Rate Of Bond Formation Research Articles

Homolytic Bond Strengths and Formation Rates in Half‐Sandwich Chromium Alkyl Complexes: Relevance for Controlled Radical Polymerization

Ein waches Auge auf Radikale: Die sterischen Eigenschaften der Arylsubstituenten von Chrom-β-Ketiminato-Komplexen lassen sich so einstellen, dass wachsende radikalische Poly(vinylacetat)-Ketten reversibel eingefangen werden (siehe Schema; V-70 = Radikalinitiator, VOAc = Vinylacetat). Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2001/2008/z801498_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Homolytic Bond Strengths and Formation Rates in Half‐Sandwich Chromium Alkyl Complexes: Relevance for Controlled Radical Polymerization

Homolytic Bond Strengths and Formation Rates in Half‐Sandwich Chromium Alkyl Complexes: Relevance for Controlled Radical Polymerization

The Kinetics of Ribosomal Peptidyl Transfer Revisited

The speed of protein synthesis determines the growth rate of bacteria. Current biochemical estimates of the rate of protein elongation are small and incompatible with the rate of protein elongation in the living cell. With a cell-free system for protein synthesis, optimized for speed and accuracy, we have estimated the rate of peptidyl transfer from a peptidyl-tRNA in P site to a cognate aminoacyl-tRNA in A site at various temperatures. We have found these rates to be much larger than previously measured and fully compatible with the speed of protein elongation for E. coli cells growing in rich medium. We have found large activation enthalpy and small activation entropy for peptidyl transfer, similar to experimental estimates of these parameters for A site analogs of aminoacyl-tRNA. Our work has opened a useful kinetic window for biochemical studies of protein synthesis, bridging the gap between in vitro and in vivo data on ribosome function.

Enhancement of L-Selectin, but Not P-Selectin, Bond Formation Frequency by Convective Flow

L-selectin-mediated leukocyte rolling has been proposed to require a high rate of bond formation compared to that of P-selectin to compensate for its much higher off-rate. To test this hypothesis, a microbead system was utilized to measure relative L-selectin and P-selectin bond formation rates on their common ligand P-selectin glycoprotein ligand-1 (PSGL-1) under shear flow. Using video microscopy, we tracked selectin-coated microbeads to detect the formation frequency of adhesive tether bonds. From velocity distributions of noninteracting and interacting microbeads, we observed that tether bond formation rates for P-selectin on PSGL-1 decreased with increasing wall shear stress, from 0.14±0.04 bonds/μm at 0.2 dyn/cm2 to 0.014±0.003 bonds/μm at 1.0 dyn/cm2. In contrast, L-selectin tether bond formation increased from 0.017±0.005 bonds/μm at 0.2 dyn/cm2 to 0.031±0.005 bonds/μm at 1.0 dyn/cm2. L-selectin tether bond formation rates appeared to be enhanced by convective transport, whereas P-selectin rates were inhibited. The transition force for the L-selectin catch-slip transition of 44 pN/bond agreed well with theoretical models (Pereverzev et al. 2005. Biophys. J. 89:1446-1454). Despite catch bond behavior, hydrodymanic shear thresholding was not detected with L-selectin beads rolling on PSGL-1. We speculate that shear flow generated compressive forces may enhance L-selectin bond formation relative to that of P-selectin and that L-selectin bonds with PSGL-1 may be tuned for the compressive forces characteristic of leukocyte-leukocyte collisions during secondary capture on the blood vessel wall. This is the first report, to our knowledge, comparing L-selectin and P-selectin bond formation frequencies in shear flow.

3′- 32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation

The analysis of reactions involving amino acids esterified to tRNAs traditionally uses radiolabeled amino acids. We describe here an alternative assay involving [3′- 32P]-labeled tRNA followed by nuclease digestion and TLC analysis that permits aminoacylation to be monitored in an efficient, quantitative manner while circumventing many of the problems faced when using radiolabeled amino acids. We also describe a similar assay using [3′- 32P]-labeled aa-tRNAs to determine the rate of peptide bond formation on the ribosome. This type of assay can also potentially be adapted to study other reactions involving an amino acid or peptide esterified to tRNA.

Peptidyl-prolyl-tRNA at the ribosomal P-site reacts poorly with puromycin

Despite remarkable recent progress in our chemical and structural understanding of the mechanisms of peptide bond formation by the ribosome, only very limited information is available about whether amino acid side chains affect the rate of peptide bond formation. Here, we generated a series of peptidyl-tRNAs that end with different tRNA-attached amino acids in the P-site of the Escherichia coli ribosome and compared their reactivity with puromycin, a rapidly A-site-accessing analog of aminoacyl-tRNAs. Among the 20 amino acids examined, proline was found to receive exceptionally slow peptidyl transfer to puromycin. These results raise a possibility that the peptidyl transferase activity of the ribosome may have some specificity with regard to the P-site amino acids.

