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J Trinick

Publications and source records attributed to J Trinick.

49 records · Page 3Linked to original sources
PubMed1986

Electron microscope study of the effect of temperature on the length of the tail of the myosin molecule.

The effect of temperature on the length of the tail of the myosin molecule has been studied by negative staining of molecules immobilized on carbon substrates at different temperatures. In buffers containing chloride as the principal anion, tail length was approximately constant up to 25 degrees C. Above this temperature, it shortened linearly with increasing temperature up to 42 degrees C, the highest temperature studied in this solvent. The amount of shortening per degree C was about 1.2 nm. A similar amount of shortening per degree C was seen in acetate-containing buffers up to 50 degrees C, but in this case it did not begin until the temperature exceeded about 40 degrees C. A large fraction of the observed shortening was localized in a region that lies roughly between the two positions in the tail where proteolysis results in production of short or long subfragment-2. Frequently, the tail had a different appearance in this region from elsewhere and could sometimes be seen to split into two strands that were separate but coiled around one another.

Macromolecular Substances↗
PubMed1986

Cryo-electron microscopy and three-dimensional reconstruction of actin filaments.

Actin filaments have been examined by electron microscopy whilst in a frozen-hydrated state. Filaments embedded in a vitreous water layer are basically similar to those prepared by negative staining and show characteristic helical substructure, where the pitches of the helices are about 70 nm and 6 nm. Variability in spacing between long pitch helix cross-over points has been observed, which is consistent with intrinsic angular disorder between successive filament subunits. Fourier transforms of the most ordered filaments show four strong layer lines that index as the first, fifth, sixth and seventh orders of a 35 nm repeat. A three-dimensional helical reconstruction, calculated to a resolution of about 4 nm, shows the individual subunits to be orientated with their long axes roughly perpendicular to the filament axis. Each subunit is somewhat curved and is resolved into two domains. Most connections between successive subunits appear to be made close to the filament axis. We also report on the performance of the specimen holder (Philips PW 5699) used in this work.

Actins↗
PubMed1985

Negative staining of myosin molecules.

A reproducible method has been developed for the negative staining of myosin molecules. The dimensions of stained molecules are in close agreement with those obtained by metal shadowing. Sharp bends in the tail, indicative of hinge regions, were observed at two positions 44 nm and 76 nm from the head-tail junction. The tail was often ill-defined at the position of the first (44 nm) bend. The bend positions may be sites of proteolytic cleavage that result in the production of long and short myosin subfragment S2. About half the molecules exhibited bending to various degrees at one or both of these positions, but cases where the tail folded back on itself in a 180 degrees bend were comparatively rare (approximately equal to 10%). However, in the absence of EGTA, a large fraction of the molecules (approximately equal to 80%) exhibited 180 degrees bends. A small region, approximately 20 nm long, at the tip of the tail often appears to be significantly different from the rest. The heads are about 19 nm long and roughly pear-shaped. Although sometimes straight, more often they show a pronounced curvature. Both senses of curvature were observed, but those curved in a clockwise manner were the most common, indicating preferential binding of one side of the head to the carbon substrate. An analysis of the different combinations of head shapes in individual molecules indicates that each head can rotate independently around its long axis. No preferred angle of orientation between the two heads in a molecule, or between either head and the tail could be found. Substructure has been observed within the heads.

Animals↗
PubMed1984

Purification and properties of native titin.

A procedure has been developed for the extraction and purification of the massive myofibrillar protein titin without exposing it to denaturing conditions. The form of the molecule that has been isolated is soluble at high ionic strength and alkaline pH, but precipitates in low salt or at pH values below 7. Sedimentation velocity experiments indicate that titin is a highly asymmetric molecule with a sedimentation coefficient of 13.4 S. This asymmetry is confirmed by electron microscopy of rotary-shadowed specimens, which shows string-like structures of diameter 40 A and lengths up to 8000 A. Significant differences were observed depending on whether the electron microscope specimens were prepared by spraying or by layering of the titin onto a mica substrate; we tentatively attribute these differences to elasticity in the titin, revealed by the high shearing forces that accompany spraying. In accord with this, the circular dichroism spectrum of titin indicates that its secondary structure is largely random coil, a conformation characteristic of elastic proteins such as elastin. Negative staining of titin again shows long string-like structures, but these can now be seen to have an appearance similar to a string of beads, where the spacing between successive beads is about 40 A. Very similar beaded strings have been observed also associated with negatively stained separated native thick filaments; these are found running alongside the cross-bridge regions and in coils near the filament ends. Since the periodicity of the strings is similar to that of end-filaments, recently identified structures at the tips of thick filaments, it is likely that end-filaments are formed from titin. Titin comprises approximately 9% of the myofibrillar mass, which means that it is the third most abundant protein in muscle. The possible role of titin in forming elastic filaments within myofibrils is discussed.

