Increased proteolytic resistance of ribonuclease A by protein engineering

Abstract

Although highly stable toward unfolding, native ribonuclease A is known to be cleaved by unspecific proteases in the flexible loop region near Ala20. With the aim to create a protease-resistant ribonuclease A, Ala20 was substituted for Pro by site-directed mutagenesis. The resulting mutant enzyme was nearly identical to the wild-type enzyme in the near-UV and far-UV circular dichroism spectra, in its activity to 2',3'-cCMP and in its thermodynamic stability. However, the proteolytic resistance to proteinase K and subtilisin Carlsberg was extremely increased. Pseudo-first-order rate constants of proteolysis, determined by densitometric analysis of the bands of intact protein in SDS-PAGE, decreased by two orders of magnitude. In contrast, the rate constant of proteolysis with elastase was similar to that of the wild-type enzyme. These differences can be explained by the analysis of the fragments occurring in proteolysis with elastase. Ser21-Ser22 was identified as the main primary cleavage site in the degradation of the mutant enzyme by elastase. Obviously, this bond is not cleavable by proteinase K or subtilisin Carlsberg. The results demonstrate the high potential of a single mutation in protein stabilization to proteolytic degradation.

Performance of protein stability predictors

“…Stabilizing mutations in active site residues can reduce enzymatic activities [Beadle and Shoichet, 2002;Counago et al, 2008;Garcia et al, 2000;Kidokoro et al, 1995;Meiering et al, 1992;Mukaiyama et al, 2006;Nagatani et al, 2007;Schreiber et al, 1994, Shoichet et al, 1995Zhi et al, 1991]. Additionally, a stabilizing mutation increased the resistance of ribonuclease A to proteolysis [Markert et al, 2001], which, for example, would be an undesirable effect if it occurred in enzymes involved in cell signaling [Fink, 2005].…”

Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy

“…Such a suggestion is in accordance with earlier observations that proteolytic and thermal stability of proteins generally are correlated because unfolded proteins expose flexible regions that are more sensitive to proteolysis (Arnold and Ulbrich-Hofmann 1997;Amin et al 2004). Furthermore, the proteolytic stabilization of certain enzymes by protein engineering was due to reduced flexibility of the protein region that was prone to proteolysis (Frenken et al 1993;Markert et al 2001).…”

Probing protein stability and proteolytic resistance by loop scanning: A comprehensive mutational analysis

“…22,39 Such linkage is apparent since both thermostability and protease susceptibility originate from the dynamics associated with the protein. 9,[22][23][24] Moreover, proteolytic susceptibility could be independent of stability when proteolysis is due to subglobal fluctuations. 24 To get better insight into the phenomenon, proteolytic resistance of the selected 17 single thermostable mutants (STMs) of lipase was probed by measuring residual activities upon incubation with a nonspecific protease, Subtilisin A.…”

“…2,6 Mutational approaches to study this relation are based on limited set of mutants, both in terms of numbers as well as positions. [7][8][9] Clearly, a systematic probing into the phenomenon of protease susceptibility/resistance and its relation to protein stability using protein wide mutations, with least possible sequence differences that alter the physical properties of native and unfolded states to minimum, will be advantageous. Proteolytic susceptibility of the protein has been exploited in structural characterization using limited proteolysis.…”