bluuebunny/crossref_metadata_2025_split huggingface dataset:
Echistatin disulfide bridges: Selective reduction and linkage assignment
Authors: Gray William R. 
Published: February 2009

AbstractEchistatin is the smallest member of the disintegrin family of snake venom proteins, containing four disulfides in a peptide chain of 49 residues. Partial assignment of disulfides has been made previously by NMR and chemical approaches. A full assignment was made by a newly developed chemical approach, using partial reduction with tris‐(2‐carboxyethyl)‐phosphine at acid pH. Reduction proceeded in a stepwise manner at pH 3, and the intermediates were isolated by high performance liquid chromatography. Alkylation of free thiols, followed by sequencer analysis, enabled all four bridges to be identified: (1) at 20 °C a single bridge linking Cys 2‐Cys 11 was broken, giving a relatively stable intermediate; (2) with further treatment at 41 °C the bridges Cys 7‐Cys 32 and Cys 8‐Cys 37 became accessible to the reagent and were reduced at approx. equal rates; (3) the two bicyclic peptides produced in this manner were less stable and could be reduced at 20 °C to a peptide that retains a single bridge linking Cys 20‐Cys 39; and (4) the monocyclic peptide can be reduced to the linear molecule at 20 °C. Some disulfide exchange occurred during alkylation of the bicyclic intermediates, but results unambiguously show the pattern to be [2–11; 7–32; 8–37; 20–39]. A comparison is made with kistrin, a longer disintegrin whose disulfide structure has been proposed from NMR analysis.
Tryptophan replacements in the trp aporepressor from escherichia coli: Probing the equilibrium and kinetic folding models
Authors: Mann Craig J. , Royer Catherine A. , Matthews C. Robert 
Published: February 2009

AbstractMutants of the dimeric Escherichia coli trp aporepressor are constructed by replacement of the two tryptophan residues in each subunit in order to assess the effects on equilibrium and kinetic fluorescence properties of the folding reaction. The three kinetic phases detected by intrinsic tryptophan fluorescence in refolding of the wild‐type aporepressor are also observed in folding of both Trp 19 to Phe and Trp 99 to Phe single mutants, demonstrating that these phases correspond to global rather than local conformational changes. Comparison of equilibrium fluorescence (Royer, C.A., Mann, C.J., & Matthews, C.R., 1993, Protein Sci. 2, 1844–1852) and circular dichroism transition curves induced by urea shows that replacement of either Trp 19 or Trp 99 results in noncoincident behavior. Unlike the wild‐type protein (Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011–7020), tertiary and/or quaternary structures are disrupted at lower denaturant concentration than is secondary structure. The equilibrium results can be interpreted in terms of enhancement in the population of a monomeric folding intermediate in which the lone tryptophan residue is highly exposed to solvent, but in which substantial secondary structure is retained. The location of both mutations at the interface between the two subunits (Zhang, R.G., et al., 1987, Nature 327, 591–597) provides a simple explanation for this phenomenon.
Design of a functional calcium channel protein: Inferences about an ion channel‐forming motif derived from the primary structure of voltage‐gated calcium channels
Authors: Grove Anne , Tomich John M. , Iwamoto Takeo , Montal Mauricio 
Published: February 2009

