Introduction
Protein engineering experiments involving fusion of different proteins/domains that do not naturally adjoin each other forces biotechnologists to have to choose suitable linker peptides, which can vary in length, complexity, and conformational characteristics. The success or failure of the fusion experiment is determined by the choice of linker peptide, for two primary reasons: (i) the linker can affect the function, conformation, and/or conformational stability of flanking proteins/domains; (ii) the linker can itself be naturally susceptible to undergoing proteolytic degradation by proteases present in the environment, or become susceptible to proteolysis (as a result of the influence of its flanking proteins/domains). In the work presented here, our objective was to focus upon differential proteolytic susceptibilities of different types of linkers, without distractions involving either (a) the effects of linkers upon conformations of flanking domains, or (b) effects of flanking domains upon conformations of the linkers, to the extent possible. Therefore, the approach we adopted was to use thermostable flanking proteins/domains that typically display autonomy of folding, as well as autonomy of conformational stabilization, in addition to high (natural) resistance to proteolysis [which is a natural corollary, because high thermal stability translates into a better-folded state, leading to lower scope for proteases to gain access to peptide bonds (Mukherjee and Guptasarma, 2005)]. In experiments presented below, through the use of thermostable flanking domains, we reduced the likelihood of proteolytic degradation in flanking domains. This allowed us to examine only the linker peptide for proteolytic degradation.
Two thermostable domains, (i) Coh2, a Type I cohesin fromClostridium thermocellum CipA, and (ii) BSX, a Bacillus sp. Xylanase, were joined. The structure of BSX consists of a single (β/α)8-fold barrel (Manikandan et al ., 2006). The structure of Coh2 consists of a nine-stranded barrel with jelly-roll fold topology, consisting of two flattened β-sheets (Carvalho et al ., 2003). Coh2 and BSX were fused with the use of five different linkers: a rigid linker, a flexible linker, and three linkers derived from the sequence of a 42 residues-long (native) linker joining the C-terminal end of Coh2, in Clostridium thermocellum CipA, to the N-terminal end of an adjoining carbohydrate binding module (CBM). Below, we show that the linker that is the most structurally-flexible and unstructured is also the linker that maximally escapes proteolysis. A systematic elimination of possibilities indicates that this counter-intuitive result owes to the linker’s facilitation of motions in its flanking domains. The motions appear to negatively affect the ability of proteases to approach the linker’s (otherwise scissile) peptide bonds.