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R Cooke

Publications and source records attributed to R Cooke.

At least 91 records · Page 5Linked to original sources
PubMed1995

Muscle cross-bridges bound to actin are disordered in the presence of 2,3-butanedione monoxime.

Electron paramagnetic resonance spectroscopy was used to monitor the orientation of muscle cross-bridges attached to actin in a low force and high stiffness state that may occur before force generation in the actomyosin cycle of interactions. 2,3-butanedione monoxime (BDM) has been shown to act as an uncompetitive inhibitor of the myosin ATPase that stabilizes a myosin.ADP.P(i) complex. Such a complex is thought to attach to actin at the beginning of the powerstroke. Addition of 25 mM BDM decreases tension by 90%, although stiffness remains high, 40-50% of control, showing that cross-bridges are attached to actin but generate little or no force. Active cross-bridge orientation was monitored via electron paramagnetic resonance spectroscopy of a maleimide spin probe rigidly attached to cys-707 (SH-1) on the myosin head. A new labeling procedure was used that showed improved specificity of labeling. In 25 mM BDM, the probes have an almost isotropic angular distribution, indicating that cross-bridges are highly disordered. We conclude that in the pre-powerstroke state stabilized by BDM, cross-bridges are attached to actin, generating little force, with a large portion of the catalytic domain of the myosin heads disordered.

Actins↗
PubMed1995

Nucleotide turnover rate measured in fully relaxed rabbit skeletal muscle myofibrils.

Steady state measurements of the ATP turnover rate of myosin crossbridges in relaxed living mammalian muscle or in in vitro systems are complicated by other more rapid ATPase activities. To surmount these problems we have developed a technique to measure the nucleotide turnover rate of fully relaxed myosin heads in myofibrils using a fluorescent analogue of ATP (mant-ATP). Rabbit myofibrils, relaxed in 1.6 mM ATP, were rapidly mixed with an equal volume of solution containing 80 microM mant-ATP and injected into a fluorimeter. As bound ADP is released, a fraction of the myosin active sites bind mant-ATP and fluorescence emission rises exponentially, defining a rate of nucleotide turnover of 0.03 +/- 0.001 s-1 at 25 degrees C (n = 17). This rate was approximately equal to one half that of purified myosin. The turnover rates for myosin and myofibrils increased between 5 degrees and 42 degrees C, reaching 0.16 +/- 0.04 s-1 and 0.06 +/- 0.005 s-1, respectively, at 39 degrees C, the body temperature of the rabbit. If the rate observed for purified myosin occurred in vivo, it would generate more heat than is observed for resting living muscle. When myosin is incorporated into the myofilament lattice, its ATPase activity is inhibited, providing at least a partial explanation for the low rate of heat production by living resting muscle.

Adenosine Triphosphate↗
PubMed1995

Reduced mtDNA diversity in the Ngöbé Amerinds of Panamá.

Mitochondrial DNA (mtDNA) haplotype diversity was determined for 46 Ngöbé Amerinds sampled widely across their geographic range in western Panamá. The Ngöbé data were compared with mtDNA control region I sequences from two additional Amerind groups located at the northern and southern extremes of Amerind distribution, the Nuu-Chah-Nulth of the Pacific Northwest and the Chilean Mapuche and from one Na-Dene group, the Haida of the Pacific Northwest. The Ngöbé exhibit the lowest mtDNA control region sequence diversity yet reported for an Amerind group. Moreover, they carry only two of the four Amerind founding lineages first described by Wallace and coworkers. We posit that the Ngöbé passed through a population bottleneck caused by ethnogenesis from a small founding population and/or European conquest and colonization. Dating of the Ngöbé population expansion using the Harpending et al. approach to the analysis of pairwise genetic differences indicates a Ngöbé expansion at roughly 6800 years before present (range: 1850-14,000 years before present), a date more consistent with a bottleneck at Chibcha ethnogenesis than a conquest-based event.

Archaeology↗
PubMed1995

The actomyosin engine.

Two recent advances have extended our knowledge of the actomyosin interaction considerably. The first of these is the determination of the crystal structures of the actin monomer and of the myosin head. The structures of these proteins were fit into the quaternary protein complexes of the actin filament, and of the actin filament decorated by the myosin head, using data from fiber diffraction or electron microscopy as constraints. The atomic models of these protein complexes have led to new hypotheses concerning the large conformational changes that must occur in these proteins in order to generate force. The second advance has been the measurements of the forces and displacements of single motor proteins, in particular, of a single myosin molecule interacting with a single actin filament. These data lead to more unambiguous interpretations than did previous mechanical data, which were obtained using ensembles of motors that worked asynchronously on their filaments. This approach brings to the motor field the equivalent of what patch clamp techniques brought to the study of membrane channels: the ability to look at the function of a single molecule.

