GTP In Solution Research Articles

A Central Interdomain Protein Joint in Elongation Factor G Regulates Antibiotic Sensitivity, GTP Hydrolysis, and Ribosome Translocation

The antibiotic fusidic acid potently inhibits bacterial translation (and cellular growth) by lodging between domains I and III of elongation factor G (EF-G) and preventing release of EF-G from the ribosome. We examined the functions of key amino acid residues near the active site of EF-G that interact with fusidic acid and regulate hydrolysis of GTP. Alanine mutants of these residues spontaneously hydrolyzed GTP in solution, bypassing the normal activating role of the ribosome. A conserved phenylalanine in the switch II element of EF-G was important for suppressing GTP hydrolysis in solution and critical for catalyzing translocation of the ribosome along mRNA. These experimental results reveal the multipurpose roles of an interdomain joint in the heart of an essential translation factor that can both promote and inhibit bacterial translation.

An Intramolecular Contact in Gα Transducin That Participates in Maintaining Its Intrinsic GDP Release Rate

Receptor mediated stimulation of the G protein-α subunit leads to exchange of GDP for GTP, activating the protein. Spontaneous GDP release from Gα can also lead to the active state, if GTP in solution binds the nucleotide binding pocket. The purpose of this study is to evaluate the molecular determinants for maintaining the spontaneous GDP release rates between two Gα subunits. Gαt has a low rate of nucleotide release, compared to Gαi1. Gαt/i1 chimeras were used to explore the molecular basis for this behavior. The C-terminal α4-helix, the N-terminal 56 residues and the Switch I/II regions of Gαt were shown to affect the low spontaneous GDP release rate in Gαt. A specific molecular contact between Asp26 and Asn191 was found in Gαt that is not present in Gαi1. In two chimeras disrupting this interaction produced an increased spontaneous GDP release; restoring the contact present in Gαt into these chimeras decreased the GDP release rate by half as compared to the original chimeras. Similarly, introduction of this contact in wild-type Gαi1 decreased the GDP release rate of Gαi1 by half. Differences in GDP release rates may reflect physiological roles these proteins play in living systems.

Inhibition of protein synthesis by didemnin B: how EF-1alpha mediates inhibition of translocation.

The antineoplastic cyclic depsipeptide didemnin B (DB) inhibits protein synthesis in cells and in vitro. The stage at which DB inhibits protein synthesis in cells is not known, although dehydrodidemnin B arrests translation at the stage of polypeptide elongation. Inhibition of protein synthesis by DB in vitro also occurs at the elongation stage, and it was shown previously that DB prevents EF-2-dependent translocation in partial reaction models of protein synthesis. This inhibition of translocation displays an absolute requirement for EF-1alpha; however, the dependence upon EF-1alpha was previously unexplained. It is shown here that DB binds only weakly to EF-1alpha/GTP in solution, but binds to ribosome. EF-1alpha complexes with a dissociation constant K(d) = 4 microM. Thus, the inhibition of protein synthesis by DB appears to involve an interaction with both EF-1alpha and ribosomes in which all three components are required. Using diphtheria toxin-mediated ADP-ribosylation to assay for EF-2, it is demonstrated that DB blocks EF-2 binding to pre-translocative ribosome.EF-1alpha complexes, thus preventing ribosomal translocation. Based on this model for protein synthesis inhibition by DB, and the proposed mechanism of action of fusidic acid, evidence is presented in support of the Grasmuk model for EF-1alpha function in which this elongation factor does not fully depart the ribosome during polypeptide elongation.

Formation of colloidal PMMA particles via free radical and group transfer dispersion polymerization of methylmethacrylate and methylmethacrylate/ethyleneglycoldimethacrylate

AbstractNon‐aqueous dispersions of colloidal PMMA microparticles with average sizes ranging from 70 to 330 nm were prepared by living polymerization of methylmethacrylate (MMA) and methylmethacrylate/ethyleneglycoldimethacrylate (EDMA) in n‐heptane diluent using 2‐methyl‐1‐methoxy‐1‐trimethylsilyloxy‐prop‐1‐ene (MTS) as initiator in the presence of tetrabutylammonium fluoride (TBAF) or tetrabutylammonium cyanide (TBAC) as catalyst, and polystyrene‐block‐poly‐(ethene‐alt‐propene) (SEP) as dispersing agent. The influence of process parameters such as concentrations of MMA, EDMA crosslinking agent, TBAF, and SEP on conversion, molecular weight, particle size, and molecular weight and particle size distributions has been investigated. In contrast to GTP in solution, group transfer dispersion polymerization (GTDP) was markedly slower and accompanied by agglomeration and drastic broadening of both molecular weight and particle size distributions with increasing MMA conversion. In a new GTDP process colloidal PMMA microparticles, obtained by free radical dispersion polymerization, were added as seeds, thus affording much narrower particle size distribution and high MMA conversion.

