Interdomain linkers tailor the stability of immunoglobulin repeats in polyproteins

Abstract

Linkers in polyproteins are considered as mere spacers between two adjacent domains. However, a series of studies using single-molecule force spectroscopy have recently reported distinct thermodynamic stability of I27 in polyproteins with varying linkers and indicated the vital role of linkers in domain stability. A flexible glycine rich linker (-(GGG)n, n≥3) featured unfolding at lower forces than regularly used arg-ser (RS) based linker. Interdomain interactions among I27 domains in Gly-rich linkers were suggested to lead to reduced domain stability. However, the negative impact of inter domain interactions on domain stability is thermodynamically counter-intuitive and demanded thorough investigations. Here, using an array of ensemble equilibrium experiments and in-silico measurements with I27 singlet and doublets with two aforementioned linkers, we delineate that the inter-domain interactions in fact raise the stability of the polyprotein with RS linker. More surprisingly, a highly flexible Gly-rich linker has no interference on the stability of polyprotein. Overall, we conclude that flexible linkers are preferred in a polyprotein for maintaining domain's independence.

Polyprotein synthesis: a journey from the traditional pre-translational method to modern post-translational approaches for single-molecule force spectroscopy

“…As interdomain linkers are known to alter the stability and flexibility of protein monomer units in polyproteins, we should have control over the sequence and length of the linker. 23,59,60 Furthermore, these linkers are a potential active site for SortaseA or ligase OaAEP1 and prevent the growth of polyprotein formation and degrade the product back to its monomers. These methods provide highly dispersed polyproteins.…”

“…A presumption in these studies is the independent response of individual domains to mechanical perturbations. However, independent research groups across the globe have recently identified cooperativity in the folding dynamics of domains in a polyprotein using force-spectroscopy, [21][22][23][24] indicating the potential of SMFS as a tool to study the physical properties of polyproteins. It is noticed that the interdomain linkers in polyprotein constructs play a pivotal role in inducing cooperativity.…”