Actomyosin↗
PubMed1995

The conformation of the active site of myosin probed using mant-nucleotides.

Changes in the conformation of the active site of myosin subfragment-1 (S1) may be linked to the production of force during the powerstroke. We probed the conformation of the nucleotide pocket by measuring the solvent accessibility of bound mant-nucleotides. Solvent accessibility was determined by measuring the quenching of fluorescence produced by the solvent phase quencher acrylamide. The fluorescent mant moiety is attached to the ribose and is located near the outside of the pocket where it is likely to be sensitive to opening of the pocket. MantADP was highly protected from the quencher when bound to the active site of S1. A similar degree of protection was also observed for mantATP during steady-state hydrolysis by S1, and for mantADP bound to acto-S1 or to myosin in myofibrils. Assuming that S1-mantATP and actoS1-mantADP represent states at the beginning and the end of the powerstroke, respectively, we conclude that the myosin nucleotide pocket does not undergo a large conformational change during the powerstroke. However, the high degree of protection seen for mant-nucleotides is not easily explained by the open structure of the nucleotide pocket in the S1-nucleotide complex observed by x-ray diffraction.

Acrylamide↗
PubMed1994

Quenching of fluorescent nucleotides bound to myosin: a probe of the active-site conformation.

The conformation of the active ATPase site of myosin subfragment 1 (S1) and actomyosin in myofibrils was probed by measuring the solvent accessibility of the bound ethenonucleotides epsilon ADP and epsilon ATP (during steady-state hydrolysis). Solvent accessibility was determined by measuring the quenching of fluorescence produced by the solvent-phase quencher acrylamide, 25-400 mM. The fraction of the nucleotides that were specifically bound to the active site was determined following sedimentation in the presence and absence of 5 mM ADP. In agreement with previous investigations, both epsilon ATP and epsilon ADP were almost completely protected from the quencher when bound to the active site of myosin. The solvent accessibility of both epsilon ADP and epsilon ATP varied with both temperature and ionic strength. The nucleotides became more accessible at higher temperatures and higher ionic strength. At 1 M KCl the quenching curve was biphasic, indicating that the nucleotide pocket of myosin can exist in both a closed form that allows little quenching and a more open form that allows considerable quenching. However, the transition between forms was not strongly coupled to the state of the nucleotide, with a similar protection observed for both epsilon ADP and for epsilon ATP during steady-state cycling. epsilon ADP bound to acto-S1 or to actomyosin in myofibrils displayed the same degree of protection as seen with S1 alone. A similar result is obtained during steady-state hydrolysis. Thus nucleotides in the myosin pocket do not become more accessible to the solvent when myosin binds to actin in either rigor-ADP or active complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylamide↗
PubMed1994

X-ray crystallographic studies of a series of penicillin-derived asymmetric inhibitors of HIV-1 protease.

In the development of a treatment for AIDS, the HIV-1 protease has been identified as a good target enzyme for inhibitor design. We previously reported a series of dimeric penicillin-derived C2-symmetric HIV-1 protease inhibitors [Humber, D., et al. (1993) J. Med. Chem. 36, 3120-3128]. In an attempt to reduce the size and optimize the binding of these C2-symmetric inhibitors, molecular modeling studies led to a novel series of monomeric penicillin-derived inhibitors of HIV-1 protease. The binding modes of these monomeric inhibitors have been characterized by X-ray crystallographic and NMR studies. Crystal structures of HIV-1 protease complexed to three inhibitors (GR123976, GR126045, and GR137615) from this series identify the molecular details of the interactions. The binding of GR123976 (IC50 = 2.3 microM) exhibits good hydrophobic contacts but few electrostatic interactions. A strategy of structure-based design and chemical synthesis led to the elaboration of GR123976 to optimize interactions with the protein. Crystallographic analysis of HIV-1 protease complexed to GR126045 and GR137615 identified these interactions with the catalytic aspartates and the protein binding pockets. The crystal structures of the three complexes confirm the presence of the major interactions modeled in order to optimize potency and reveal details of the molecular recognition by HIV-1 protease of this novel series of nonpeptidic inhibitors.