Theoretical problems related to the attachment of microtubules to kinetochores.

A possible model is analyzed for the maintenance of attachment of a shortening microtubule (MT) to a kinetochore. In this model it is assumed that a MT is inserted and held in a sleeve or channel of the outer layer of a kinetochore while subunits are lost from the MT tip through the central layer of the kinetochore. A second problem considered is the elementary bioenergetics of MT growth and shortening, as associated with the presence or absence of a GTP cap on the MT ends. The free-energy source is the hydrolysis of GTP in solution. The third problem discussed is the kinetics of capture of a centrosomal MT by a target (e.g., a kinetochore).

Kinetics and thermodynamics of the interaction of elongation factor Tu with elongation factor Ts, guanine nucleotides, and aminoacyl-tRNA.

The exchange of elongation factor Tu (EF-Tu)-bound GTP in the presence and absence of elongation factor Ts (EF-Ts) was monitored by equilibrium exchange kinetic procedures. The kinetics of the exchange reaction were found to be consistent with the formation of a ternary complex EF-Tu X GTP X EF-Ts. The equilibrium association constants of EF-Ts to the EF-Tu X GTP complex and of GTP to EF-Tu X EF-Ts were calculated to be 7 X 10(7) and 2 X 10(6) M-1, respectively. The dissociation rate constant of GTP from the ternary complex was found to be 13 s-1. This is 500 times larger than the GTP dissociation rate constant from the EF-Tu X GTP complex (2.5 X 10(-2) s-1). A procedure based on the observation that EF-Tu X GTP protects the aminoacyl-tRNA molecule from phosphodiesterase I-catalyzed hydrolysis was used to study the interactions of EF-Tu X GTP with Val-tRNAVal and Phe-tRNAPhe. Binding constants of Phe-tRNAPhe and Val-tRNAVal to EF-Tu X GTP of 4.8 X 10(7) and 1.2 X 10(7)M-1, respectively, were obtained. The exchange of bound GDP with GTP in solution in the presence of EF-Ts was also examined. The kinetics of the reaction were found to be consistent with a rapid equilibrium mechanism. It was observed that the exchange of bound GDP with free GTP in the presence of a large excess of the latter was accelerated by the addition of aminoacyl-tRNA. On the basis of these observations, a complete mechanism to explain the interactions among EF-Tu, EF-Ts, guanine nucleotides, and aminoacyl-tRNA has been developed.

PROPERTIES OF THE PROTEIN SUBUNIT OF CENTRAL-PAIR AND OUTER-DOUBLET MICROTUBULES OF SEA URCHIN FLAGELLA

The subunit protein has been isolated from the central-pair and outer-doublet microtubules of sea urchin sperm tails. Both proteins have a sedimentation constant of 6S and a molecular weight of 120,000. Both are converted to a 60,000 molecular weight species by denaturation in 6 M guanidine hydrochloride and reduction with mercaptoethanol. The reduced-alkylated proteins have the same R(f) on disc electrophoresis, and the same amino acid composition, which is very similar to that of muscle actin. The central-pair protein has one binding site for colchicine per 120,000 g. Both proteins appear to have a guanine nucleotide binding site, but the ability to bind GTP in solution has been demonstrated only for the central-pair protein. Although 1 mole of guanine nucleotide is bound per 60,000 g to outer-doublet tubules, the protein obtained by dissolving the doublets at pH 10.5 has lost the guanine nucleotide-binding site and also shows little or no colchicine-binding activity. Comparison of the properties of the isolated protein with electron microscopic evidence on structure of microtubules suggests that the chemical subunit (M = 120,000) consists of two of the 40 A morphological subunits.