Metal-Catalyzed Asymmetric Synthesis of P-Stereogenic Phosphines

This account summarizes our attempts to develop metal-catalyzed asymmetric syntheses of P-stereogenic phosphines. While such phosphines undergo pyramidal inversion slowly at room temperature, inversion is rapid in metal-phosphido com­plexes (M-PR2). These observations were the basis for catalytic, dynamic kinetic resolution processes in which racemic secondary phosphines [PR(R′)H] were converted into enantioenriched tertiary phosphines [PR(R′)(R′′)] by platinum-catalyzed asymmetric hydrophosphination of acrylonitrile or related Michael acceptors, by ­palladium-catalyzed asymmetric phosphination of aryl iodides using secondary phosphines or phosphine-boranes, and by platinum-catalyzed asymmetric alkylation of secondary phosphines. The key intermediates were diastereomeric phosphido complexes with chiral ancillary ligands (Ln*-M-PRR′). Their relative rates of P-inversion and phosphorus-carbon bond formation controlled the enantioselectivity of product formation, whether the phosphorus-carbon bonds were formed by reductive elimination (for Pd), or by the reaction of a platinum-phosphido complex with an electrophile (an alkene in hydrophosphination, or a benzyl bromide in alkylation). The results of mechanistic studies and their use in the design of improved catalytic reactions are described.

Generalized Lifshits-Slezov-Wagner distribution

The particle (cluster) size distribution function and the temporal variation of the middle (critical) and maximum particle dimensions have been calculated assuming that two mechanisms of mass transfer—those controlled by (i) the volume diffusion and (ii) the rate of chemical bond formation on the particle surface—are simultaneously operative. A comparison of the theoretical curves to experimental histograms shows that the proposed mechanism of particle growth can be realized in practice in the course of the Ostwald ripening.

A Barley Xyloglucan Xyloglucosyl Transferase Covalently Links Xyloglucan, Cellulosic Substrates, and (1,3;1,4)-β-D-Glucans

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-beta-D-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-beta-D-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-beta-D-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-beta-D-glucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-beta-D-glucan:xyloglucan conjugate by a specific (1,3;1,4)-beta-D-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.

Exploring the mechanism of protein synthesis with modified substrates and novel intermediate mimics

Translation, the synthesis of proteins from individual amino acids based on genetic information, is a cornerstone biological process. During ribosomal protein synthesis, new peptide bonds form through aminolysis of the peptidyl–tRNA ester bond by the alpha-amino group of the A-site amino acid. The rate of this reaction is accelerated at least 107-fold in the ribosome, but the catalytic mechanism has remained controversial. We have used a combination of synthetic chemistry, biochemical, and structural biology approaches to characterize the mechanism of the peptidyl transfer reaction and the configuration of the reaction's tetrahedral intermediate. Substitution of the P-site tRNA A76 2′ OH with 2′ H or 2′ F results in at least a 106-fold reduction in the rate of peptide bond formation, but does not affect binding of the modified substrates. This indicates that the 2′-OH is essential to the reaction through participation in substrate assisted catalysis. A series of novel mimics of the tetrahedral intermediate were examined to distinguish between possible regio- and stereoisomeric forms of the intermediate. The determination of these parameters has important implications for the configuration of the substrates and intermediate within the ribosomal active site, and thus which functional groups are properly positioned to play various roles in promoting the reaction. Our results contribute to an emerging model of the peptidyl transfer reaction in which the ribosomal active site positions the substrates in an orientation specifically designed to promote the reaction, wherein the A76 2′-OH serves as a proton shuttle to enable critical proton transfers in the formation of the final peptide product.

Perturbing Hemostasis by Altering the Kinetics of the Interactions between GPIb Apha and the A1 Domain of VWF: A Mouse Tale.

