Detecting Disordered Regions in Proteins by Limited Proteolysis

Abstract

The limited proteolysis technique can be used to analyse protein structure and dynamics and, in particular, to identify disordered sites or regions within otherwise folded globular proteins. The approach relies on the fact that the proteolysis of a polypeptide substrate requires its binding and adaptation at the protease’s active site and thus enhanced backbone flexibility or local unfolding of the site of proteolytic attack. A striking correlation was found between sites of limited proteolysis and sites of enhanced chain flexibility of the polypeptide chain, this last evaluated by the crystallographically determined B-factor. It is herewith shown that often limited proteolysis occurs at chain regions characterized by missing electron density, thus indicating that protein disorder occurs at these regions. It is concluded that limited proteolysis is a very useful and reliable experimental technique that can be used to probe protein structure and dynamics and, in particular, to detect sites of disorder in proteins, thus complementing the results that can be obtained by the use of other physicochemical and computational approaches. The peculiar advantages of this simple biochemical technique include the requirement of very minute amounts of protein sample, thus when other experimental techniques cannot be used

Inhibition of Amyloid Aggregation by Formation of Helical Assemblies

“…Proteolytic cleavage generally occurs at solvent-exposed surfaces and flexible regions of a peptide, while compact hydrogen-bond-stabilized regular structures such as a-helices and b-sheets remain intact. [70] The amyloid core in particular shows resistance towards proteolytic cleavage because of its tightly packed b-strands. By adding different proteases to amyloid aggregates it is therefore possible to obtain valuable information regarding their structural features and dynamics.…”

Multiple Intrinsically Disordered Sequences Alter DNA Binding by the Homeodomain of the Drosophila Hox Protein Ultrabithorax

“…A protease will act primarily at unfolded, solvent-accessible peptides at least 10 amino acids long and containing its recognition sequence (38). Thus, unfolded or disordered regions can be identified by their extreme sensitivity to proteases relative to stable, folded proteins (55,56). Three proteases with different target sequences were tested to probe different sites within the UbxIb sequence and to ensure that global results are not dependent on the optimal buffer of the protease.…”

“…Three proteases with different target sequences were tested to probe different sites within the UbxIb sequence and to ensure that global results are not dependent on the optimal buffer of the protease. In order to monitor the progress of UbxIb proteolysis, the enzyme/substrate ratio had to be reduced to 1:1000 (w/w), a low ratio even for disordered proteins (38,55). For all three enzymes, full-length UbxIb was significantly digested within 5 min (Fig.…”

Comparative analysis of malate synthase G from Mycobacterium tuberculosis and E. coli: Role of ionic interaction in modulation of structural and functional properties

“…For this reason limited proteolysis has been effectively used to monitor structural domains in proteins, ligand-induced conformational changes, and protein folding/unfolding studies [16]. Limited proteolysis of MtbMS resulted in two fragment of molecular mass of about 48 and 30 kDa where as in case of ecMS three prominent fragments of about 50, 40 and 35 kDa, respectively along with many smaller fragments were observed (Fig.…”