Animals↗
PubMed1984

Structure of the myosin projections on native thick filaments from vertebrate skeletal muscle.

Rabbit psoas muscle filaments, isolated in relaxing buffer from non-glycerinated muscle, have been applied to hydrophilic carbon films and stained with uranyl acetate. Electron micrographs were obtained under low-dose conditions to minimize specimen damage. Surrounding the filament backbone, except in the bare zone, is a fringe of clearly identifiable myosin heads. Frequently, both heads of individual myosin molecules are seen, and sometimes a section of the tail can be seen connecting the heads to the backbone. About half the expected number of heads can be counted, and they are uniformly distributed along the filament. The majority of heads appear curved. The remainder could be curved heads viewed from another aspect. Three times as many heads curve in a clockwise sense than in an anticlockwise sense, suggesting a preferential binding of one side of the head to the carbon film. The two heads of myosin molecules exhibit all the possible combinations of clockwise, anticlockwise and straight heads, and analysis of their relative frequencies suggests that the heads rotate freely and independently. The heads also adopt a wide range of angles of attachment to the tail. The lengths of heads cover a range of 14 to 26 nm, with a peak at 19 nm. The average maximum width is 6.5 nm. Both measurements are in excellent agreement with values for shadowed molecules. Since our data are from heads adsorbed to the film in relaxing conditions and the shadowed molecules were free of nucleotide, gross shape changes are not likely to be produced by nucleotide binding. The length of the link between the heads and the backbone was found to vary between 10 nm and 52 nm, with a broad peak at about 25 nm. Thus, the hinge point detected in the tail of isolated molecules was not usually the point from which the crossbridges swung out from the filament surface. The angle made by the link to the filament axis was between 20 degrees and 80 degrees, with a broad maximum around 45 degrees. These lengths and angles concur with our observation of an average limit of the crossbridges from the filament surface of 30 nm. This is sufficient to enable heads in the myofibril lattice to reach out beyond the nearest thin filament and should allow considerable flexibility for stereospecific binding to actin in active muscle.

Animals↗
PubMed1984

Binding and location of AMP deaminase in rabbit psoas muscle myofibrils.

It is shown that an interaction exists between AMP deaminase (EC 3.5.4.6) and myofibrils that is sufficiently strong (Kd congruent to 10(-10) M) for more than 99% of the binding sites for the enzyme to be filled in vivo. The binding is not strong enough, however, to stop removal of the enzyme during the extensive washing normally used in the preparation of myofibrils. Fluorescent antibodies to the enzyme label myofibrils close to the junction of the A- and I-bands. The invariance of the position of the antibody stripes at this site, over a range of sarcomere lengths, indicates that the enzyme is attached to the A-band. The intensity of the fluorescence declines in parallel with dissociation of the enzyme. In this muscle, the number of AMP deaminase binding sites per thick filament is approximately six, suggesting that the enzyme is located at a single axial position in each half A-band. Electron microscopy of negatively stained, antibody-labelled myofibrils reveals the distance between the AMP deaminase sites at opposite ends of an A-band to be 1.69(+/- 0.02 micron). Since the length of the A-band is 1.57 micron, the binding site for the enzyme must be significantly beyond where thick filaments have previously been thought to end.

AMP Deaminase↗
PubMed1982

Effect of substrate on freeze-dried and shadowed protein structures.

Muscle thick filaments have been prepared for electron microscopy by a method involving freeze-drying and shadowing. The same method applied to filaments on three different substrates gives rise to quite different appearances. The filaments on one of the substrates, hydrophilic carbon, show new detail not previously observed.

Aluminum Silicates↗
PubMedDate not supplied

A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray.

We describe here a second generation apparatus for studying transient reaction conformations in macromolecules and their complexes by electron cryo-microscopy. Reactions are trapped by rapid freezing in times ranging from a few milliseconds to tens of seconds after initiation. Blotting of the electron microscope grid and freezing it in liquid ethane uses computer controlled microstepping motors. For the fastest time resolution, a blotted grid containing a thin film of one reactant is sprayed with small droplets containing a second reactant just before freezing. The spray is produced electrically (electrospray), which gives a dense cloud of droplets <1 microm in diameter from the 1-2 microl of solution required per grid. A second method in which two solutions are first mixed by turbulent flow and then blotted prior to freezing is used for reactions with time courses >1s.

Biophysical Phenomena↗