Effective hemostasis relies on ability of circulating platelets to be rapidly deposited at sites of vascular injury. Critical to this process is the formation of adhesive bonds between the platelet receptor GPIb alpha and the A1 domain of VWF, an interaction that is rapid and reversible due to the relatively high rate of bond formation and dissociation. The importance of maintaining the kinetic properties of this interaction within a critical range is suggested by the bleeding phenotype that results from gain- or loss-of function points mutations associated with VWD. To demonstrate the an alteration in bond kinetics can have a dramatic effect of hemostasis, we generated a mouse in which a single point mutation was introduced into the vWF-A1 domain that results in a shortening of the time it can maintain contact with GPIb alpha (aka - increase in off-rate). The mutation was chosen based on our crystal structure data of the recombinant mouse A1 domain protein and its effect on mouse platelet adhesion to the immobilized mutant protein under flow conditions. Homozygous animals were viable and exhibited normal Mendelian ratios. Both VWF antigen levels as determined by ELISA and multimer banding pattern were identical to WT littermate controls. Moreover, transcription of the A1-A2-A3 region of VWF was comparable to controls as determined by RT-PCR. Despite the similarities in biochemical and molecular parameters to WT littermates, mutant A1 domain mice had a significant prolongation in bleeding time as measured by severing a small portion of their tail. Moreover, perfusion of whole blood from these animals over surface-immobilized collagen resulted in >50% reduction in platelet accumulation at 1600 s-1. This abnormality in platelet adhesion was also observed in vivo. Although mutant A1 domain mice were able to form thrombi upon laser-induced injury to arterioles located within the cremaster muscle, these thrombi were unstable and rapidly dissolved. In conclusion, we are the first to demonstrate that an alteration in the kinetic properties of a receptor-ligand bond can have a significant effect on biological processes such as hemostasis. Our results also provide insight into mechanisms by which point mutations associated with certain types of VWD may result in bleeding in afflicted individuals.

Crystal Structure of the bb′ Domains of the Protein Disulfide Isomerase ERp57

The synthesis of proteins in the endoplasmic reticulum (ER) is limited by the rate of correct disulfide bond formation. This process is carried out by protein disulfide isomerases, a family of ER proteins which includes general enzymes such as PDI that recognize unfolded proteins and others that are selective for specific proteins or classes. Using small-angle X-ray scattering and X-ray crystallography, we report the structure of a selective isomerase, ERp57, and its interactions with the lectin chaperone calnexin. Using isothermal titration calorimetry and NMR spectroscopy, we show that the b' domain of ERp57 binds calnexin with micromolar affinity through a conserved patch of basic residues. Disruption of this binding site by mutagenesis abrogates folding of RNase B in an in vitro assay. The relative positions of the ERp57 catalytic sites and calnexin binding site suggest that activation by calnexin is due to substrate recruitment rather than a direct stimulation of ERp57 oxidoreductase activity.

A novel real‐time confocal imaging technique for examining host–implant interfacial shear failure patterns

In clinical practice, implant failure usually occurs at the biomaterial-host tissue interface, typically involving both biomechanical and biochemical mechanisms. By definition, any new 'bioactive' material will bond to living bone but, prior to clinical use, interface formation, performance, longevity and failure pattern characterizations are necessary. The common missing link in many biomaterial interface investigations is imaging at the point of presumed loaded failure. The novel real-time confocal technique described here allows bond strength, formation rate, longevity and bone-material interface failure pattern characterization for a wide range of biomaterials capable of forming tissue interfaces, in one real-time imaged microshear stress process, conducted using imaging frame matched load/displacement data acquisition under relatively normal near in vivo environmental conditions. The technique, validated by post-failure scanning electron microscopy imaging, revealed that more slowly reacting melt-derived 45S5 glass materials produced stronger and more stable long-term interfaces than faster reacting microporous bioactive sol-gel glasses.

DISSECTING INDIVIDUAL LIGAND–RECEPTOR BONDS WITH A LAMINAR FLOW CHAMBER

The most important function of proteins may well be to bind to other biomolecules. It has long been felt that kinetic rates of bond formation and dissociation between soluble receptors and ligands might account for most features of the binding process. Only theoretical considerations allowed to predict the behaviour of surface-attached receptors from the properties of soluble forms. During the last decade, experimental progress essentially based on flow chambers, atomic force microscopes or biomembrane force probes allowed direct analysis of biomolecule interaction at the single bond level and gave new insight into previously ignored features such as bond mechanical properties or energy landscapes. The aim of this review is (i) to describe the main advances brought by laminar flow chambers, including information on bond response to forces, multiplicity of binding states, kinetics of bond formation between attached structures, effect of molecular environment on receptor efficiency and behaviour of multivalent attachment, (ii) to compare results obtain by this and other techniques on a few well defined molecular systems, and (iii) to discuss the limitations of the flow chamber method. It is concluded that a new framework may be needed to account for the effective behaviour of biomolecule association.

Peptide bond formation does not involve acid-base catalysis by ribosomal residues

Ribosomes catalyze the formation of peptide bonds between aminoacyl esters of transfer RNAs within a catalytic center composed of ribosomal RNA only. Here we show that the reaction of P-site formylmethionine (fMet)-tRNA(fMet) with a modified A-site tRNA substrate, Phelac-tRNA(Phe), in which the nucleophilic amino group is replaced with a hydroxyl group, does not show the pH dependence observed with small substrate analogs such as puromycin and hydroxypuromycin. This indicates that acid-base catalysis by ribosomal residues is not important in the reaction with the full-size substrate. Rather, the ribosome catalyzes peptide bond formation by positioning the tRNAs, or their 3' termini, through interactions with rRNA that induce and/or stabilize a pH-insensitive conformation of the active site and provide a preorganized environment facilitating the reaction. The rate of peptide bond formation with unmodified Phe-tRNA(Phe) is estimated to be >300 s(-1).

