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.

X-ray diffraction patterns were obtained from skinned rabbit psoas muscle under relaxing and rigor conditions over a wide range of ionic strengths (50-170 mM) and temperatures (1 degree C-30 degrees C). For the first time, an intensification of the first actin-based layer line is observed in the relaxed muscle. The intensification, which increases with decreasing ionic strength at various temperatures, including 30 degrees C, parallels the formation of weakly attached cross-bridges in the relaxed muscle. However, the overall intensities of the actin-based layer lines are low. Furthermore, the level of diffuse scattering, presumably a measure of disorder among the cross-bridges, is little affected by changing ionic strength at a given temperature. The results suggest that the intensification of the first actin layer line is most likely due to the cross-bridges weakly bound to actin, and that the orientations of the weakly attached cross-bridges are hardly distinguishable from the detached cross-bridges. This suggests that the orientations of the weakly attached cross-bridges are not precisely defined with respect to the actin helix, i.e., nonstereospecific. Intensities of the myosin-based layer lines are only marginally affected by changing ionic strength, but markedly by temperature. The results could be explained if in a relaxed muscle the cross-bridges are distributed between a helically ordered and a disordered population with respect to myosin filament structure. Within the disordered population, some are weakly attached to actin and others are detached. The fraction of cross-bridges in the helically ordered assembly is primarily a function of temperature, while the distribution between the weakly attached and the detached within the disordered population is mainly affected by ionic strength. Some other notable features in the diffraction patterns include a approximately 1% decrease in the pitch of the myosin helix as the temperature is raised from 4 degrees C to 20 degrees C.

By using synchrotron radiation and an imaging plate for recording diffraction patterns, we have obtained high-resolution x-ray patterns from relaxed rabbit psoas muscle at temperatures ranging from 1 degree C to 30 degrees C. This allowed us to obtain intensity profiles of the first six myosin layer lines and apply a model-building approach for structural analysis. At temperatures 20 degrees C and higher, the layer lines are sharp with clearly defined maxima. Modeling based on the data obtained at 20 degrees C reveals that the average center of the cross-bridges is at 135 A from the center of the thick filament and both of the myosin heads appear to wrap around the backbone. At 10 degrees C and lower, the layer lines become very weak and diffuse scattering increases considerably. At 4 degrees C, the peak of the first layer line shifts toward the meridian from 0.0047 to 0.0038 A(-1) and decreases in intensity approximately by a factor of four compared to that at 20 degrees C, although the intensities of higher-order layer lines remain approximately 10-15% of the first layer line. Our modeling suggests that as the temperature is lowered from 20 degrees C to 4 degrees C the center of cross-bridges extends radially away from the center of the filament (135 A to 175 A). Furthermore, the fraction of helically ordered cross-bridges decreases at least by a factor of two, while the isotropic disorder (the temperature factor) remains approximately unchanged. Our results on the order/disordering effects of temperature are in general agreement with earlier results of Wray [Wray, J. 1987. Structure of relaxed myosin filaments in relation to nucleotide state in vertebrate skeletal muscle. J. Muscle Res. Cell Motil. 8:62a (Abstr.)] and Lowy et al. (Lowy, J., D. Popp, and A. A. Stewart. 1991. X-ray studies of order-disorder transitions in the myosin heads of skinned rabbit psoas muscles. Biophys. J. 60:812-824). and support Poulsen and Lowy's hypothesis of coexistence of ordered and disordered cross-bridge populations in muscle (Poulsen, F. R., and J. Lowy. 1983. Small angle scattering from myosin heads in relaxed and rigor frog skeletal muscle. Nature (Lond.). 303:146-152.). However, our results added new insights into the disordered population. Present modeling together with data analysis (Xu, S., S. Malinchik, Th. Kraft, B. Brenner, and L. C. Yu. 1997. X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle. Biophys. J. 73:000-000) indicate that in a relaxed muscle, cross-bridges are distributed in three populations: those that are ordered on the thick filament helix and those that are disordered; and within the disordered population, some cross-bridges are detached and some are weakly attached to actin. One critical conclusion of the present study is that the apparent order <--> disorder transition as a function of temperature is not due to an increase/decrease in thermal motion (temperature factor) for the entire population, but a redistribution of cross-bridges amon...

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)

Previously we have shown that cross-bridge attachment to actin and the radial position of the myosin heads surrounding the thick filament backbone affect the equatorial x-ray diffraction intensities in different ways (Yu, 1989). In the present study, other factors frequently encountered experimentally are analyzed by a simple model of the filament lattice. It is shown that the ordering/disordering of filaments, lattice spacing changes, the azimuthal redistributions of cross-bridges, and variations in the ordered/disordered population of cross-bridges surrounding the thick filaments can distinctly affect the equatorial intensities. Consideration of Fourier transforms of individual components of the unit cell can provide qualitative explanations for the equatorial intensity changes. Criteria are suggested that can be used to distinguish the influence of some factors from others.