Structural analysis of Alzheimer's beta(1-40) amyloid: protofilament assembly of tubular fibrils.

Detailed structural studies of amyloid fibrils can elucidate the way in which their constituent polypeptides are folded and self-assemble, and exert their neurotoxic effects in Alzheimer's disease (AD). We have previously reported that when aqueous solutions of the N-terminal hydrophilic peptides of AD beta-amyloid (A beta) are gradually dried in a 2-Tesla magnetic field, they form highly oriented fibrils that are well suited to x-ray fiber diffraction. The longer, more physiologically relevant sequences such as A beta(1-40) have not been amenable to such analysis, owing to their strong propensity to polymerize and aggregate before orientation is achieved. In seeking an efficient and inexpensive method for rapid screening of conditions that could lead to improved orientation of fibrils assembled from the longer peptides, we report here that the birefringence of a small drop of peptide solution can supply information related to the cooperative packing of amyloid fibers and their capacity for magnetic orientation. The samples were examined by electron microscopy (negative and positive staining) and x-ray diffraction. Negative staining showed a mixture of straight and twisted fibers. The average width of both types was approximately 70 A, and the helical pitch of the latter was approximately 460 A. Cross sections of plastic-embedded samples showed a approximately 60-A-wide tubular structure. X-ray diffraction from these samples indicated a cross-beta fiber pattern, characterized by a strong meridional reflection at 4.74 A and a broad equatorial reflection at 8.9 A. Modeling studies suggested that tilted arrays of beta-strands constitute tubular, 30-A-diameter protofilaments, and that three to five of these protofilaments constitute the A beta fiber. This type of structure--a multimeric array of protofilaments organized as a tubular fibril--resembles that formed by the shorter A beta fragments (e.g., A beta(6-25), A beta(11-25), A beta(1-28)), suggesting a common structural motif in AD amyloid fibril organization.

Interpretation of the X-ray diffraction pattern from relaxed skeletal muscle and modelling of the thick filament structure.

The first part of this paper is devoted to the model-building studies of our high resolution meridional X-ray diffraction patterns (in the region from 1/500 to 1/50 A-1) obtained from relaxed frog muscle. A one-dimensional model of thick filament was proposed which basically consists of two symmetrical arrays of 50 crossbridge crown projections. In the proximate and central zones of the filament the crossbridge crowns are regularly shifted with a 429 A period and appear as triplets with a 130 A distance between crowns, while the crowns in the distal parts of filament are regularly ordered with a 143 A repeat. The centre-to-centre distance between regions with crossbridge perturbations is 7050 A. The length of each crown projection is about 125 A. The model includes also (1) C-protein component represented in each half of the filament by seven stripes of about 350 A long and located 429 A apart, (2) a uniform density of filament backbone of about 1.5 micron length, and (3) 13 high density stripes in a central zone located with 223 A period. The final model explains very well the positions and intensities of the main meridional reflections. A three-dimensional model of crossbridge configuration is described in the second part of the work. The model was constructed by using the intensity profiles of the first six myosin layer lines of the X-ray pattern from stretched muscle and taking into account the crossbridge perturbations and the axial size of crossbridge crown obtained from the one-dimensional studies.(ABSTRACT TRUNCATED AT 250 WORDS)

[Interpretation of the pictures of meridional diffraction on X-rays of membrane-free rabbit spinal muscles in a state of rigor and their dependence on physico-chemical parameters of rigorising solution]. / Interpretatsiia kartin meridional'noi difraktsii na rentgenogrammakh demembranizirovannoi spinnoi myshtsy krolika v sostoianii rigora i ikh zavisimosti ot fiziko-khimicheskikh parametrov rigorizuiushchego rastvora.

Results of the studies of structural and mechanical properties of rabbit psoas muscle by means of X-ray diffraction and physiological technique are summarised along the following lines: localisation of minor proteins in the thick filaments and the origin of meridional reflections in the X-ray diffraction patterns of rigorised rabbit psoas muscle; molecular basis of the effects of pH, ionic strength, pyrophosphate, ethyleneglycol and pyrophosphate-ethyleneglycol mixture on the value of rigor tension and their dependence on the sarcomerès length and concentration of Mg2+; effect of selective extraction of minor proteins from M- and P-zones of the thick filaments on meridional reflections; structure of the thick filaments in the state of "Ca2+-free rigor"; attachment of cross-bridges to actin filaments in relaxing solution (is the model of "sterick brocking" correct?); role of structural transitions in S-2-segments of myosin in force generation.