Amino Acid Sequence↗
PubMed1994

Mobility and orientation of spin probes attached to nucleotides incorporated into actin.

Each actin molecule contains a nucleotide, tightly bound in a deep cleft that divides the molecule. To probe conformational changes within this region of the molecule, we have incorporated two spin label analogues of ATP into actin. In both analogs the spin label was attached to the 6 position on the adenine ring, either directly (6nSLATP) or via a longer thioacetamido linker (6sSLATP). Electron paramagnetic resonance spectra of randomly oriented actin filaments showed that both the probes possessed considerable rotational mobility relative to the protein surface. The 6nSLADP has two degrees of rotational mobility that can be approximately modeled by rapid diffusion within cones with half angles of 30 +/- 1 degrees and 42 +/- 1.5 degrees. The 6sSLADP displayed one degree of rotational mobility approximated by rapid motion within a cone with a half-angle of 38 +/- 1 degrees. The rotational mobility of the probes is determined by the protein structure surrounding them, and changes in this structure should alter the mobility. The mobility of the probes was unchanged by addition of 20 mM Pi, which forms an ADP-Pi complex. However, binding of myosin heads (S1) shifted the population of 6nSLADP toward the more highly restricted cone, while binding of DNase-I shifted it toward the less restricted cone. We conclude that this region of actin is unchanged by binding of phosphate, while the binding of S1 or DNase-I produces only a modest shift in conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1994

A C-terminal conformational equilibrium in thymidylate synthase observed by electron paramagnetic resonance spectroscopy.

A spin-label was attached to the C-terminal side chain of Lactobacillus casei thymidylate synthase (TS, EC2.1.1.45), and EPR spectroscopy was used to study the change in conformational equilibrium that occurs when the enzyme binds nucleotides or the methylenetetrahydrofolate analog CB3717. The C244T/V316C mutant TS has only two cysteines, the active site Cys-198 and an engineered cysteine which replaces valine as the C-terminal residue. dUMP was used to block the active-site cysteine while the C-terminus was reacted with the spin-label 4-maleimido-2,2,6,6- tetramethylpiperidinyl-1-oxy. Exclusive attachment of the label to the C-terminal cysteine was verified by a study of the labeled enzyme's reaction with 5,5'-dithiobis(2-nitrobenzoic acid). EPR spectra of the labeled enzyme and its complexes were composed of two components corresponding to populations of both flexible and more immobilized forms of the C-terminus (tau C = 1 and 9.7 ns, respectively). Ligand binding increased the population of the more immobilized form of the C-terminus with the following series: free enzyme < E.dUMP approximately dTMP approximately E.FdUMP < E.CB3717 < E.dUMP.CB3717. Ligand-induced perturbation of the conformational equilibrium was titratable and indicated approximate Kd values of 3 and 13 microM for formation of the E.dUMP and E.CB3717 binary complexes, respectively, and 7 microM for the binding of CB3717 to the E.dUMP complex. Immobilization of the spin-label correlated well with crystallographic B-factors of the C-terminal residue in corresponding TS crystal structures. These results show that TS has two major conformations which are in equilibrium, and the position of the equilibrium changes in the presence of ligands.

Amino Acid Sequence↗
PubMed1994

The orientation of spin-probes attached to Cys374 on actin in oriented gels.

Changes in protein orientation are thought to be involved in the generation of force that occurs in active muscle. To address the possibility that such changes occur in the actin filament, a spin label was attached to Cys374 of actin and its orientation was measured for filaments that were aligned in capillaries. In the best aligned samples the label was found in two populations, one with a narrow distribution of angles centered at an average angle of 36 (+/- 4) degrees, and a second in a broad distribution centered at an average angle of 45(+/- 4) degrees. The birefringence of the samples was high and X-ray diffraction patterns showed that the actin filaments were well aligned with average angles similar to the population of the better aligned spin labels, suggesting that the more disordered population of probes arose from either disorder of the probe relative to the protein, or from disorder of a local portion of the protein. When myosin subfragment-1 (S-1) was also included with the actin, molar ratio 1 S-1 to 4 actins, there was little change in the angular distribution of either of the two probe populations. We conclude that the binding of the myosin head does not produce appreciable alterations in the orientation of this domain.

Actins↗
PubMed1994

A model of the release of myosin heads from actin in rapidly contracting muscle fibers.

