First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value

Abstract

The EC `Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, ∼170 structures have been determined to date. Here, the impact of high‐throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.

SPINE high-throughput crystallization, crystal imaging and recognition techniques: current state, performance analysis, new technologies and future aspects

“…Experience with SPINE and from the Joint Center for Structural Genomics (JCSG) suggests that around one-third of proteins fall into this category Page et al, 2003) and in these cases the only way forward is some sort of modification to the protein itself. One common approach is the production of multiple constructs for targets of high value, although this is expensive and poses challenges in construct design (see Banci et al, 2006). A second approach is some sort of post-translational modification.…”

Honing the in silico toolkit for detecting protein disorder

“…However, since many structures in the PDB are partly disordered, the PDB is the largest source of disorder data and the basis for most analyses. In many cases where disorder is suspected, the best strategy for crystal production is to work with multiple constructs in parallel (see, for example, Banci et al, 2006), and disorder prediction has become an essential tool in the construct design process.…”

“…It may be noted that in CASP5, one of the submitted fully disordered proteins, Target 145 (Melamud & Moult, 2003), was in fact the cytoplasmic domain of the Drosophila adhesion protein gliotactin (Zeev-Ben-Mordehai et al, 2003), one of the targets included in SPINE workpackage 10 (Human Proteins of Biomedical Relevance; Banci et al, 2006). In CASP5, six methods were tested, whereas in 2004, for the CASP6 trial, 20 methods were evaluated.…”

The impact of protein characterization in structural proteomics

“…The first of these is particularly severe for the laboratories involved in the Structural Proteomics In Europe (SPINE) consortium, since many of the proteins targeted for structure determination by these groups are eukaryotic and the major 'workhorse' expression system is Escherichia coli. Indeed, this is reflected in the overall statistics for the SPINE project, which show some 30% of constructs yield soluble protein (Banci et al, 2006), rising to 89% for the expression of carefully chosen bacterial proteins (Alzari et al, 2006). Similarly, less than 30% of the total set of SPINE targets which express as soluble protein either crystallize or are suitable for NMR analysis (Banci et al, 2006), although success rates for subsets of favourable targets rise to 60% (for example, anthrax proteins; see Au et al, 2006).…”

“…Indeed, this is reflected in the overall statistics for the SPINE project, which show some 30% of constructs yield soluble protein (Banci et al, 2006), rising to 89% for the expression of carefully chosen bacterial proteins (Alzari et al, 2006). Similarly, less than 30% of the total set of SPINE targets which express as soluble protein either crystallize or are suitable for NMR analysis (Banci et al, 2006), although success rates for subsets of favourable targets rise to 60% (for example, anthrax proteins; see Au et al, 2006). Given this attrition rate, it is clearly vital to do everything possible to optimize the sample entered into crystallization (or NMR) screening.…”