[Direct proof of the existence of Ca2+-induced structural changes in miosin-containing thick filaments of vertebrate skeletal muscles]. / Dokazatel'stvo sushchestvovaniia Ca2+-indutsiruemogo strukturnogo perekhoda v stvolakh miozinsoderzhashchikh nitei skeletnykh myshts pozvonochnykh.

Frog sartorius muscles were skinned with the help of detergent Triton X-100 in relaxing solution and then rigorized in the solution free of ATP and calcium (Ca2+-free rigor). Some of the X-ray diffraction patterns obtained from such muscles differed drastically from the patterns of usual "calcium" rigor state of the muscle. Analysis of the X-ray diffraction patterns of Ca2+-free rigor state of the muscle allows the following conclusions to be made. (1) It is proved that the so-called forbidden meridional reflections localised in groups at the successive orders of the repeat period of about 430 A are due to diffraction on the lattice formed by minor proteins of the thick filaments. This must be taken into account while interpreting the intensity changes of the meridional 143 and 215 A reflections in the contracting muscle. (2) The shafts of the myosin-containing filaments may exist in two different structural states; the transition between these states takes place on the change of calcium concentration. (3) The deflection of myosin cross-bridges from the shafts of the filaments is possible at either of two conformations of the myosin shafts. (4) The formation of actomyosin rigor-complexes does not necessarily leads to a structural change in the myosin filament. (5) Existence of Ca2+-induced structural changes in the thick filaments indicates possible myosin-linked regulations in the vertebrate skeletal muscle alongside with the actin-linked ones.

[Localization of minor proteins and structural changes in the myosin filaments of vertebrate striated muscle]. / Lokalizatsiia minornykh belkov i strukturnye izmeneniia v miozinovykh poperechno-polosatykh myshts pozvonochnykh.

The origin of meridional reflections in the X-ray diffraction patterns of vertebrate skeletal muscles in resting and rigor states was studied. The main results may be summarized as follows. 1. Most of the meridional reflections localized in groups at the positions of successive orders of the repeat period of about 430 A are contributed mainly by the C-protein component of thick filaments. 2. The meridional reflections at about 143 and 72 A in the X-ray diffraction pattern of the resting muscle are contributed mainly by the cross-bridge axial repeat period, while in the X-ray diffraction patterns of the rigorized muscle the reflections at approximately the same positions are contributed mainly by C-protein. The change in the positions of these particular reflections accompanying the transition of the muscle from rest to rigor and from rest to contraction cannot be considered as an indication of a change in the axial repeat period of the cross-bridges, as it was earlier suggested by some authors. 3. The transition of the muscle from resting to rigor state is accompanied by substantial changes in the positions of the meridional reflections contributed my minor proteins, which is indicative of the structural transition in the thick filaments. The observed changes may be interpreted as the result of the thick filaments elongation by about 1.5% or, alternatively, as a consequence of the redistribution of electron density of the meridional reflections 215 and 143 A during a single twitch of the muscle (Huxley et al., Nature, 1980 284, 140) may be interpreted as a natural consequence of the structural change in the thick filaments. It is concluded therefore that on stimulation of the vertebrate skeletal muscle the thickness filaments undergo a reversible structural change which may reflect the existence of myosin-linked regulation in that type of muscle.

[Partial activation of thin filaments in resting frog skeletal muscle fibers]. / Chastichnaia aktivatsiia tonkikh nitei v pokoiashchikhsia skeletnykh myshtsakh liagushki.

X-ray patterns from frog skeletal muscles at rest show a series of relatively weak meridional reflections which may be indexed as the 5, 7, 9, 11 and 13 orders of the repeat period of about 2 x 385 A. According to the model of the thin filaments structure, suggested by V. V. Lednev and G. M. Frank (1977), this period is specific for the activated or "switched on" state of the actin--containing filaments. At the same time, according to the generally accepted model (suggested in 1972), the axial repeat period of the thin filament structure is approximately equal to 385 A and does not depend on the functional state of the muscle. The existence of the repeat period of about 2 x 385 A in the thin filaments of a resting muscle suggests that even at rest the thin filaments of vertebrate skeletal muscle are not completely inhibited. It may be suggested that partial activation of the thin filaments in a resting muscle is the result of formation of long life rigorlike crossbridges, the existence of which was postulated by D. K. Hill in 1968 on the basis of his studies on resting tension in the frog skeletal muscle.