AbstractTo identify sequence‐specific motifs associated with the formation of an ionic pore, we systematically evaluated the channel‐forming activity of synthetic peptides with sequence of predicted transmembrane segments of the voltage‐gated calcium channel. The amino acid sequence of voltage‐gated, dihydropyridine (DHP)‐sensitive calcium channels suggests the presence in each of four homologous repeats (I–IV) of six segments (S1–S6) predicted to form membrane‐spanning, α‐helical structures. Only peptides representing amphipathic segments S2 or S3 form channels in lipid bilayers. To generate a functional calcium channel based on a four‐helix bundle motif, four‐helix bundle proteins representing IVS2 (T4CaIVS2) or IVS3 (T4CaIVS3) were synthesized. Both proteins form cation‐selective channels, but with distinct characteristics: the single‐channel conductance in 50 mM BaCl2 is 3 pS and 10 pS. For T4CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba2+, Ca2+, and Sr2+ are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T4CaIVS2 does not saturate up to 150 mM salt. Whereas T4CaIVS3 is blocked by μM Ca2+ and Cd2+, T4CaIVS2 is not blocked by divalent cations. Only T4CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T4CaIVS3 exhibits pore properties characteristic also of authentic calcium channels. The designed functional calcium channel may provide insights into fundamental mechanisms of ionic permeation and drug action, information that may in turn further our understanding of molecular determinants underlying authentic pore structures.
Isolation and characterization of a resistant core peptide of recombinant human granulocyte‐macrophage colony‐stimulating factor (gm‐csf); confirmation of the gm‐csf amino acid sequence by mass spectrometry
Authors: Tsarbopoulos Anthony , Pramanik Birendra N. , Labdon James E. , Reichert Paul , Gitlin Gerry , Patel Suman , Sardana Vinod , Nagabhushan Tattanahalli L. , Trotta Paul P. 
Published: February 2009

AbstractA trypsin‐resistant core peptide of recombinant human granulocyte‐macrophage colony‐stimulating factor (rhGM‐CSF) was isolated and analyzed by high‐energy Cs+ liquid secondary‐ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high‐mass disulfide‐linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM‐CSF provided rapid confirmation of the cDNA‐derived sequence and determination of the existing disulfide bonds between cysteine residues 54–96 and 88–121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with β‐mercaptoethanol.
Heme biosynthesis in mammalian systems: Evidence of a schiff base linkage between the pyridoxal 5′‐phosphate cofactor and a lysine residue in 5‐aminolevulinate synthase
Authors: Ferreira Gloria C. , Neame Peter J. , Dailey Harry A. 
Published: February 2009

Abstract5‐Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in nonplant higher eukaryotes. Murine erythroid 5‐aminolevulinate synthase has been purified to homogeneity from an Escherichia coli overproducing strain, and the catalytic and spectroscopic properties of this recombinant enzyme were compared with those from nonrecombinant sources (Ferreira, G.C. & Dailey, H.A., 1993, J. Biol. Chem. 268, 584–590). 5‐Aminolevulinate synthase is a pyridoxal 5′‐phosphate‐dependent enzyme and is functional as a homodimer.The recombinant 5‐aminolevulinate synthase holoenzyme was reduced with tritiated sodium borohydride and digested with trypsin. A single peptide contained the majority of the label. The tritiated peptide was isolated, and its amino acid sequence was determined; it corresponded to 15 amino acids around lysine 313, to which pyridoxal 5′‐phosphate is bound. Significantly, the pyridoxyllysine peptide is conserved in all known cDNA‐derived 5‐aminolevulinate synthase sequences and is present in the C‐terminal (catalytic) domain.Mutagenesis of the 5‐aminolevulinate synthase residue, which is involved in the Schiff base linkage with pyridoxal 5′‐phosphate, from lysine to alanine or histidine abolished enzyme activity in the expressed protein.
Amide proton exchange rates of oxidized and reduced saccharomyces cerevisiae iso‐1‐cytochrome c
Authors: Marmorino Jennifer L. , Auld Douglas S. , Betz Stephen F. , Doyle Donald F. , Young Gregory B. , Pielak Gary J. 
Published: February 2009

AbstractProton NMR spectroscopy was used to determine the rate constant, kobs, for exchange of labile protons in both oxidized (Fe(III)) and reduced (Fe(II)) iso‐1‐cytochrome c. We find that slowly exchanging backbone amide protons tend to lack solvent‐accessible surface area, possess backbone hydrogen bonds, and are present in regions of regular secondary structure as well as in Ω‐loops. Furthermore, there is no correlation between kobs and the distance from a backbone amide nitrogen to the nearest solvent‐accessible atom. These observations are consistent with the local unfolding model. Comparisons of the free energy change for denaturation, ΔGd, at 298 K to the free energy change for local unfolding, ΔGop, at 298 K for the oxidized protein suggest that certain conformations possessing higher free energy than the denatured state are detected at equilibrium. Reduction of the protein results in a general increase in ΔGop. Comparisons of ΔGd to ΔGop for the reduced protein show that the most open states of the reduced protein possess more structure than its chemically denatured form. This persistent structure in high‐energy conformations of the reduced form appears to involve the axially coordinated heme.
Application of molecular dynamics and free energy perturbation methods to metalloporphyrin‐ligand systems ii: Co and dioxygen binding to myoglobin
Authors: Lopez Marco A. , Kollman Peter A. 
Published: February 2009

