Jean Witz

The crystallization of biological macromolecules from precipitates: evidence for Ostwald ripening

Abstract Crystals were obtained by different methods under conditions where nucleation and growth occur from precipitated macromolecular material. The phenomenon was observed with compounds of different size and nature, such as thaumatin, concanavalin A, an α-amylase, a thermostable aspartyl-tRNA synthetase, the nucleo-protein complex between a tRNA Asp transcript and its cognate yeast aspartyl-tRNA synthetase, and tomato bushy stunt virus. In each system, after a rather rapid precipitation step at high supersaturation lasting one to several days, a few microcrystals appear after prolonged equilibration at constant temperature. With α-amylase, the virus and the thermostable synthetase, crystallization is accompanied by appearance of depletion zones around the growing crystals and growth of the largest crystals at the expense of the smaller ones. These features are evidences for crystal growth by Ostwald ripening. In the case of thaumatin, concanavalin A and the nucleo-protein complex, crystallization occurs by a phase transition mechanism since it is never accompanied by the disappearance of the smallest crystals. A careful analysis with thermostable aspartyl-tRNA synthetase indicates that its crystallization at 4°C under high supersaturation starts by a phase transition mechanism with the formation of small crystals within an amorphous protein precipitate. Ostwald ripening follows over a period of up to three/four months with a growth rate of about 0.8 A/s that is 13 times slower than that of crystals growing at 20°C in the absence of precipitate without ripening. At the end of the ripening process at 4°C, only one unique synthetase crystal remains per microassay with dimensions as large as 1 mm.

A neutron scattering study of the structure of compact and swollen forms of southern bean mosaic virus

Neutron small-angle scattering in H2O/D20 buffers has been used to investigate the radial distribution of protein and RNA in the compact and swollen forms of southern bean mosaic virus (SBMV). The architecture of the particle is described by a set of four concentric shells. Compact SBMV was found to consist of a tightly packed outer protein shell, with the RNA and about 15% of the protein at the interior of the particle. The protein penetrates as deeply as the RNA toward the center. This structure resembles that of tomato bushy stunt virus although the average distribution of the protein is much less bilobal. There is no major reorganisation of the architecture of the virion upon swelling induced by the removal of divalent ions at pH 8.5: the radii of all four shells increase by about 15 A, with a concomittant decrease of the packing density. Possible correlations between the mechanism of swelling and the architecture and stability of small isometric virions are discussed.

Divalent ion-dependent reversible swelling of tomato bushy stunt virus and organization of the expanded virion.

Abstract The divalent ion-dependent swelling of tomato bushy stunt virus at neutral pH has been investigated by a combination of analytical ultracentrifugation, fluorescence techniques and small-angle X-ray and neutron scattering. In the presence of EDTA, the virions swell by about 12% (in radius) in a narrow pH interval centered at about pH 7.0. Native virus is progressively changed into swollen particles with increasing pH, and no intermediate component could be observed. Upon swelling, virions exhibit a twofold increase of their intrinsic fluorescence; they remain resistant to RNAase but the viral RNA becomes accessible to ethidium bromide, which intercalates at about the same number of sites as in free RNA. The radial distribution of RNA and protein resembles that of the native virus, all radii being increased by 22 A. The protein remains clustered into two concentric shells separated by a 30 A thick shell containing most of the viral RNA. Swelling of native tomato bushy stunt virus at pH 7 is under the specific control of very strongly bound calcium ions, which may be antagonized by magnesium ions. Swollen virons can be recompacted in the presence of calcium ions at pH > 7.0, or by dialysis against EDTA-containing buffers at pH ≤ 6. Recompaction is progressive, from about pH 6.6 to 6.2. Throughout the transition, small-angle X-ray scattering curves are demonstrably not linear combinations of the two extremes, showing that intermediate particles must exist. Neutron and X-ray small-angle scattering curves of recompacted particles are identical to those of native virus, providing evidence that the gross features of the architecture have been recovered. It is only if recompaction takes place at pH 5 that virions behave.

Structural polymorphism of bromegrass mosaic virus: A neutron small angle scattering investigation

Abstract Neutron small angle scattering has been used to investigate the architecture of bromegrass mosaic virus (BMV) in solution as a function of pH, ionic strength, and nature of the polyvalent ions at room temperature: The radial extensions of RNA and protein were determined from measurements performed in buffers containing suitable amounts of heavy water (D20) to match the scattering of either component. Our results confirm the polymorphism of BMV known from the variation of its overall size ( N. L. Incardona and P. Kaesberg, 1964 , Biophys. J. 4, 11–21; M. Zulauf, 1977 , J. Mol. Biol. 114, 259–266) and extend it to the RNA. Indeed, we found that although at pH 5.5 the overall size of compact BMV remains constant, measurable changes in the radial extension of RNA take place under the compacting action of high ionic strength or spermine. At neutral pH both the overall size of BMV and the extension of RNA vary in a similar way and depend strongly upon the environment: Swelling is maximal in the absence of polyvalent cations (spermine, Ca2+, Mg2+), and the compacting action of at least Mg2+ has been shown to be partially reversed by increasing the concentration of K+. The thickness of the protein shell does not change significantly upon swelling. Back-titration of swollen BMV is reversible only in the presence of divalent ions. The lack of reversibility extends to both RNA and overall sizes and is accompanied by an increase of the thickness of the capsid. The scattering curve of empty capsids obtained by self-assembly of dissociated BMV protein differs qualitatively from that of the capsid of the virions, implying not only that the size is intermediate between those of compact and swollen BMV but also that the clustering of the protein subunits is probably different at the periphery of the particle. Implications on the relative importance of protein-protein and protein-RNA interactions as well as on the action of ions on the stability of BMV are discussed, and mechanism is proposed for the lack of reversibility of BMV upon back-titration.

