalysis.
Molecular dynamics studies of a DNA‐binding protein: 2. An evaluation of implicit and explicit solvent models for the molecular dynamics simulation of the Escherichia coli trp repressor
Authors: Guenot Jeanmarie , Kollman Peter A. 
Published: February 2009

AbstractAlthough aqueous simulations with periodic boundary conditions more accurately describe protein dynamics than in vacuo simulations, these are computationally intensive for most proteins. Trp repressor dynamic simulations with a small water shell surrounding the starting model yield protein trajectories that are markedly improved over gas phase, yet computationally efficient. Explicit water in molecular dynamics simulations maintains surface exposure of protein hydrophilic atoms and burial of hydrophobic atoms by opposing the otherwise asymmetric protein‐protein forces. This properly orients protein surface side chains, reduces protein fluctuations, and lowers the overall root mean square deviation from the crystal structure. For simulations with crystallographic waters only, a linear or sigmoidal distance‐dependent dielectric yields a much better trajectory than does a constant dielectric model. As more water is added to the starting model, the differences between using distance‐dependent and constant dielectric models becomes smaller, although the linear distance‐dependent dielectric yields an average structure closer to the crystal structure than does a constant dielectric model. Multiplicative constants greater than one, for the linear distance‐dependent dielectric simulations, produced trajectories that are progressively worse in describing trp repressor dynamics. Simulations of bovine pancreatic trypsin were used to ensure that the trp repressor results were not protein dependent and to explore the effect of the nonbonded cutoff on the distance‐dependent and constant dielectric simulation models. The nonbonded cutoff markedly affected the constant but not distance‐dependent dielectric bovine pancreatic trypsin inhibitor simulations. As with trp repressor, the distance‐dependent dielectric model with a shell of water surrounding the protein produced a trajectory in better agreement with the crystal structure than a constant dielectric model, and the physical properties of the trajectory average structure, both with and without a nonbonded cutoff, were comparable.
Accelerated high‐sensitivity microsequencing of proteins and peptides using a miniature reaction cartridge
Authors: Totty Nicholas F. , Hsuan J. Justin , Waterfield Michael D. 
Published: February 2009

AbstractGreatly reduced automated protein sequencing degradation times of less than 25 min with concurrent on‐line phenylthiohydantoin (PTH) derivative analysis times of less than 16 min have been achieved by using a miniaturized reaction cartridge with optimized chemical and analytical cycles. Using this method a wide range of standard and novel peptides and proteins have been sequenced with reproducibly high initial and repetitive cycle yields. In these accelerated analyses the recovery of the more labile PTH derivatives was markedly improved by using elevated pressure during cleavage steps and temperature programming throughout the Edman cycle.
Unfolding domains of recombinant fusion αα‐tropomyosin
Authors: Ishii Yoshiharu , Hitchcock‐Degregori Sarah , Mabuchi Katsuhide , Lehrer Sherwin S. 
Published: February 2009

AbstractThe thermal unfolding of the coiled‐coil α‐helix of recombinant αα‐tropomyosin from rat striated muscle containing an additional 80‐residue peptide of influenza virus NS1 protein at the N‐terminus (fusion‐tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion‐tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35 °C involving the middle of the molecule; (2) a major transition at 46 °C involving no more than 36% of the helix from the C‐terminus; (3) a major transition at 56 °C involving about 46% of the helix from the N‐terminus; and (4) a transition from the nonhelical fusion domain at about 70 °C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56 °C or 70 °C transition. The very stable fusion unfolding domain of fusion‐tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a crosslink to stabilize the adjacent N‐terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C‐terminal domain at 46 °C from the stabilized N‐terminal unfolding domain at 56 °C. Thus, strong localized interchain interactions in coiled‐coil molecules can increase the stability of neighboring domains.
A new subfamily of bacterial ABC‐type transport systems catalyzing export of drugs and carbohydrates
Authors: Reizer Jonathan , Reizer Aiala , Saier Milton H. 
Published: February 2009

AbstractSequence comparison studies revealed that the drug resistance transporter of Streptomyces peucetius (DrrAB) and two nodulation gene products (NodIJ) of Rhizobium leguminosarum are homologous to proteins encoded by three sets of genes that comprise capsular polysaccharide export systems in gram‐negative bacteria: KpsTM of Escherichia coli, BexABC of Haemophilus influenzae, and CtrDCB of Neisseria meningitidis. These five systems comprise a new subfamily within the family of ATP binding cassette (ABC)‐type transporters. We have termed this subfamily the ABC‐2 subfamily. For three of the systems comprising this subfamily (Drr, Nod, and Kps) only one integral membrane constituent has been identified, whereas for the other two systems (Bex and Ctr) two dissimilar integral membrane constituents have been found. This observation suggests that the transmembrane channels of ABC‐2‐type transporters can be formed of homo‐ or heterooligomers as is true of several other classes of transport systems.