Rate Of Bond Formation Research Articles

Significance of thermal fluctuations and hydrodynamic interactions in receptor-ligand-mediated adhesive dynamics of a spherical particle in wall-bound shear flow.

The dynamics of adhesion of a spherical microparticle to a ligand-coated wall, in shear flow, is studied using a Langevin equation that accounts for thermal fluctuations, hydrodynamic interactions, and adhesive interactions. Contrary to the conventional assumption that thermal fluctuations play a negligible role at high Péclet numbers, we find that for particles with low surface densities of receptors, rotational diffusion caused by fluctuations about the flow and gradient directions aids in bond formation, leading to significantly greater adhesion on average, compared to simulations where thermal fluctuations are completely ignored. The role of wall hydrodynamic interactions on the steady-state motion of a particle, when the particle is close to the wall, has also been explored. At high Péclet numbers, the shear induced force that arises due to the stresslet part of the Stokes dipole plays a dominant role, reducing the particle velocity significantly and affecting the states of motion of the particle. The coupling between the translational and rotational degrees of freedom of the particle, brought about by the presence of hydrodynamic interactions, is found to have no influence on the binding dynamics. On the other hand, the drag coefficient, which depends on the distance of the particle from the wall, plays a crucial role at low rates of bond formation. A significant difference in the effect of both the shear force and the position-dependent drag force on the states of motion of the particle is observed when the Péclet number is small.

Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis

Studying the kinetics of translocation of mRNA and tRNAs on the translating ribosome is technically difficult since the rate-limiting steps involve large conformational changes without covalent bond formation or disruption. Here, we have developed a unique assay system for precise estimation of the full translocation cycle time at any position in any type of open reading frame (ORF). Using a buffer system optimized for high accuracy of tRNA selection together with high concentration of elongation factor G, we obtained in vivo compatible translocation rates. We found that translocation was comparatively slow early in the ORF and faster further downstream of the initiation codon. The maximal translocation rate decreased from the in vivo compatible value of 30s−1 at 1mM free Mg2+ concentration to the detrimentally low value of 1s−1 at 6mM free Mg2+ concentration. Thus, high and in vivo compatible accuracy of codon translation, as well as high and in vivo compatible translocation rate, required a remarkably low Mg2+ concentration. Finally, we found that the rate of translocation deep inside an ORF was not significantly affected upon variation of the standard free energy of interaction between a 6-nt upstream Shine-Dalgarno (SD)-like sequence and the anti-SD sequence of 16S rRNA in a range of 0–6kcal/mol. Based on these experiments, we discuss the optimal choice of Mg2+ concentration for maximal fitness of the living cell by taking its effects on the accuracy of translation, the peptide bond formation rate and the translocation rate into account.

Hydrogen implantation in silicates: The role of solar wind in SiOH bond formation on the surfaces of airless bodies in space

AbstractHydroxyl on the lunar surface revealed by remote measurements has been thought to originate from solar wind hydrogen implantation in the regolith. The hypothesis is tested here through experimental studies of the rate and mechanisms of OH bond formation due to H+ implantation of amorphous SiO2 and olivine in ultrahigh vacuum. The samples were implanted with 2–10 keV H+, in the range of solar wind energies, and the OH absorption band at ~2.8 µm measured by transmission Fourier transform infrared spectroscopy. For 2 keV protons in SiO2, the OH band depth saturated at fluences F ~5 × 1016 H+/cm2 to a maximum 0.0032 absorption band depth, corresponding to a column density ηs = 1.1 × 1016 OH/cm2. The corresponding values for 5 keV protons in olivine are >2 × 1017/cm2, 0.0067, and 4.0 × 1016 OH/cm2. The initial conversion rate of implanted H+ into hydroxyl species was found to be ~90% and decreased exponentially with fluence. There was no evidence for molecular water formation due to proton irradiation. Translating the laboratory measurements in thin plate samples to the granular lunar regolith, it is estimated that the measurements can account for a maximum of 17% relative OH absorption in reflectance spectroscopy of mature soils, consistent with spacecraft observations in the infrared of the Moon.