AbstractThe protein contribution to the relative binding affinity of the ligands CO and O2 toward myoglobin (Mb) has been simulated using free energy perturbation calculations. The tautomers of the His E7 residue are different for the oxymyoglobin (MbO2) and carboxymyoglobin (MbCO) systems. This was modeled by performing two‐step calculations that mutate the ligand and mutate the His E7 tautomers in separate steps. Differences in hydrogen bonding to the O2 and CO ligands were incorporated into the model. The O2 complex was calculated to be 2–3 kcal/mol more stable than the corresponding CO complex when compared to the same difference in an isolated heme control. This value agrees well with the experimental value of 2.0 kcal/mol. In qualitative agreement with experiments, the Fe‐C‐O bond is found to be bent (θ = 159.8°) with a small tilt (ϕ = 6.2°). The contributions made by each of the 29 residues — within the 9.0‐Å radius of the iron atom — to the free energy difference are separated into van der Waals and electrostatic contributions; the latter contributions are dominant. Aside from the proximal histidine and the heme group, the residues having the largest difference in free energy in mutating MbO2 → MbCO are His E7, Phe CD1, Phe CD4, Val E11, and Thr E10.
Thermodynamics of bpti folding
Authors: Makhatadze George I. , Kim Key‐Sun , Woodward Clare , Privalov Peter L. 
Published: February 2009

AbstractA calorimetric study of the basic pancreatic trypsin inhibitor (BPTI) has been performed using the new generation of the adiabatic scanning microcalorimeters, operating in an extended temperature range of 5–130 °C. Precise measurements of the heat capacities of the native and unfolded states of BPTI show that the heat capacity change upon unfolding strongly depends on temperature; its value is maximal at about 50 °C and diminishes as the temperature is increased. The temperature dependencies of the enthalpy and entropy changes upon BPTI unfolding were found to be similar to those normally observed for other small globular proteins. The stability of BPTI has been correlated with its structure.
Thermodynamics of unfolding for turkey ovomucoid third domain: Thermal and chemical denaturation
Authors: Swint Liskin , Robertson Andrew D. 
Published: February 2009

AbstractWe have used thermal and chemical denaturation to characterize the thermodynamics of unfolding for turkey ovomucoid third domain (OMTKY3). Thermal denaturation was monitored spectroscopically at a number of wavelengths and data were subjected to van't Hoff analysis; at pH 2.0, the midpoint of denaturation (Tm) occurs at 58.6 + 0.4 °C and the enthalpy of unfolding at this temperature (ΔHm) is 40.8 + 0.3 kcal/mol. When Tm was perturbed by varying pH and denaturant concentration, the resulting plots of ΔHm versus Tm yield a mean value of 590 + 120 cal/(mol·K) for the change in heat capacity upon unfolding (ΔCp). A global fit of the same data to an equation that includes the temperature dependence for the enthalpy of unfolding yielded a value of 640 + 110 cal/(mol·K). We also performed a variation of the linear extrapolation method described by Pace and Laurents, which is an independent method for determining ΔCp (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 2520–2525). First, OMTKY3 was thermally denatured in the presence of a variety of denaturant concentrations. Linear extrapolations were then made from isothermal slices through the transition region of the denaturation curves. When extrapolated free energies of unfolding (ΔGu) were plotted versus temperature, the resulting curve appeared linear; therefore, ΔCp could not be determined. However, the data for ΔGu versus denaturant concentration are linear over an extra ordinarily wide range of concentrations. Moreover, extrapolated values of ΔGu in urea are identical to values measured directly.
Structure and orientation of the antibiotic peptide magainin in membranes by solid‐state nuclear magnetic resonance spectroscopy
Authors: Bechinger Burkhard , Zasloff Michael , Opella Stanley J. 
Published: February 2009