We describe a model that relates the maximum shortening velocity of a muscle fiber, Vm, to the kinetics of the dissociation of a myosin head from actin. At Vm, the positive work exerted by cross-bridges attached in the powerstroke must be balanced by cross-bridges that have been carried by movement of the filaments into a region where they exert a negative force. This balance allows one to relate Vm and the rate of cross-bridge detachment. Studies of actomyosin kinetics suggest that at high substrate, detachment should be limited by a slow protein isomerization (approximately 50 s-1) that precedes ADP release. This rate is too slow to be easily accommodated in existing models. However, a slow rate for cross-bridge dissociation, similar to that of the isomerization, is predicted if previous models are modified to include rapid detachment of cross-bridges that have been carried so far into the negative force region that their free energy exceeds that of the detached state. The model also explains another aspect of muscle contraction: at high shortening velocities, the observed rate of ATP hydrolysis is low, because a cross-bridge can interact with multiple actin binding sites before releasing the hydrolysis products and binding another ATP.

Actins↗
PubMed1994

Temperature dependence of the inhibitory effects of orthovanadate on shortening velocity in fast skeletal muscle.

We have investigated the effects of the orthophosphate (P(i)) analog orthovanadate (Vi) on maximum shortening velocity (Vmax) in activated, chemically skinned, vertebrate skeletal muscle fibers. Using new "temperature-jump" protocols, reproducible data can be obtained from activated fibers at high temperatures, and we have examined the effect of increased [Vi] on Vmax for temperatures in the range 5-30 degrees C. We find that for temperatures < or = 20 degrees C, increasing [Vi] inhibits Vmax; for temperatures > or = 25 degrees C, increasing [Vi] does not inhibit Vmax. Attached cross-bridges bound to Vi are thought to be an analog of the weakly bound actin-myosin.ADP-P(i) state. The data suggest that the weakly bound Vi state can inhibit velocity at low temperature, but not at high temperature, with the transition occurring over a narrow temperature range of < 5 degrees C. This suggests a highly cooperative interaction. The data also define a Q10 for Vmax of 2.1 for chemically skinned rabbit psoas fibers over the temperature range of 5-30 degrees C.

Actins↗
PubMed1994

Radioimmunoassay of cytidine 3',5'-cyclic monophosphate: unambiguous assay by means of an optimized protocol incorporating a trilayer column separation to obviate cross-reactivity problems.

Previous assays for cytidine 3', 5'-cyclic monophosphate (cyclic CMP) have been criticized as being ambiguous. Here a modified RIA protocol, in which the production of assay components has been optimized and a novel trilayer chromatography column separation introduced which successfully separates cyclic CMP from compounds, endogenous to living tissues, which cross-react with anti-cyclic CMP sera, is described. The assay is capable of assaying cyclic CMP between 0.1 and 5 pmol, can be increased in sensitivity by means of an additional acetylation step, and enables the separation of cyclic CMP, cyclic AMP and cyclic GMP so that all three can be estimated in a single sample.

Animals↗
PubMed1994

Effect of series elasticity on delay in development of tension relative to stiffness during muscle activation.

Experimental data have indicated that during activation, the attachment of myosin to actin, measured by mechanical stiffness, precedes tension generation by 10-30 ms. Using computer simulation, we have investigated the effect of a series elastic element on the lag between stiffness and tension development during muscle activation. Two versions of the two-state cross-bridge model originally proposed by Huxley and a three-state model were considered. After simulated activation, stiffness and tension increased with rates that were strongly dependent on the series elastic strain. In the absence of a series elastic element, the rise in stiffness preceded, lagged, or was coincident with the increase in tension, depending on the model. For large elastic strains, tension lagged stiffness for all models. Lags of 10-30 ms could be obtained with elastic strains of 0.3-1% of the muscle length. This is a realistic value in experiments without sarcomere length servocontrol, suggesting that series elasticity may be an important contributor to the experimentally observed lag between tension and stiffness.

Animals↗
PubMed1993

A series of penicillin-derived C2-symmetric inhibitors of HIV-1 proteinase: structural and modeling studies.

The binding modes of a series of penicillin-derived C2 symmetric dimer inhibitors of HIV-1 proteinase were investigated by NMR, protein crystallography, and molecular modeling. The compounds were found to bind in a symmetrical fashion, tracing and S-shaped course through the active site, with good hydrophobic interactions in the S1/S1' and S2/S2' pockets and hydrogen bonding of inhibitor amide groups. Interactions with the catalytic aspartates appeared poor and the protein conformation was very similar to that seen in complexes with peptidomimetics, in spite of the major differences in ligand structure.

Amino Acid Sequence↗