The interface between Escherichia coli elongation factor Tu and aminoacyl-tRNA.

Nineteen of the highly conserved residues of Escherichia coli (E. coli) Elongation factor Tu (EF-Tu) that form the binding interface with aa-tRNA were mutated to alanine to better understand how modifying the thermodynamic properties of EF-Tu–tRNA interaction can affect the decoding properties of the ribosome. Comparison of ΔΔGo values for binding EF-Tu to aa-tRNA show that the majority of the interface residues stabilize the ternary complex and their thermodynamic contribution can depend on the tRNA species that is used. Experiments with a very tight binding mutation of tRNATyr indicate that interface amino acids distant from the tRNA mutation can contribute to the specificity. For nearly all of the mutations, the values of ΔΔGo were identical to those previously determined at the orthologous positions of Thermus thermophilus (T. thermophilus) EF-Tu indicating that the thermodynamic properties of the interface were conserved between distantly related bacteria. Measurement of the rate of GTP hydrolysis on programmed ribosomes revealed that nearly all of the interface mutations were able to function in ribosomal decoding. The only interface mutation with greatly impaired GTPase activity was R223A which is the only one that also forms a direct contact with the ribosome. Finally, the ability of the EF-Tu interface mutants to destabilize the EF-Tu–aa-tRNA interaction on the ribosome after GTP hydrolysis were evaluated by their ability to suppress the hyperstable T1 tRNATyr variant where EF-Tu release is sufficiently slow to limit the rate of peptide bond formation (kpep) . In general, interface mutations that destabilize EF-Tu binding are also able to stimulate kpep of T1 tRNATyr, suggesting that the thermodynamic properties of the EF-Tu–aa-tRNA interaction on the ribosome are quite similar to those found in the free ternary complex.

The effects of codon context on in vivo translation speed.

We developed a bacterial genetic system based on translation of the his operon leader peptide gene to determine the relative speed at which the ribosome reads single or multiple codons in vivo. Low frequency effects of so-called “silent” codon changes and codon neighbor (context) effects could be measured using this assay. An advantage of this system is that translation speed is unaffected by the primary sequence of the His leader peptide. We show that the apparent speed at which ribosomes translate synonymous codons can vary substantially even for synonymous codons read by the same tRNA species. Assaying translation through codon pairs for the 5′- and 3′- side positioning of the 64 codons relative to a specific codon revealed that the codon-pair orientation significantly affected in vivo translation speed. Codon pairs with rare arginine codons and successive proline codons were among the slowest codon pairs translated in vivo. This system allowed us to determine the effects of different factors on in vivo translation speed including Shine-Dalgarno sequence, rate of dipeptide bond formation, codon context, and charged tRNA levels.

A residue-free production of biaryls using supported gold nanoparticles

Small gold nanoparticles in the presence of O2 are able to cleave CH bonds of non-activated arenes and facilitate catalytic formation of biaryls under mild conditions. This procedure avoids the common requirement of iodine, acids, bases, and/or other stoichiometrical additives to accomplish multiple turnovers and results in a zero-waste synthetic process. A number of unactivated arenes such as benzene, toluene, p-xylene, nitrobenzene, chlorobenzene, or phenol are selectively converted to the corresponding biaryls, with accumulated turnover numbers greater than 600. Kinetic measurements, in combination with IR, HRTEM, and STEM-HAADF data, suggest that the reaction takes place on gold atoms that are metallic in character, with the highest CC bond formation rate provided by gold nanoparticles of approximately 3nm of diameter. A radical-free reaction mechanism is postulated on the basis of the kinetic measurements. The results represent a first stone for the waste-free preparation of biaryls using supported gold catalysts.

Bis-Aliphatic Hydrazone-Linked Hydrogels Form Most Rapidly at Physiological pH: Identifying the Origin of Hydrogel Properties with Small Molecule Kinetic Studies

Rheological and small molecule kinetic studies were performed to study the formation and hydrolysis of the bis-aliphatic hydrazone bond. The rate of gelation was found to correspond closely with the rate of bond formation and the rate of gel relaxation with the rate of hydrolysis, indicating that small molecule kinetic studies can play an important role in material design. Furthermore, unlike aryl or acyl hydrazone bonds, the bis-aliphatic hydrazone bond forms rapidly under physiological conditions without requiring aniline catalysis yet maintains a pH-dependent rate of hydrolysis. These results suggest the bis-aliphatic hydrazone bond should find use alongside existing bioorthogonal click chemistries for bioconjugation, biomaterial synthesis, and controlled release applications.