AbstractMagainin 2 is a 23‐residue peptide that forms an amphipathic α‐helix in membrane environments. It functions as an antibiotic and is known to disrupt the electrochemical gradients across the cell membranes of many bacteria, fungi, and some tumor cells, although it does not lyse red blood cells. One‐ and two‐dimensional solid‐state 15N NMR spectra of specifically 15N‐labeled magainin 2 in oriented bilayer samples show that the secondary structure of essentially the entire peptide is α‐helix, immobilized by its interactions with the phospholipids, and oriented parallel to the membrane surface.
Structural prototypes for an extended family of flavoprotein reductases: Comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin
Authors: Correll Carl C. , Ludwig Martha L. , Bruns Christopher M. , Karplus P. Andrew 
Published: February 2009

AbstractThe structure of phthalate dioxygenase reductase (PDR), a monomeric iron‐sulfur flavoprotein that delivers electrons from NADH to phthalate dioxygenase, is compared to ferredoxin‐NADP+ reductase (FNR) and ferredoxin, the proteins that reduce NADP+ in the final reaction of photosystem I. The folding patterns of the domains that bind flavin, NAD(P), and [2Fe‐2S] are very similar in the two systems. Alignment of the X‐ray structures of PDR and FNR substantiates the assignment of features that characterize a family of flavoprotein reductases whose members include cytochrome P‐450 reductase, sulfite and nitrate reductases, and nitric oxide synthase. Hallmarks of this subfamily of flavoproteins, here termed the FNR family, are an antiparallel β‐barrel that binds the flavin prosthetic group, and a characteristic variant of the classic pyridine nucleotide‐binding fold. Despite the similarities between FNR and PDR, attempts to model the structure of a dissociable FNR:ferredoxin complex by analogy with PDR reveal features that are at odds with chemical crosslinking studies (Zanetti, G., Morelli, D., Ronchi, S., Negri, A., Aliverti, A., & Curti, B., 1988, Biochemistry 27, 3753–3759).Differences in the binding sites for flavin and pyridine nucleotides determine the nucleotide specificities of FNR and PDR. The specificity of FNR for NADP+ arises primarily from substitutions in FNR that favor interactions with the 2′ phosphate of NADP+. Variations in the conformation and sequences of the loop adjoining the flavin phosphate affect the selectivity for FAD versus FMN.The midpoint potentials for reduction of the flavin and [2Fe–2S] groups in PDR are higher than their counterparts in FNR and spinach ferredoxin, by about 120 mV and 260 mV, respectively. Comparisons of the structure of PDR with spinach FNR and with ferredoxin from Anabaena 7120, along with calculations of electrostatic potentials, suggest that local interactions, including hydrogen bonds, are the dominant contributors to these differences in potential.
Modeling the αIIbβ3 integrin solution conformation
Authors: Rocco Mattia , Spotorno Bruno , Hantgan Roy R. 
Published: February 2009

AbstractThe αIIbβ3 platelet integrin is the prototypical member of a widely distributed class of transmembrane receptors formed by the noncovalent association of α and β subunits. Electron microscopic (EM) images of the αIIbβ3 complex show an asymmetric particle with a globular domain from which two extended regions protrude to contact the lipid bilayer. Distance constraints provided by disulfide bond patterns, epitope mapping, and ligand mimetic cross‐linking studies rather suggest a somewhat more compact conformation for the αIIbβ3 complex.We have studied the shape of detergent‐solubilized αIIbβ3 by employing a low‐resolution modeling procedure in which each polypeptide has been represented as an array of interconnected, nonoverlapping spheres (beads) of various sizes. The number, size, and three‐dimensional relationships among the beads were defined either solely by dimensions obtained from published EM images of integrin receptors (EM models, 21 beads), or solely by interdomain constraints derived from published biochemical data (biochemical model, 37 beads). Interestingly, although no EM data were employed in its construction, the resulting overall shape of the biochemical model was still compatible with the EM data. Both kinds of models were then evaluated for their calculated solution properties. The more elongated EM models have diffusion and sedimentation coefficients that differ, at best, by +2% and ‐18% from the experimental values, determined, respectively, in octyl glucoside and Triton X‐100. On the other hand, the parameters calculated for the more compact biochemical model showed a more consistent agreement with experimental values, differing by ‐7% (octyl glucoside) to ‐6% (Triton X‐100).Thus, it appears that using the biochemical constraints as a starting point has resulted in not only a more detailed model of the detergent‐solubilized αIIbβ3 complex, where the relative spatial location of specific domains the size of 5–10 kDa can be tentatively mapped, but in a model that can also reconcile the electron microscopy with the biochemical and the solution data.
Human immunodeficiency virus type‐1 reverse transcriptase and ribonuclease h as substrates of the viral protease
Authors: Tomasselli Alfredo G. , Sarcich Jean L. , Barrett Linda J. , Reardon Ilene M. , Howe W. Jeffrey , Evans David B. , Sharma Satish K. , Heinrikson Robert L. 
Published: February 2009

