Biomedical subjects
Publications and source records attributed to C Berthet-Colominas.
Crystals have been obtained of seryl-tRNA synthetase from the extreme thermophile Thermus thermophilus, using mixed solutions of ammonium sulphate and methane pentane diol. The crystals are very stable and diffract to at least 2 A. The crystals are monoclinic (space group P21) with cell parameters a = 87.1 A, b = 126.9 A, c = 63.5 A and beta = 109.7 degrees.
Acid-soluble and pepsin-soluble type I collagen from calf skin and pepsin-soluble type II collagen from bovine articular cartilage were precipitated in fibrillar form by various methods. Reconstituted native-like fibrils were analysed by low-angle X-ray diffraction, and the patterns were compared with those obtained with native type I (rat tail tendon) and type II fibrils (bovine articular cartilage). For both orientated and disorientated forms of these samples, we measured the ratio of the first/third-order intensities of the meridional diffraction peaks which are associated with the gap-filling. The values obtained with the reconstituted native-like fibrils (types I and II) were double and 20-times the values, respectively, measured for rat tail tendon and bovine articular cartilage. These differences reflect the extent of specific interactions of other components (proteoglycans) at the gap level along the collagen fibrils in the two tissues.
Crystals of seryl-tRNA synthetase from Escherichia coli can be grown from ammonium sulphate/octyl glucoside solutions in two days. The crystals appear to be very suitable for X-ray analysis, diffracting to at least 2.8 A resolution and being resistant to radiation damage. The crystals are monoclinic (space group C2) with cell parameters a = 148.2 A, b = 90.6 A, c = 69.5 A and beta = 119.0 degrees. Depending on whether the asymmetric unit is the enzyme monomer (Mr 48,414) or dimer the Vm value would be either 4.12 or 2.10 A3/dalton. Although the former would indicate a rather high solvent content, other proteins crystallized in the presence of octyl glucoside have Vm values similar to this.
A comparative analysis, by low-angle X-ray diffraction and electron microscopy, of bovine articular cartilage either submitted or not to chemical (0.5-2 M CaCl2, 4 M guanidinium chloride) or enzymatic (hyaluronidase, trypsin) treatments is reported. An analysis of the micrographs using a filtering program on the Fourier transform patterns reveals the absence of modification or alteration of the fibrils after treatment, whereas the X-ray diffraction patterns change. The ratio of the first/third orders intensities increases when the tissue proteoglycans content decreases. These results indicate that proteoglycans are regularly ordered on the type II collagen fibrils in articular cartilage.
Crystals of the fibre protein of adenovirus type 2 have been grown and studied by electron microscopy and X-ray powder diffraction. The molecular packing and density of the crystals suggest that the fibre is dimeric.
Turnip yellow mosaic virus (TYMV) RNA escapes from viral capsids after freezing and thawing the virus, and the remaining capsids look very similar to natural capsids in the electron microscope after negative staining [Katouzian-Safadi, M., Favre, A., and Haenni, A. L. (1980) Eur. J. Biochem. 112, 478-486]. In order to understand how an RNA of 2 X 10(6) Da (33% virus by weight) can escape from a compact protein shell we have compared artificial capsids formed after freezing TYMV and natural capsids produced in vivo in infected plants. We have used various physicochemical techniques including analytical ultracentrifugation, X-ray scattering, X-ray diffraction and orientation in a magnetic field. From the combination of these results we conclude that the escape of the RNA is accompanied by the formation of a hole in the capsid surface. The size of such a hole is estimated to 5-9 coat protein subunits.
Small-angle neutron and X-ray scattering have been used to investigate various aspects of the structural organization of adenovirus type 2. Neutron scattering allows the determination of the radial distribution of DNA and protein, which because of the highly icosahedral form of the virus allows it to be described in terms of three icosahedral shells. X-ray scattering shows that the distance between the major coat proteins (hexons) in the capsid is 100 +/- A. Evidence was also observed for an organization in the nucleoprotein core that gives rise to a maximum in the X-ray scattering at 1/29 A-1.
The self-assembly of brome mosaic virus capsid has been studied kinetically by means of X-ray and neutron scattering. It appears to be a very fast process: for the concentrations used (5 to 8 mg/ml) the forward scattering reaches 50% of its maximal value in less than one second. Further, the assembly seems to proceed through intermediate states whose nature is still speculative.
A combined biochemical, X-ray and electron microscopic study is presented on the molecular structure of bovine and human intervertebral disc. Positively stained electron micrographs of Type I and Type II collagen are shown to have slightly but significantly different banding patterns. New low-angle meridional patterns from different anatomical parts of the intervertebral disc are reported from native and trypsin-extracted discs. Qualitative measurements of these intensities lead to the conclusion that in the nucleus pulposus of the disc, another molecule, probably proteoglycan, is ordered on the collagen fibrils at the axial level of the gap region.
X-ray diffraction was used to monitor the effects of electron microscope fixation, staining, and embedding procedures on the preservation of the three-dimensional crystalline order in collagen fibrils of rat tail tendon. A procedure is described in which the characteristic 3.8-nm lateral spacing is preserved, with increased contrast, in the diffraction pattern of the embedded fiber. This spacing is correlated with the separation between the tangentially oriented equally spaced lines of density observed in electron microscope ultrathin fibril cross sections of the same material. Optical diffraction of electron micrographs gives an objective measure of the periodicity and suggests that the fibril is composed of concentrically oriented crystalline domains. These observations, when combined with a recent interpretation of the native x-ray diffraction data [Hulmes, D. J. S. & Miller, A. (1979) Nature (London) 282, 878-880] suggest a tentative model for the three-dimensional structure of collagen fibrils.
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