Ester Formation and Hydrolysis during Wet–Dry Cycles: Generation of Far-from-Equilibrium Polymers in a Model Prebiotic Reaction

Biopolymers exist within living cells as far-from-equilibrium metastable polymers. Living systems must constantly invest energy for biopolymer synthesis. In the earliest stages of life on Earth, the complex molecular machinery that contemporary life employs for the synthesis and maintenance of polymers did not exist. Thus, a major question regarding the origin of life is how the first far-from-equilibrium polymers emerged from a prebiotic “pool” of monomers. Here, we describe a proof-of-principle system, in which l-malic acid monomers form far-from-equilibrium, metastable oligoesters via repeated, cyclic changes in hydration and temperature. Such cycles would have been associated with day–night and/or seasonal cycles on the early Earth. In our model system, sample heating, which promotes water evaporation and ester bond formation, drives polymerization. Even though periodic sample rehydration and heating in the hydrated state promotes ester bond hydrolysis, successive iterations of wet–dry cycles result in polymer yields and molecular weight distributions in excess of that observed after a single drying cycle. We term this phenomenon a “polymerization ratchet”. We have quantitatively characterized the “ratchet” of our particular system. Ester bond formation rates and oligoester hydrolysis rates were determined for temperatures ranging from 60 to 95 °C. Based on these rates, a mathematical model was developed using polycondensation kinetics, from which conditions were predicted for oligoester growth. This model was verified experimentally by the demonstration that l-malic acid monomers subjected to multiple wet–dry cycles form oligoesters, which reach a steady-state concentration and mean length after several cycles. The concentration of oligoesters that persist between subsequent steady-state cycles depends on the temperature and durations of the dry and wet phases of the cycle. These results provide insights regarding the potential for very simple systems to exhibit features that would have been necessary for initiation of polymer evolution, before the emergence of genomes or enzymes.

Aminoacylating Urzymes Challenge the RNA World Hypothesis

We describe experimental evidence that ancestral peptide catalysts substantially accelerated development of genetic coding. Structurally invariant 120-130-residue Urzymes (Ur = primitive plus enzyme) derived from Class I and Class II aminoacyl-tRNA synthetases (aaRSs) acylate tRNA far faster than the uncatalyzed rate of nonribosomal peptide bond formation from activated amino acids. These new data allow us to demonstrate statistically indistinguishable catalytic profiles for Class I and II aaRSs in both amino acid activation and tRNA acylation, over a time period extending to well before the assembly of full-length enzymes and even further before the Last Universal Common Ancestor. Both Urzymes also exhibit ∼60% of the contemporary catalytic proficiencies. Moreover, they are linked by ancestral sense/antisense genetic coding, and their evident modularities suggest descent from even simpler ancestral pairs also coded by opposite strands of the same gene. Thus, aaRS Urzymes substantially pre-date modern aaRS but are, nevertheless, highly evolved. Their unexpectedly advanced catalytic repertoires, sense/antisense coding, and ancestral modularities imply considerable prior protein-tRNA co-evolution. Further, unlike ribozymes that motivated the RNA World hypothesis, Class I and II Urzyme·tRNA pairs represent consensus ancestral forms sufficient for codon-directed synthesis of nonrandom peptides. By tracing aaRS catalytic activities back to simpler ancestral peptides, we demonstrate key steps for a simpler and hence more probable peptide·RNA development of rapid coding systems matching amino acids with anticodon trinucleotides.

