Biomedical subjects
Publications and source records attributed to K C Holmes.
Present evidence on crossbridge behaviour is reviewed from the viewpoint of the Eisenberg-Hill concepts. We conclude that a cartoon illustrating the operation of these ideas in practice should be drawn so as to include morphological changes in the body of the myosin molecule as part of the power-stroke. The bulk of structural evidence on the operation of crossbridges in intact muscle supports such a description.
The Moessbauer method was applied to obtain information on a suggested hydrogen bond in tobacco mosaic virus (TMV), between the hydroxyl group of Tyr 139 and a carboxyl oxygen of Glu 22 in a neighbouring subunit. Spectra of 129I were taken of 3,5-di-iodo-L-tyrosine as a free amino acid and in situ in TMV. The increase of the pK value of 3,5-di-iodo-L-tyrosine by 0.8 units at position 139 in TMV compared to the free value is a strong argument in favour of the existence of a hydrogen bond via the relevant hydroxyl group. The reported study demonstrates the surprising sensitivity of the observable Moessbauer parameters to details of the electronic configuration in the neighbourhood of the probe nucleus.
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The binding of rabbit muscle myosin subfragment 1 (S1) to glycerinated insect flight muscle fibers has been studied by low-angle x-ray diffraction, quantitative sodium dodecyl sulfate gel electrophoresis, quantitative interference microscopy, and electron microscopy. Changes induced in the rigor x-ray diffraction pattern are consistent with the idea that vacant myosin-binding sites on thin filaments are filled by exogenous S1. Electron microscopy indicates that S1 permeates and labels fibers and fibrils completely. Electron micrographs also show that cross-bridges are not displaced by exogenous S1 under the conditions used, and this is supported by the unchanged mechanical stiffness of the S1-labeled fibers. The amount of bound S1, as measured by gel electrophoresis and interference microscopy, together with the magnitude of the intensity changes in the x-ray diffraction pattern, is consistent with a thick filament structure that contains four molecules of endogenous myosin per 14.5 nm of its length, but does not agree well with earlier estimates of six myosins per crown. Lack of information on possible inhibition of S1-binding by factors other than the presence of cross-bridges, e.g., troponin, render uncertain calculations of the number of attached cross-bridges in the rigor state. However, it appears that at least 75% of the endogenous myosin heads are attached. Occupancy of binding sites on thin filaments after incubation with S1 is high, probably greater than 85%, so that x-ray scattering from those parts of the structure that adhere to the symmetry of the thin filaments can be treated as diffraction from S1-decorated thin filaments. In addition, we show in thin flared X cross sections that exo-S1 heads bind to actin with the geometry described in decorated actin by Taylor, K.A., and L.A. Amos.
Evidence from sequence studies and from proteolysis suggests that S1 consists of three domains. Cross-linking studies show that one S1 can bind to two actin monomers which may lie in different strands of the actin long helix. The S1-actin interaction comprises two states "weak" and "strong". We suggest there are distinct hinged binding sites, "weak" and "rigor", of which only the rigor site is sensitive to tropomyosin control. If one takes the weak binding domain to be a "nose-cone" which is attached to the rest of the S1 by a flexible covalent hinge allowing the rigor link to be formed independently a number of structural phenomena observed in fibres may be explained.
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Tobacco mosaic virus (TMV) particles are rod-like, 300 nm long and 18 nm in diameter. TMV consists of 2140 protein subunits, each with a relative molecular mass of 17420 (158 residues), arranged on a helix of pitch 2.3 nm with 16 1/3 subunits per turn. Winding through this helix is a single strand of RNA 6400 nucleotides long. Three bases are bound to each protein subunit. TMV has a central hole of diameter 4.0 nm. Assembly of TMV occurs by the threading of the RNA through the central hole of the growing rodlet of viral coat protein and involves a preassembled double disk as intermediate. Given the structure of the subunit, such a mechanism requires that the segment of polypeptide chain which separates the nucleic acid binding site from the lumen of the cylinder should be able to move out of the way during the assembly process. Evidence from diffraction studies and from proton nuclear magnetic resonance spectroscopy points to a segment of about 20 amino acid residues being very flexible in the disk. In the helical virus these residues take on a well-defined conformation which completely shields the nucleic acid from the central channel.