Translational Fidelity Mutations in 18S rRNA Affect the Catalytic Activity of Ribosomes and the Oxidative Balance of Yeast Cells

The function of mutations rdn1A, rdn1T, and rdn2 in 18S rRNA of Saccharomyces cerevisiae is investigated. The mutations correspond to substitutions C1054A, C1054U in helix 34, and G517A in helix 18 of 16S rRNA in Escherichia coli, respectively, in which the first and third mutations caused nonsense suppression, while C1054U caused no suppression. In yeast, rdn1A caused phenotypic suppression at nonsense codons, whereas rdn1T and rdn2 caused antisuppression. We provide in vitro evidence that, in addition, rdn1A decreases translational accuracy at sense codons as well, by a factor of 8, accompanied by extreme sensitivity to paromomycin, compatible with its error-prone character. Mutations rdn1T andrdn2 exhibit hyperaccuracy and paromomycin resistance. Thus, mutations in conserved rRNA regions may affect the same functions in the various species but in opposite directions. Mutation rdn1A, but not rdn1T or rdn2, affected also the catalytic activity of the ribosome, a 60S subunit activity. The rate of peptide bond formation was reduced to half its normal value, indicating a communication between the two subunits. Moreover, error-prone mutation rdn1A was less susceptible to oxidative modifications than wild type, indicated by decreased lipid peroxidation and nonprotein/protein disulfides, as well as by increased protein thiols. In contrast, hyperaccurate mutations rdn1T and rdn2 displayed increased oxidative stress. Our results suggest that the cells may consume more energy to achieve hyperaccuracy leading to increased oxidative modifications.

Opsin is present as dimers in COS1 cells: Identification of amino acids at the dimeric interface

Rhodopsin in the disk membranes of rod outer segments serves as the dim-light photoreceptor and is a prototypic member of a G protein-coupled receptor family. Electron and atomic-force microscopy indicate that rhodopsin is present as dimers in the native membranes. Here, we have expressed the protein, opsin, in COS1 cells and have studied its molecular state by using FRET and by intermolecular cross-linking after site-directed cysteine mutagenesis. To observe FRET, the ends of the genes corresponding to the N termini of the cyan or yellow fluorescent proteins were fused to the ends of the genes corresponding to the C terminus of the opsin and the resulting fused genes were expressed in COS1 cells. The emission spectra in situ of the expressed proteins were recorded, and FRET was then calculated. The result indicated intermolecular interaction between opsin molecules in COS1 cells. To identify the amino acids involved in the interaction, those predicted by molecular modeling to be at the dimer interface were mutated one at a time to cysteine, and dimer formation was measured by the rate of disulfide bond formation in the presence of cupric orthophenanthroline. The mutants W175C and Y206C formed the dimers most rapidly, showing that the two amino acids were at the dimer interface.

The Position 68(E11) Side Chain in Myoglobin Regulates Ligand Capture, Bond Formation with Heme Iron, and Internal Movement into the Xenon Cavities

After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (-15 --> 65 degrees C). This approach was applied to a series of double mutants, myoglobin CO (H64L/V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68(E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B --> A, C --> A, and D --> A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68(E11) side chain. The relative amplitudes of the B --> A and C --> A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly.

Essential Mechanisms in the Catalysis of Peptide Bond Formation on the Ribosome

Peptide bond formation is the main catalytic function of the ribosome. The mechanism of catalysis is presumed to be highly conserved in all organisms. We tested the conservation by comparing mechanistic features of the peptidyl transfer reaction on ribosomes from Escherichia coli and the Gram-positive bacterium Mycobacterium smegmatis. In both cases, the major contribution to catalysis was the lowering of the activation entropy. The rate of peptide bond formation was pH independent with the natural substrate, amino-acyl-tRNA, but was slowed down 200-fold with decreasing pH when puromycin was used as a substrate analog. Mutation of the conserved base A2451 of 23 S rRNA to U did not abolish the pH dependence of the reaction with puromycin in M. smegmatis, suggesting that A2451 did not confer the pH dependence. However, the A2451U mutation alters the structure of the peptidyl transferase center and changes the pattern of pH-dependent rearrangements, as probed by chemical modification of 23 S rRNA. A2451 seems to function as a pivot point in ordering the structure of the peptidyl transferase center rather than taking part in chemical catalysis.

Spontaneous Thermal Motion of the GABAA Receptor M2 Channel-lining Segments

The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.