Sacrificial bonds and hidden length in biomaterials: A kinetic constitutive description of strength and toughness in bone

Sacrificial bonds and hidden length in structural molecules account for the greatly increased fracture toughness of biological materials compared to synthetic materials without such structural features by providing a molecular-scale mechanism for energy dissipation. One example is in the polymeric glue connection between collagen fibrils in animal bone. In this paper we propose a simple kinetic model that describes the breakage of sacrificial bonds and the release of hidden length, based on Bell's theory. We postulate a master equation governing the rates of bond breakage and formation. This enables us to predict the mechanical behavior of a quasi-one-dimensional ensemble of polymers at different stretching rates. We find that both the rupture peak heights and maximum stretching distance increase with the stretching rate. In addition, our theory naturally permits the possibility of self-healing in such biological structures.

Kinetic Characterization of Exonuclease-Deficient Staphylococcus aureus PolC, a C-family Replicative DNA Polymerase

PolC is the C-family replicative polymerase in low G+C content Gram-positive bacteria. To date several structures of C-family polymerases have been reported, including a high resolution crystal structure of a ternary complex of PolC with DNA and incoming deoxynucleoside triphosphate (dNTP). However, kinetic information needed to understand the enzymatic mechanism of C-family polymerases is limited. For this study we have performed a detailed steady-state and pre-steady-state kinetic characterization of correct dNTP incorporation by PolC from the Gram-positive pathogen Staphylococcus aureus, using a construct lacking both the non-conserved N-terminal domain and the 3′–5′ exonuclease domain (Sau-PolC-ΔNΔExo). We find that Sau-PolC-ΔNΔExo has a very fast catalytic rate (kpol 330 s−1) but also dissociates from DNA rapidly (koff ∼150 s−1), which explains the low processivity of PolC in the absence of sliding clamp processivity factor. Although Sau-PolC-ΔNΔExo follows the overall enzymatic pathway defined for other polymerases, some significant differences exist. The most striking feature is that the nucleotidyl transfer reaction for Sau-PolC-ΔNΔExo is reversible and is in equilibrium with dNTP binding. Simulation of the reaction pathway suggests that rate of pyrophosphate release, or a conformational change required for pyrophosphate release, is much slower than rate of bond formation. The significance of these findings is discussed in the context of previous data showing that binding of the β-clamp processivity factor stimulates the intrinsic nucleotide incorporation rate of the C-family polymerases, in addition to increasing processivity.

State-Dependent Etomidate Occupancy of Its Allosteric Agonist Sites Measured in a Cysteine-Substituted GABAA Receptor

A central axiom of ligand-receptor theory is that agonists bind more tightly to active than to inactive receptors. However, measuring agonist affinity in inactive receptors is confounded by concomitant activation. We identified a cysteine substituted mutant γ-aminobutyric acid type A (GABAA) receptor with unique characteristics allowing the determination of allosteric agonist site occupancy in both inactive and active receptors. Etomidate, the allosteric agonist, is an anesthetic that activates or modulates α1β2γ2L GABAA receptors via transmembrane sites near β2M286 residues in M3 domains. Voltage-clamp electrophysiology studies of α1β2M286Cγ2L receptors show that GABA is an efficacious agonist and that etomidate modulates GABA-activated activity, but direct etomidate agonism is absent. Quantitative analysis of mutant activity using an established Monod-Wyman-Changeux (MWC) allosteric model indicates that the intrinsic efficacy of etomidate, defined as its relative affinity for active versus inactive receptors, is lower than in wild-type receptors. Para-chloromercuribenzene sulfonate covalently modifies β2M286C side-chain sulfhydryls, irreversibly altering GABA-induced currents. Etomidate concentration dependently reduces the apparent rate of β2M286C-pCMBS bond formation, tracked electrophysiologically. High etomidate concentrations completely protect the β2M286C suflhydryl from covalent modification, suggesting close steric interactions. The 50% protective etomidate concentration (PC50) is 14 μM in inactive receptors and 1.1 to 2.2 μM during GABA-activation, experimentally demonstrating that activated receptors bind etomidate more avidly than do inactive receptors. The experimental PC50 values are remarkably close to, and therefore validate, MWC model predictions for etomidate dissociation constants in both inactive and active receptors. Our results support MWC models as valid frameworks for understanding the agonism, coagonism, and modulation of ligand-gated ion channels.