AbstractA study has been made of the susceptibility of recombinant constructs of reverse transcriptase (RT) and ribonuclease H (RNase H) from human immunodeficiency virus type 1 (HIV‐1) to digestion by the HIV‐1 protease. At neutral pH, the protease attacks a single peptide bond, Phe440‐Tyr441, in one of the protomers of the folded, active RT/RNase H (p66/p66) homodimer to give a stable, active heterodimer (p66/p51) that is resistant to further hydrolysis (Chattopadhyay, D., et al., 1992, J. Biol. Chem. 267, 14227–14232). The COOH‐terminal p15 fragment released in the process, however, is rapidly degraded by the protease by cleavage at Tyr483‐Leu484 and Tyr532. Leu533. In marked contrast to this p15 segment, both p66/p51 and a folded RNase H construct are stable to breakdown by the protease at neutral pH. It is only at pH values around 4 that these latter proteins appear to unfold and, under these conditions, the heterodimer undergoes extensive proteolysis. RNase H is also hydrolyzed at low pH, but cleavage takes place primarily at Gly436‐Ala437 and at Phe440‐Tyr441, and only much more slowly at residues 483, 494, and 532. This observation can be reconciled by inspection of crystallographic models of RNase H, which show that residues 483, 494, and 532 are relatively inaccessible in comparison to Gly436 and Phe440. Our results fit a model in which the p66/p66 homodimer exists in a conformation that mirrors that of the heterodimer, but with a p15 segment on one of the protomers that is structurally disordered to the extent that all of its potential HIV protease cleavage sites are accessible for hydrolysis.
P22 Arc repressor: Enhanced expression of unstable mutants by addition of polar C‐terminal sequences
Authors: Milla Marcos E. , Brown Bronwen M. , Sauer Robert T. 
Published: February 2009

AbstractMany mutant variants of the P22 Arc repressor are subject to intracellular proteolysis in Escherichia coli, which precludes their expression at levels sufficient for purification and subsequent biochemical characterization. Here we examine the effects of several different C‐terminal extension sequences on the expression and activity of a set of Arc mutants. We show that two tail sequences, KNQHE (st5) and H6KNQHE (st11), increase the expression levels of most mutants from 10‐ to 20‐fold and, in some cases, result in restoration of biological activity in the cell. A third tail sequence, HHHHHH (st6), was not as effective in increasing mutant expression levels. All three tail sequences are functionally and structurally silent, as judged by their lack of effects on the DNA binding activity and stability of otherwise wild‐type Arc. The properties of the st11 tail sequence make it an efficient system for the expression and purification of mutant Arc proteins, both because mutant expression levels are increased and because the proteins can be rapidly purified using nickel‐chelate affinity chromatography. Arc mutants containing the EA28, RL31, and SA32 mutations were purified in the st11 background. The thermodynamic stability of the EA28 mutant (ΔΔGu ‐0.4 kcal/mol) is reduced modestly compared to the st11 parent, whereas the RL31 mutant (ΔΔGu ‐3.0 kcal/mol) and SA32 mutant (ΔΔGu ‐3.3 kcal/mol) are substantially less stable.