Investigating the role of linkers in the mechanoresponse of multidomain proteins

Abstract

Mechanical force is a critical cue in biology that regulates various cellular processes. Cells utilize mechanotransduction mechanisms to convert mechanical signals into biochemical responses, which is crucial for maintaining proper cellular functions and responding to environmental changes. Mechanosensitive proteins act as essential mediators in the process of mechanotransduction, allowing cells to sense and respond to mechanical forces. Understanding the mechanism and function of mechanosensitive proteins is essential for elucidating how these forces shape the biological outcomes. Mechanosensitive proteins are typically organized in a modular fashion, with one domain connected to another by a linker region. While the roles of these domains in power generation and elasticity are well understood in mechanosensing proteins, the significance of inter domain linkers (IDLs) not widely acknowledged. This research emphasizes the physiological significance of linkers in force-sensing proteins, as mutations within the linker region have been associated with disease phenotypes, thereby emphasizing the pivotal role that linkers play in the overall functionality of these proteins. In-silico studies and biophysical techniques such as SAXS, CD, and fluorescence spectroscopy were employed to evaluate the conformational variations and thermodynamic stability of proteins in the presence of different linker variants. Additionally, single-molecule magnetic tweezers measurements were utilized to elucidate how linkers regulate the mechanoresponse of proteins during both constant and oscillatory force perturbations. We investigated three engineered constructs each with varying IDL region, regulating the flexibility and interdomain interactions in proteins. Notably, the more cooperative variant with inter-domain interactions endures higher tensile forces and shows faster folding kinetics. However, it gets fatigued faster than the rest under a long-term oscillatory force perturbation. Overall, our findings indicate that adjusting the chemical and physical nature of peptide linkers may have been favored by natural selection for regulating the mechanical characteristics of proteins.