Structural dynamics, an intrinsic property of viral capsids.

Summary. In this review, we emphasize that high-resolution models of the structures of small plant and animal viruses obtained by X-ray crystallography are static and insufficient to describe the behavior of these virions. Viral capsids are highly flexible and may undergo conformational changes under physiological conditions without collapse of the virions. This flexibility plays a key role in the process of infection.

The three-dimensional distribution of RNA and protein in the interior of tomato bushy stunt virus: a neutron low-resolution single-crystal diffraction study

BACKGROUND The published high-resolution model of the isometric T = 3 plant virus tomato bushy stunt virus (TBSV) shows the packing in three different environments (A, B, C) of the 180 coat protein subunits of the capsid. It does not, however, account for the localization of either the viral RNA or approximately 25% of the amino acids of the protein subunits, although at least the RNA is rigidly linked to the viral capsid. Solution studies have shown that most of the missing protein is located in an inner shell, and that most of the RNA is sandwiched between the two protein shells. RESULTS We have determined the organization of TBSV at 16 A resolution, using neutron single-crystal diffraction. Connections between the two protein shells are confined to the 20 three-fold axes of the virion, where three C-type subunits meet. Much more RNA density is located under the 30 C-C dimers than under the 60 A-B dimers, where we could even identify lagoons of solvent. CONCLUSIONS Our results emphasize the importance of the amino termini of the 60 C-type protein subunits not only in the RNA-protein interactions but also in the organization of the coat protein, and, probably, in the assembly of the virion. The lack of equivalence between subunits of classes A or B and subunits of class C is even more pronounced in the interior of the virion than in the outer shell, which possesses icosahedral symmetry.

Low pH RNA-protein interactions in turnip yellow mosiac virus: III. Reassociation experiments with other viral RNAs and chemically modified TYMV-RNA

Abstract Dissociation-reassociation experiments were performed with turnip yellow mosaic virus in the presence of RNAs extracted from two related viruses: belladona mottle virus and eggplant mosaic virus, containing 32% and 38% cytosine, respectively. Both RNAs interact specifically at low pH with “nascent capsids” of TYMV obtained by dissociation of the virus in 8 M urea, 1 M NaCl, 0.01 M sodium phosphate buffer, pH 7, in the presence of MgCl 2 or spermidine. Up to 80% of the cytosine residues of TYMV-RNA (which contains 38% C) can be modified by bisulfite treatment. But modified TYMV-RNA containing only 32% C (or less) failed to interact with “nascent capsids” at low pH. Bisulfite treatment can also be achieved in the virion and modifies the stability of the particle. It is concluded that it is not the average cytosine content of the RNA that is responsible for the specific low pH RNA-protein interaction, but rather the presence of a special class of cytosine residues located in single-stranded segments of TYMV-RNA. Some peculiarities of the secondary and tertiary structure of TYMV-RNA are also discussed.

Laser seeding for biomolecular crystallization

Crystal seeding, which permits the decoupling of crystal nucleation and growth, is a potentially powerful tool for biomolecular crystallization. However, its usefulness is severely limited by the inability of current techniques to manipulate single seeds and it is often considered as a last resort in protein crystal growth trials. Remarkably, current practice relies on the cumbersome technique of entraining seed crystals on animal whiskers. Seeding by whisker entrainment is not reproducible and does not allow the observation, selection or individual transfer of seeds. Here, we present a novel approach in which optical forces generated by a focused laser beam are used to select and transfer single microscopic seed crystals in a growth solution. The technique permits the non-mechanical, in situ manipulation of individual seed crystals as small as one micron. The seed transfer is simple and efficient, and can be completed in a few minutes. To demonstrate the procedure we have successfully seeded and grown crystals of tomato bushy stunt virus (TBSV). Applications to other situations which require the selection and manipulation of delicate microcrystals are discussed briefly.

The structure of eggplant mosaic virus: Evidence for the presence of low molecular weight RNA in top component

Abstract Three nucleoproteins were found when purified preparations of eggplant mosaic virus (EMV) were submitted to cesium chloride density gradient centrifugation. Bottom component corresponded to a density ϱ = 1.45 g/ml and a sedimentation coefficient of 106 S. It was infectious. Its RNA fixed valine and lysine, in agreement with the results of Pinck M., et al. ((1974). Biochimie56, 423–428), indicating that high molecular weight EMV-RNA fixes valine and its associated low molecular weight RNA fixes lysine. Top component corresponded to ϱ = 1.26 g/ml and s = 53 S. It is not an empty protein shell. On the contrary each particle contains on the average two to three molecules of RNA of the size of transfer RNA. These RNAs are able to bind alanine, arginine, histidine, leucine, lysine, phenylalanine, tyrosine and valine. Lysine, arginine and valine accounted, respectively, for 70, 18 and 7% of the total amino acid charge. Small amounts of a third component, sedimenting between bottom and top (ϱ = 1.38 g/ml; s = 70 S) have also been found reproducibly in our preparations of EMV. The nature of this component is not yet known. The biological significance of these findings is discussed.

The spherically averaged structure of a DNA isometric plant virus: Cauliflower mosaic virus

The organization of cauliflower mosaic virus was reinvestigated by neutron small-angle scattering in buffers containing various amounts of D2O. The molecular weight of the virions and their DNA content were determined and found to agree well with the known primary structure of the nucleic acid. The capsid probably possesses a T = 7 icosahedral organization. The exceptionally low packing density of the outer protein shell may account for some of the unusual properties of these very stable virions.