Use of Limited Proteolysis to Identify Protein Domains Suitable for Structural Analysis

Abstract

Publisher Summary This chapter explains the use of limited proteolysis to identify protein domains suitable for structural analysis. The field of structural proteomics arose with the availability of complete genomic sequences for several organisms and high-throughput methods to purify recombinant forms of their proteins. The aim of this research area is to solve the structure of every protein using either experimental or computational methods. Moreover, there is an overall bias in these studies with the vast majority of solved structures representing peptides less than 30 kDa in size. This bias may reflect the initial selection process of candidate genes or the greater propensity for smaller peptides to yield samples suitable for crystallography or nuclear magnetic resonance (NMR) spectroscopy. This chapter illustrates that for NMR analyses intrinsic limitations on the size of molecules that can be studied often necessitate analysis of individual domains. Protein domains have additional features that render them attractive targets for structural analysis. Finally, this chapter demonstrates that domain mapping by limited proteolysis is a powerful complement to the more common methods of sequence conservation and deletion mutagenesis. The process is rapid, requires minimal sample handling, and is highly adaptable to high-throughput analysis.