Crystal structure of PAV1-137: a protein from the virus PAV1 that infects Pyrococcus abyssi.

Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 Å. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four- α -helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.

Production and crystallization of protein domains: how useful are disorder predictions ?

The failure to produce and/or crystallize proteins is often due to their modular structure. There exists therefore considerable interest to develop strategies for tailoring proteins into crystallizable domains. In the framework of a Structural Genomics Project on soluble yeast proteins, we have tested the expression of numerous genetic constructs of our targets in order to produce and crystallize proteins and protein domains and solve their three-dimensional structure. In some cases, the choice of the domain boundaries was guided by prediction from sequence using various software packages, including Prelink, a home-made prediction method for detecting unfolded regions. In other cases, large numbers of constructs were generated using molecular biology or biochemical methods. In this paper, we analyze the results of the over-expression in E. coli and crystallization of these constructs, and compare these with the predictions that can be obtained from our software and from others.

Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE.

The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation.

Recombinant protein expression and solubility screening in Escherichia coli: a comparative study.

Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His(6)-tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore ;expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization.

Crystal structure of the S. cerevisiae D-ribose-5-phosphate isomerase: comparison with the archaeal and bacterial enzymes.

Ribose-5-phosphate isomerase A has an important role in sugar metabolism by interconverting ribose-5-phosphate and ribulose-5-phosphate. This enzyme is ubiquitous and highly conserved among the three kingdoms of life. We have solved the 2.1 A resolution crystal structure of the Saccharomyces cerevisiae enzyme by molecular replacement. This protein adopts the same fold as its archaeal and bacterial orthologs with two alpha/beta domains tightly packed together. Mapping of conserved residues at the surface of the protein reveals strong invariability of the active site pocket, suggesting a common ligand binding mode and a similar catalytic mechanism. The yeast enzyme associates as a homotetramer similarly to the archaeal protein. The effect of an inactivating mutation (Arg189 to Lys) is discussed in view of the information brought by this structure.

Functional regions in the essential light chain of smooth muscle myosin as revealed by the mutagenesis approach.

The endogenous essential light chain (LC17) of myosin from intestine smooth muscle was replaced with mutated essential light chains prepared using recombinant techniques. Complete exchange was observed with histidine-tagged derivatives of LC17a, LC17b and E122A-LC17a (LC17a and LC17b are the usual constituants of smooth muscle myosin), with small changes in the ATPase activity of reconstituted myosins. Much less exchange was observed with the light-chain derivative lacking the last 12 amino acid residues, demonstrating the importance of this segment, which may act as one arm of a pair of pincers to bind the myosin heavy chain.

Role of the C-terminal extremities of the smooth muscle myosin heavy chains: implication for assembly properties.

The two light meromyosin isoforms from rabbit smooth muscle were prepared as recombinant proteins in Escherichia coli. These species which differed only by their C-terminal extremity showed the same circular dichroism spectra and endotherms in measurements of differential scanning calorimetry. Their solubility properties were different at pH 7.0 in the absence of monovalent salts. Their paracrystals formed at low pH differed by their aspect and number. These data suggest a role for the C-terminal extremity of myosin heavy chains in the assembly of myosin molecules in filaments and consequently in the contractility of smooth muscles.