Gerd Rosenbaum

Cross-bridge conformation as revealed by x-ray diffraction studies on insect flight muscles with ATP analogues.

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.

Co-ordinated electron microscopy and X-ray studies of glycerinated insect flight muscle. I. X-ray diffraction monitoring during preparation for electron microscopy of muscle fibres fixed in rigor, in ATP and in AMPPNP

SummarySynchrotron radiation was used for low-angle X-ray diffraction to monitor structural changes produced in insect flight muscle during fixation, dehydration and embedding for electron microscopy of thin sections. Fibre bundles were fixed by cold glutaraldehyde in one of three states, namely rigor, ATP or AMPPNP, followed by additional cross-linking treatment. No heavy metals were used before embedding. During fixation-embedding, all specimens lost the continuous actin layer lines of spacing 11–5 nm, shrank 18–21% in lattice spacing, shrank 0.5–2.5% in axial spacings and showed equatorial intensity changes which were similar for all three states, while the well-sampled inner layer lines (39–13 nm) were preserved with different fidelity in each state, highest for rigor and lowest for ATP. In different AMPPNP bundles, these layer lines indicated different degrees of unexplained shift (from slight to total) towards the structure of muscle fixed in ATP. Fixation in ATP caused obvious gain of intensity on 39, 19 and 13 nm layer lines, which can be interpreted as trapping of myosin crossbridge attachments to actin; this artifact was unchanged by seven variations in fixation conditions. Fixation in rigor gave no indication of crossbridge detachment nor of the presence or alteration of any significant population of non-bridging myosin heads. X-ray monitoring allowed selection of best-preserved samples for subsequent electron microscopy. The rapid pattern-recording possible with synchrotron X-ray intensity allowed us to complete and compare experiments with many fibre bundles from a single glycerinatedLethocerus muscle.

Small-Angle Diffraction of X Rays and the Study of Biological Structures

Specimens fall broadly into three categories, crystals, fibers, or solutions, depending on the degree of order. The categories crystals and solutions are the subject of Chapters 17 and 15, respectively, so that they will not be considered in detail here. However, the experimental conditions necessary for the registration of diffraction patterns from crystals and fibers with large unit cells (ca. 50 nm) are similar and thus the two will be considered together in the following discussion. In the main, similar considerations apply to solution studies. However, as they need additionally very low parasitic scattering and as the size of specimen is not generally limiting, design strategy is considerably different. For crystals and fibers the specimens are usually very small (100–500 μm). A limited amount of parasitic scattering in defined directions is tolerable. In the following analysis we do not explicitly consider the parasitic scattering. In critical situations the rules for solution scattering apply.

An Investigation of the Cross-bridge Cycle Using ATP Analogues and Low-angle X-ray Diffraction from Glycerinated Fibres of Insect Flight Muscle

The interdigitating filament arrays of muscle contain as their major components the proteins actin and myosin. Globular actin molecules aggregate to form the “thin” filaments. Myosin molecules consist of fully α-helical tails and enzymatically active heads. The molecules spontaneously aggregate to form bipolar filaments (the “thick” filaments) with the heads protruding to form the “cross-bridges”. Two heads may constitute one cross-bridge. The interaction between actin and myosin is mediated by the cross-bridges. By limited proteolytic digestion the enzymatically active fragments of myosin S1 (single heads) and HMM (two heads with a length of tail) may be prepared. The heads (S1) are joined to the tails by a more flexible part of the tail known as the S2 region. (For details and references see Huxley, this volume.)