ONIOM and ab-initio calculations on the mechanism of uncatalyzed peptide bond formation

Finding a proper transition structure for the peptide bond formation process can lead one to a better understanding of the role of ribosome in catalyzing this reaction. Using computer simulations, we performed the potential energy surface scan on the ester bond dissociation of P-site aminoacyl-tRNA and the peptide bond formation of P-site and A-site amino acids. The full fragments of initiator tRNA(i)(met) and elongator tRNA(phe) are attached to both cognate and non-cognate amino acids as the P-site substrate. The A-site amino acid for all four calculations is methionine. We used ONIOM calculations to reduce the computational cost. Our study illustrates the reduced rate of peptide bond formation for misacylated tRNA(i)(met) in the absence of ribosomal bases. The misacylated elongator tRNA(phe), however, did not show any difference in its PES compared with that for the phe-tRNA(phe). This demonstrates the structural specification of initiator tRNA(i)(met) for the amino acids side chain.

A High-Throughput Technique Reveals the Load- and Site Density-Dependent Kinetics of E-Selectin.

The kinetics of bond rupture between receptors and ligand are critically dependent on applied mechanical force. Force spectroscopy of single receptor-ligand pairs to measure kinetics is a laborious and time-consuming process that is generally performed using individual force probes and making one measurement at a time when typically hundreds of measurements are needed. A high-throughput approach is thus desirable. We report here a magnetic bond puller that provides high-throughput measurements of single receptor-ligand bond kinetics. Electromagnets are used to apply pN tensile and compressive forces to receptor-coated magnetic microspheres while monitoring their contact with a ligand-coated surface. Bond lifetimes and the probability of forming a bond are measured via videomicroscopy, and the data are used to determine the load dependent rates of bond rupture and bond formation. The approach is simple, customizable, relatively inexpensive, and can make dozens of kinetic measurements simultaneously. We used the device to investigate how compressive and tensile forces affect the rates of formation and rupture, respectively, of bonds between E-selectin and sialyl Lewisa (sLea), a sugar on P-selectin glycoprotein ligand-1 to which selectins bind. We confirmed earlier findings of a load-dependent rate of bond formation between these two molecules, and that they form a catch-slip bond like other selectin family members. We also make the novel observation of an "ideal" bond in a highly multivalent system of this receptor-ligand pair.

Regulation of chaperone activity in the endoplasmic reticulum

The synthesis and folding 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 large family of ER proteins. The PDI family includes general enzymes such as PDI‐1 that recognize unfolded proteins, and others, including ERp57 and ERp72, that are selective for specific proteins. PDI‐1 consists of four thioredoxin‐like domains termed a‐b‐ b’‐a’. The a and a’ domains contain catalytic CxxC motifs that confer the disulfide oxidoreductase activity, while the b and b’ domains contain the principal binding site for unfolded proteins. Here, we determined the crystal structure of a dimeric form of a bb’ fragment of human PDI at 2.3 Å resolution. The structure shows that the b’ domain is mediates substrate interactions through a hydrophobic binding site for unfolded proteins. In ERp57, this site is conserved but binds lectin co‐chaperones that are specific for the folding of glycoproteins. Recent results with PDI‐1 suggest the hydrophobic binding site of PDI‐1 may also be involved in regulation of the redox state of ER chaperones.

Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

BackgroundThe protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor) or a larger protein (α-amylase).ResultsHAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress.ConclusionsApparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.

Zinc enhances adiponectin oligomerization to octadecamers but decreases the rate of disulfide bond formation

