Biomedical subjects

Roger Cooke

Publications and source records attributed to Roger Cooke.

5 recordsLinked to original sources

PubMed2002

Holding two heads together: stability of the myosin II rod measured by resonance energy transfer between the heads.

Myosin, similar to many molecular motors, is a two-headed dimer held together by a coiled-coiled rod. The stability of the coiled coil has implications for head-head interactions, force generation, and possibly regulation. Here we used two different resonance energy transfer techniques to measure the distances between probes placed in the regulatory light chain of each head of a skeletal heavy meromyosin, near the head-rod junction (positions 2, 73, and 94). Our results indicate that the rod largely does not uncoil when myosin is free in solution, and at least beyond the first heptad, the subfragment 2 rod remains relatively intact even under the relatively large strain of two-headed myosin (rigor) binding to actin. We infer that uncoiling of the rod likely does not play a role in myosin II motility. To keep the head-rod junction intact, a distortion must occur within the myosin heads. This distortion may lead to different orientations of the light-chain domains within the myosin dimer when both heads are attached to actin, which would explain previously puzzling observations and require reinterpretation of others. In addition, by comparing resonance energy transfer techniques sensitive to different dynamical time scales, we find that the N terminus of the regulatory light chain is highly flexible, with possible implications for regulation. An intact rod may be a general property of molecular motors, because a similar conclusion has been reached recently for kinesin, although whether the rod remains intact will depend on the relative stiffness of the coiled coil and the head in different motors.

Actins↗

PubMed2002

A novel restricted photoaffinity spin-labeled non-nucleoside ATP analogue as a covalently attached reporter group of the active site of Myosin subfragment 1.

The photoaffinity spin-labeled non-nucleoside ATP analogue, 2-(4-azido-2-nitrophenyl)amino-2,2-(1-oxyl-2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene)ethyl triphosphate (SSL-NANTP), has been shown to be a substrate for skeletal mysoin subfragment 1 (S1) that can be photoincorporated at the active site of S1 [Chen, X., et al. (2000) Bioconjugate Chem. 11, 725-733]. Electron paramagnetic resonance spectroscopy shows that the probe undergoes restricted motion with respect to the protein. The parent compound, NANTP (2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate), is specifically photoincorporated at Trp-130 on the amino-terminal 23 kDa tryptic fragment in rabbit skeletal myosin. Surprisingly, amino acid sequence analysis shows that SSL-NANTP is photoincorporated on the carboxy-terminal 20 kDa tryptic fragment at Lys-681 on the side opposite Trp-130 in the nucleotide pocket. This is the first direct evidence showing that this residue in the 20 kDa tryptic fragment is close enough to the active site to be photolabeled by trapped ATP analogues. After actin treatment in the presence of MgATP, SSL-NANDP-labeled myosin S1 had normal ATPase activity, indicating that photolabeling did not significantly alter the enzymatic properties of S1. Photoincorporated SSL-NANDP was bound inside the nucleotide site of S1, with an effective concentration of 20 mM as judged by the concentration of MgADP needed to displace it. Molecular dynamics simulations suggest that the ability of NANTP and SSL-NANTP to photolabel different sites results from different orientations of the phenyl ring in the active site. For SSL-NANTP, the p-azido group on the phenyl ring points toward Lys-681. For NANTP, it points in the opposite direction toward Trp-130.

Adenosine Triphosphate↗

PubMed2002

A classical and ab initio study of the interaction of the myosin triphosphate binding domain with ATP.

We used classical molecular mechanics (MM) simulations and quantum mechanical (QM) structural relaxations to examine the active site of myosin when bound to ATP. Two conformations of myosin have been determined by x-ray crystallography. In one, there is no direct interaction between switch 2 and the nucleotide (open state). In the other (closed state), the universally conserved switch 2 glycine forms a hydrogen bond with a gamma-phosphate oxygen. MM simulations indicate that the two states are thermodynamically stable and allow us to investigate the extent to which the P-loop, switch 1, and switch 2 are involved in hydrolysis. We find that the open structure has a higher affinity for ATP than the closed structure, and that ATP is distorted toward a transition state by interactions with the protein. We also examine how the structure of the binding site changes with either MgATP or CaATP as the nucleotide in myosin in the open conformer. Our analyses suggest that higher CaATPase rates occur because the leaving phosphate (P(i)) group is more weakly bound and dissociation occurs faster. Finally, we validate the use of a particular formulation of a QM methodology (Car-Parrinello) to further refine the structures of the active site.

Adenosine Triphosphate↗

PubMed2000

Electron paramagnetic resonance: a new techniquein skin research.

BACKGROUND/AIM: Electron paramagnetic resonance (EPR) spectra of nitroxide spin probes have been used for studying biological membranes and chemical-membrane interaction. We have investigated the influence of surfactants on the intercellular lipid structure of cadaver stratum corneum and the possibility of EPR spectral measurements on the stripped stratum corneum utilizing cyanoacrylate resin, which might reflect the actual skin lipid conditions. CONCLUSION: EPR spectra are useful in evaluating the fluidity measurement of stratum corneum of cadaver skin and stripped stratum corneum.

Journal Article↗

PubMed2000

Electron paramagnetic resonance study utilizing stripping method on normal human stratum corneum.

BACKGROUND/AIM: Electron paramagnetic resonance (EPR) spectra of nitroxide spin probes are useful for studying biological membranes, and chemical-membrane interaction. The objective of this study was to assess the fluidity of stripped stratum corneum (SC) of normal human skin with EPR. METHODS: We quantified the fluidity of stripped SC (S-SC) by a single stripping with cyanoacrylate onto quartz glass, cover glass or transparent film. Spectra were compared with those of the SC of cadaver skin (C-SC). RESULTS: All S-SC spectra were similar to those of C-SC. There were no statistical differences in the order parameters S between C-SC and those on quartz cell and cover glass, but those of transparent film were significantly higher. Spectra after a month storage at room temperature at 5 degrees C and at -20 degrees C were unchanged. The order parameter S had decreased after a 3-month storage in all conditions. CONCLUSION: This method is useful for the evaluation of the fluidity measurement utilizing EPR spectra of S-SC.

Journal Article↗