Yeast phosphoglycerate kinase is a structurally well-characterized enzyme consisting of 415 amino acids without disulfide bonds. Anion-induced refolding from its acid-unfolded state gives rise to the formation of worm-like amyloid fibrils with a persistence length of 73 nm. Electron microscopy and small-angle X-ray scattering data indicate that the fibrils have an elliptical cross-section with dimensions of 10.2 nm x 5.1 nm. About half of all amino acids are organized in form of cross-beta structure which gives rise to typical infrared spectra, X-ray diffraction and yellow-green birefringence after Congo red staining. The kinetics of amyloid formation, monitored by infrared spectroscopy, dynamic light scattering and X-ray scattering, was found to be strongly dependent on protein concentration. The infrared data indicate that the formation of cross-beta structure practically comes to an end already after some hours, whereas the length-growth of the amyloid fibrils, monitored by small-angle X-ray scattering, was not yet completed after 1,300 hours.
The dependence on environmental conditions of the assembly of barstar into amyloid fibrils was investigated starting from the nonnative, partially folded state at low pH (A-state). The kinetics of this process was monitored by CD spectroscopy and static and dynamic light scattering. The morphology of the fibrils was visualized by electron microscopy, while the existence of the typical cross-beta structure substantiated by solution X-ray scattering. At room temperature, barstar in the A-state is unable to form amyloid fibrils, instead amorphous aggregation is observed at high ionic strength. Further destabilization of the structure is required to transform the polypeptide chain into an ensemble of conformations capable of forming amyloid fibrils. At moderate ionic strength (75 mM NaCl), the onset and the rate of fibril formation can be sensitively tuned by increasing the temperature. Two types of fibrils can be detected differing in their morphology, length distribution and characteristic far UV CD spectrum. The formation of the different types depends on the particular environmental conditions. The sequence of conversion: A-state-->fibril type I-->fibril type II appears to be irreversible. The transition into fibrils is most effective when the protein chain fulfills particular requirements concerning secondary structure, structural flexibility and tendency to cluster.
Abstract:The structure of maltodextrin gels was investigated by small angle X-ray scattering. The inhomogeneities in the maltodextrin gels are molecular aggregates with maximum dimensions of almost 300 nm. Their shape can be approximated by oblate ellipsoids of revolution with an axial ratio of 0.1. The radius of gyration of the aggregates amounts to about 90 nm. For this reason in the nomenclature of Papkov the gels are polymer gels of the 2 na type. The melting of these aggregates is measured by SAXS with a position sensitive detector in the range near 56 ~
X-ray small-and medium-angle scattering of partially ordered or semicrystalline materials is composed of background scattering from the form scattering of the components and from the amorphous phase and of peaks from the scattering of the crystallites. By the slit geometry of X-ray diffractometers constructed for registration of small-and medium-angle X-ray scattering, the diffuse scattering and the peaks are distorted and the peak positions and half-widths are changed. A program module based on the Akima interpolation [Akima (1970). J. Assoc. Comput. Math. 17,[589][590][591][592][593][594][595][596][597][598][599][600][601][602] is proposed for calculation of the first derivative of the complete smeared scattering curve, which is then explicitly used in direct collimationcorrection procedures. The desmearing of scattering curves from semicrystalline starch samples proves the convenience of the method for low-noise conditions and exhibits a significant gain of measuring time in comparison with data of comparable accuracy but measured with SoUer-slit collimation systems or desmeared with direct methods using frequency filtering.
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