Peter T. Beernink

Applications of Functional Protein Microarrays

The use of protein arrays and their importance in proteomic applications continues to be at the forefront of scientific discovery and innovative technology development. To date, array-based approaches have proven to be a powerful tool for protein expression profiling, novel biomarker discovery, and the examination of protein, DNA, and small molecule interactions. Our laboratory has developed several approaches for characterizing protein-protein interactions using protein microarrays for a variety of different biological applications. Here we describe the identification of protein-protein interactions using a microarray format.

Application of In Vitro Protein Expression to Human Prote

INTRODUCTION. A critical step in many proteomics projects is the identification of genes that express proteins in sufficient quantities for subsequent studies. LLNL houses a collection of >200,000 human cDNAs, known as the IMAGE Consortium (http://image.llnl.gov)[1], including some full-length enriched cDNA libraries. We have developed an integrated system that exploits coupled in vitro transcription/translation (IVT) to identify human IMAGE cDNAs that express proteins of the predicted mass. Moreover, this expression screening can be conducted without plasmid DNA preparation or subcloning. IVT-produced proteins are used directly in functional assays or the cDNAs are transported to a bacterial expression system for large-scale protein production.

Application of Cell-free Expression Systems to Proteomic Studies

The promise of proteomics is to identify and characterize the physical and functional properties of proteins and protein complexes in parallel. Many proteomics efforts require the production of large numbers of purified proteins for biochemical or physical analyses. In particular, structural proteomics requires milligram quantities of highly purified proteins. However it has been established by pilot studies that soluble protein expression is one of the bottlenecks in structural proteomics processes (Fig. 12.1) [1]. Many proteins are inherently poorly expressed, insoluble, cytotoxic or subject to proteolysis, which results in low soluble expression in vivo. Cell-free protein expression strategies can overcome some of these problems and yield a larger number of expressed proteins [2]. Cell-free expression can also be used to identify rapidly well-expressed proteins and to obtain proteins for biochemical and structural studies.

Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: Implications for allosteric regulation

A central problem in understanding enzyme regulation is to define the conformational states that account for allosteric changes in catalytic activity. ForEscherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) the active, relaxed (R state) holoenzyme is generally assumed to be represented by the crystal structure of the complex of the holoenzyme with the bisubstrate analogN-phosphonacetyl-L-aspartate (PALA). It is unclear, however, which conformational differences between the unliganded, inactive, taut (T state) holoenzyme and the PALA complex are attributable to localized effects of inhibitor binding as contrasted to the allosteric transition. To define the conformational changes in the isolated, nonallosteric C trimer resulting from the binding of PALA, we determined the 1.95-Å resolution crystal structure of the C trimer-PALA complex. In contrast to the free C trimer, the PALA-bound trimer exhibits approximate threefold symmetry. Conformational changes in the C trimer upon PALA binding include ordering of two active site loops and closure of the hinge relating the N- and C-terminal domains. The C trimer-PALA structure closely resembles the liganded C subunits in the PALA-bound holoenzyme. This similarity suggests that the pronounced hinge closure and other changes promoted by PALA binding to the holoenzyme are stabilized by ligand binding. Consequently, the conformational changes attributable to the allosteric transition of the holoenzyme remain to be defined.