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J J Harford

Publications and source records attributed to J J Harford.

8 recordsLinked to original sources
PubMed2003

Modelling muscle motor conformations using low-angle X-ray diffraction.

New results on myosin head organization using analysis of low-angle X-ray diffraction patterns from relaxed insect flight muscle (IFM) from a giant waterbug, building on previous studies of myosin filaments in bony fish skeletal muscle (BFM), show that the information content of such low-angle diffraction patterns is very high despite the 'crystallographically low' resolution limit (65 A) of the spacings of the Bragg diffraction peaks being used. This high information content and high structural sensitivity arises because: (i) the atomic structures of the domains of the myosin head are known from protein crystallography; and (ii) myosin head action appears to consist mainly of pivoting between domains which themselves stay rather constant in structure, thus (iii) the intensity distribution among diffraction peaks in even the low resolution diffraction pattern is highly determined by the high-resolution distribution of atomically modelled domain mass. A single model was selected among 5000+ computer-generated variations as giving the best fit for the 65 reflections recorded within the selected resolution limit of 65 A. Clear evidence for a change in shape of the insect flight muscle myosin motor between the resting (probably like the pre-powerstroke) state and the rigor state (considered to mimic the end-of-powerstroke conformation) has been obtained. This illustrates the power of the low-angle X-ray diffraction method. The implications of these new results about myosin motor action during muscle contraction are discussed.

Journal Article↗
PubMed2000

Conformation of the myosin motor during force generation in skeletal muscle.

Myosin motors drive muscle contraction, cytokinesis and cell locomotion, and members of the myosin superfamily have been implicated in an increasingly diverse range of cell functions. Myosin can displace a bound actin filament several nanometers in a single interaction. Crystallographic studies suggest that this 'working stroke' involves bending of the myosin head between its light chain and catalytic domains. Here we used X-ray fiber diffraction to test the crystallographic model and measure the interdomain bending during force generation in an intact single muscle fiber. The observed bending has two components: an elastic distortion and an active rotation that generates force. The average bend of the force-generating myosin heads in a muscle fiber is intermediate between those in crystal structures with different bound nucleotides, and the C-terminus of the head is displaced by 7 nm along the actin filament axis compared with the in vitro conformation seen in the absence of nucleotide.

Actins↗
PubMed1997

Myosin head configuration in relaxed fish muscle: resting state myosin heads must swing axially by up to 150 A or turn upside down to reach rigor.

The arrangement and shape of myosin heads in relaxed muscle have been determined by analysis of low-angle X-ray diffraction data from a very highly ordered vertebrate muscle in bony fish. This reveals the arrangement and interactions between the two heads of the same myosin molecule, the shape of the resting myosin head (M.ADP.Pi) assuming a putative hinge between the myosin catalytic domain and the light chain binding-domain, and the way that the actin-binding sites on myosin are arrayed around the actin filaments in the bony fish muscle A-band cell unit. The results are discussed in terms of possible force-generating mechanisms. Changes in myosin head shape or tilt have been implicated in the mechanism of force generation. The myosin head arrangement, including perturbations from perfect helical symmetry, has all heads oriented roughly the same way up (there is only a small range of rotations around the head long axis). X-ray data do not define the absolute polarity of the myosin head array. The resting head rotation is either similar to (65 degrees difference) or opposite to (115 degrees difference) the rotation in the rigor state. If the rotations are similar, probably the more likely possibility, then the average relative axial displacement of the inner and outer ends of the heads from the resting state to rigor is about 140 to 150 A. If (less likely) the resting head rotation is opposite to rigor, then the heads would need to turn over (i.e. rotate about 115 degrees around their own long axes) and the mean relative axial displacement from relaxed to rigor would only be 20 to 30 A.

Actins↗
PubMed1994

Molecular movements in contracting muscle: towards "muscle--the movie".

The recent publication of the crystal structures of G-actin and of myosin subfragment-1, together with analysis of a time-resolved series of well sampled low-angle 2D X-ray diffraction patterns from bony fish muscle permits the study of the molecular movements in muscle that are associated with generation and regulation of contractile force. Here it is shown that even though low-angle (i.e. low resolution) X-ray diffraction patterns are being used, these patterns are sensitive, for example, to sub-domain movements of as little as 3 A or 4 degrees within the actin monomers of actin filaments. Actin filament diffraction patterns from whole muscle are being used to define actin domain and tropomyosin movements involved in regulation. Myosin and actin filament diffraction patterns are being used together to start to show how the complete "quasi-crystalline" unit cell in the bony fish muscle A-band can be modelled as a series of time-slices through a typical tetanic contraction of the muscle. In this way, the time sequence of images can be used to create "muscle--the movie".

Actins↗
PubMed1992

Evidence for structurally different attached states of myosin cross-bridges on actin during contraction of fish muscle.