Muscle contraction takes place by the cyclic interaction of two-stranded helical actin filaments with protuberances from the myosin filaments known as cross-bridges. The nature of the contractile process demands that the myosin cross-bridge be highly polymorphic. Biochemical evidence points to the cross-bridge having a domain structure such that contraction may be a manifestation of the rearrangement of these subunits. An essential step in contraction may be the initial binding of a small hinged domain of the cross-bridge (the 'nose-cone') to actin, followed by a series of changes ending in the formation of a second union (the rigor bond) with an actin monomer in the neighbouring strand of the actin helix.
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A composite data set in Fourier space has been produced from electron micrographs of negatively stained decorated actin and X-ray fibre diffraction patterns of glycerinated insect muscle "decorated" with rabbit myosin cross-bridges. It is shown that a simple model of actin and myosin may be adjusted to fit this data. The model conforms to the Taylor-Amos geometry for the acto-myosin interaction in accordance with the idea that the cross-bridge binds to two neighbouring actin monomers on different strands.
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A review of the structural studies of tobacco mosaic virus (TMV) is given. TMV is essentially a flat helical microcrystal with 16 1/3 subunits per turn. A single strand of RNA runs along the helix and is deeply embedded in the protein. The virus particles form oriented gels from which high-resolution X-ray fiber diffraction data can be obtained. This may be interpreted by the use of six heavy-chain derivatives to give an electron density map at 0.4 nm resolution from which the RNA configuration and the form of the inner part of the protein subunit may be determined. In addition, the protein subunits form a stable 17-fold two-layered disk which is involved in virus assembly and which crystallizes. By the use of noncrystallographic symmetry and a single heavy-atom derivative, it has been possible to solve the structure of the double disk to 0.28 nm resolution. In this structure one sees that an important structural role is played by four alpha-helices, one of which (the LR helix) appears to form the main binding site for the RNA. The main components of the binding site appear to be hydrophobic interactions with the bases, hydrogen bonds between aspartate groups and the sugars, and arginine salt bridges to the phosphate groups. The binding site is between two turns of the virus helix or between the turns of the double disk. In the disk, the region proximal to the RNA binding site is in a random coil until the RNA binds, whereupon the 24 residues involved build a well-defined structure, thereby encapsulating the RNA.
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Viruses played an important role in analysing macromolecular structures. Structure and function in living systems are arranged hierarchically. An important purpose of biology is to describe the correlation between the structural hierarchy and the observed assembly and to analyse the embryological program of the organism.
The electron density distribution of tobacco mosaic virus has been determined to 6.7 A resolution by analysis of the X-ray diffraction pattern given by oriented gels of the virus. This has been achieved by separation of overlapping Bessel function terms by a technique analogous to crystallographic isomorphous replacement. The course of the polypeptide chain of the coat protein may be traced for a large part of its length.
The effects of three ATP analogues, alpha,beta-methylene-ATP [ATP(alpha,beta-CH1)], adenosine 5'-0-(3-thiotrophosphate) [ATP(gamma-S)], and beta,gamma-amino-ATP [ATP(beta,gamma-NH)] at various concentrations and temperatures on the X-ray fiber diagrams of glycerinated flight muscles from a water bug (Lethocerus maximus) have been investigated. It is shown that the "relaxed" state can be obtained with all three analogues at high concentrations, the result being particularly clear with ATP(gamma-S). It is inferred that the binding of an ATP-like molecule suffices to produce the relaxed state. At low concentrations ATP(beta,gamma-NH) produces state intermediate between rigor and relaxed which is not simply a mixture of the two. The possible nature of the intermediate is discussed.
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