Adiponectin, a hormone secreted from adipocytes, has been shown to protect against development of insulin resistance, ischemia-reperfusion injury, and inflammation. Adiponectin assembles into multiple oligomeric isoforms: trimers, hexamers and several higher molecular weight (HMW) species. Of these, the HMW species are selectively decreased during the onset of type 2 diabetes. Despite the critical role of HMW adiponectin in insulin responsiveness, its assembly process is poorly understood. In this report, we investigated the role of divalent cations in adiponectin assembly. Purified adiponectin 18mers, the largest HMW species, did not collapse to smaller oligomers after treatment with high concentrations of EDTA. However, treatment with EDTA or another chelator DTPA inhibited the oligomerization of 18mers from trimers in vitro. Zn(2+) specifically increased the formation of 18mers when compared with Cu(2+), Mg(2+), and Ca(2+). Distribution of adiponectin oligomers secreted from zinc chelator TPEN-treated rat adipocytes skewed toward increased proportions of hexamers and trimers. While we observed presence of zinc in adiponectin purified from calf serum, the role of zinc in disulfide bonding between oligomers was examined because the process is critical for 18mer assembly. Surprisingly, Zn(2+) inhibited disulfide bond formation early in the oligomerization process. We hypothesize that initial decreases in disulfide formation rates could allow adiponectin subunits to associate before becoming locked in fully oxidized conformations incapable of further oligomerization. These data demonstrate that zinc stimulates oligomerization of HMW adiponectin and possibly other disulfide-dependent protein assembly processes.

The Two-Dimensional Kinetics of Binding and Unbinding are Both Regulated by Myosin's Actin-Binding Loop

Binding of myosin to actin is a multi-step, ionic strength-sensitive process for which the kinetic properties of bond formation and rupture have been studied in solution. However, the solution (three dimensional) environment is a poor model of the pseudo-two dimensional geometry in which myofilament proteins interact in vivo. We therefore investigated the rate of actomyosin bond formation and rupture in a two-dimensional setting using dynamic force spectroscopy at physiological and reduced ionic strength. We previously showed that catch bond rupture is slowed at physiologic ionic strength (145 mM KCl) when compared to low ionic strength (25 mM), and that bond lifetime is maximal at the isometric force generated by a single myosin molecule. In the current study we confirmed this result and sought to determine whether this difference is mirrored in the rate of actomyosin bond formation. A laser trap was used to measure the time to bond formation (tb) between actin filaments and nucleotide-free heavy meromyosin (HMM) over a range of compressive loads. Two dimensional on-rates were determined from tb and were increasingly force-dependent with decreasing ionic strength. We examined the effect of ionic strength on the initial long-range ionic interaction constituting the first step of actomyosin binding by targeted tryptic digestion of HMM’s actin-binding loop. Our data at physiologic ionic strength suggest that the actin-binding loop contributes to the decreased 2D rate and load-dependence of bond formation. This is matched by reduced rates of actomyosin unbinding. These data provide useful parameters for modeling studies and suggest that the actin-binding loop of myosin contributes to both the binding and unbinding of myosin from actin.

Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

The protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor) or a larger protein (α-amylase). HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress. Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.

Structural insights into complete metal ion coordination from ternary complexes of B family RB69 DNA polymerase.

We have captured a preinsertion ternary complex of RB69 DNA polymerase (RB69pol) containing the 3' hydroxyl group at the terminus of an extendable primer (ptO3') and a nonhydrolyzable 2'-deoxyuridine 5'-α,β-substituted triphosphate, dUpXpp, where X is either NH or CH(2), opposite a complementary templating dA nucleotide residue. Here we report four structures of these complexes formed by three different RB69pol variants with catalytically inert Ca(2+) and four other structures with catalytically competent Mn(2+) or Mg(2+). These structures provide new insights into why the complete divalent metal-ion coordination complexes at the A and B sites are required for nucleotidyl transfer. They show that the metal ion in the A site brings ptO3' close to the α-phosphorus atom (Pα) of the incoming dNTP to enable phosphodiester bond formation through simultaneous coordination of both ptO3' and the nonbridging Sp oxygen of the dNTP's α-phosphate. The coordination bond length of metal ion A as well as its ionic radius determines how close ptO3' can approach Pα. These variables are expected to affect the rate of bond formation. The metal ion in the B site brings the pyrophosphate product close enough to Pα to enable pyrophosphorolysis and assist in the departure of the pyrophosphate. In these dUpXpp-containing complexes, ptO3' occupies the vertex of a distorted metal ion A coordination octahedron. When ptO3' is placed at the vertex of an undistorted, idealized metal ion A octahedron, it is within bond formation distance to Pα. This geometric relationship appears to be conserved among DNA polymerases of known structure.