Using data from fast time-resolved x-ray diffraction experiments on the synchrotrons at Daresbury and (Deutsches Elektronen Synchrotron [DESY]), it is shown that during contraction of fish muscle there are at least two distinct configurations of myosin cross-bridges on actin, that they appear to have different tension producing properties and that they probably differ in the axial tilt of the cross-bridges on actin. Evidence is presented for newly observed myosin-based layer lines in patterns from active fish muscle, together with intensity changes of the actin layer lines. On the equator, the 110 reflection changes much faster (time for 50% change t1/2 = 21 +/- 4 ms after activation) than the 100 reflection (t1/2 = 35 +/- 8 ms) and tension (t1/2 = 41 +/- 3 ms) during the rising phase of tetanic contractions. These and higher order reflections have been used to show the time course of mass attachment at actin during this rising phase. Mass arrival (t1/2 = 25 ms) precedes tension by approximately 15 ms. Analysis has been carried out to evaluate the effects of changes in sarcomere length during the tetanus. It is shown that any such effects are very small. Difference "equatorial" electron density maps between active muscle at a time when mass arrival at actin is just complete, but the tension is still rising, and at a later time well into the tension plateau, show that the structural difference between the lower and higher force states corresponds to mass movement consistent with axial swinging of heads from a nonstereospecific actin attached state (low force) to a more stereospecific (high force) state.

Actins↗
PubMed1991

Crossbridge states in isometrically contracting fish muscle: evidence for swinging of myosin heads on actin.

We have previously shown that time-resolved X-ray diffraction studies of the 2-D pattern from isometrically contracting flatfish (turbot) fin muscle have considerable advantages over similar studies of other vertebrate muscles due to the simple lattice and the better long-range order in these muscles (5, 24). Here we show not only that two structurally different myosin head to actin attached states must exist in the crossbridge cycle but that we are also able to define the likely crossbridge configurations in these states. A non-force producing "weak-binding" state is evident from the lead of the (11) equatorial reflections (and actin mass) time-course relative to that of tension (and the (10) equatorial reflection decrease) by about 30 msec. The first myosin layer line at 429 A has a weakened but altered intensity distribution, with no change in axial spacing, in patterns from active muscle. We show this to be consistent with myosin heads binding in the non-specific manner envisaged for the "weak-binding" state. Evidence for the second force-producing attached state, or series of states, comes from the observation of only a small increase in the intensity of the 360 A actin layer line between resting and active muscle patterns. It might be thought that a substantial increase in this layer line would be expected if myosin heads were even transiently attached to the thin filaments in a force-producing state. However, this is not so because internal changes in the structure of the thin filaments in active muscle have the opposite effect of causing this layer line to decrease in intensity. Observation of a small net intensity increase is therefore evidence for myosin head attachment with the symmetry of the actin helix. From the equatorial diffraction pattern, Fourier synthesis maps were computed at 10 msec intervals throughout the isometric tetanus, enabling changes in the mass distribution to be visualised between the force- and non-force producing populations of crossbridges. This difference map shows that in the force-producing state myosin heads have their centres of mass on average at a smaller radius from the thin filament axis compared to the case for non-force producing myosin heads. Since there is good evidence that there is no substantial change in myosin head shape during contraction (30) these observations are consistent with myosin heads swinging on actin as fairly rigid structures.

Actins↗
PubMed1988

Actin filament organization and myosin head labelling patterns in vertebrate skeletal muscles in the rigor and weak binding states.

The structures of vertebrate skeletal muscles (particularly from frog and fish) in the rigor state are analysed in terms of the concept of target areas on actin filaments. Assuming that 100% of the heads are to be attached to actin in rigor, then satisfactory qualitative low-resolution modelling of observed X-ray diffraction data is obtained if the outer ends of these myosin heads can move axially (total range about 200A) and azimuthally (total range less than 60 degrees) from their original lattice sites on the myosin filament surface to attach in defined target areas on the actin filaments. On this basis, each actin target area comprises about four actin monomers along one of the two long-pitched helical strands of the actin filament (about 200 A) or an azimuthal range of actin binding sites of about 100 degrees around the thin filament axis. If myosin heads simply label in a non-specific way the nearest actin monomers to them, as could occur with non-specific transient attachment in a 'weak binding' state, then the predicted X-ray diffraction pattern would comprise layer lines at the same axial spacings (orders of 429 A) as those seen in patterns from resting muscle. It is shown that actin target areas in vertebrate skeletal muscles are probably arranged on an approximate 62 (right-handed) helix of pitch (P) of about 720 A, subunit translation P/6 and near repeat P/2. Troponin position need not be considered in defining the labelling pattern of cross-bridges on this 62 helix of target areas; the target areas appear to be defined solely by the azimuthal position of the actin binding sites. The distribution of actin filament labelling patterns could be regular in fish muscle which has a 'crystalline' A-band, but will be irregular in higher vertebrate muscles such as frog sartorius muscle.

Actin Cytoskeleton↗