The Y9P Variant of the Titin I27 Module: Structural Determinants of Its Revisited Nanomechanics

Abstract

The titin I27 module from human cardiac titin has become a standard in protein nanomechanics. A proline-scanning study of its mechanical clamp found three mechanically hypomorphic mutants and a paradoxically hypermorphic mutant (I27Y9P). Both types of mutants have been commonly used as substrates of several protein unfoldase machineries in studies relating protein mechanostability to translocation or degradation rates. Using single-molecule force spectroscopy based on atomic force microscopy, polyprotein engineering, and steered molecular dynamics simulations, we show that, unexpectedly, the mechanostability of the Y9P variant is comparable to the wild type. Furthermore, the NMR analysis of homomeric polyproteins of this variant suggests that these constructs may induce slight structural perturbations in the monomer, which may explain some minor differences in this variant's properties; namely the abolishment of the mechanical unfolding intermediate and a reduced thermal stability. Our results clarify a previously reported paradoxical result in protein nanomechanics and contribute to refining our toolbox for understanding the unfolding mechanism used by translocases and degradation machines.

Influence of Fluorination on Single-Molecule Unfolding and Rupture Pathways of a Mechanostable Protein Adhesion Complex

“…Additionally, TFL incorporation decreased thermal denaturation temperatures of both XMod-Doc­(WT)-TFL and XMod­(XL2V)-Doc-TFL by ∼8 °C. This significant decrease (15% on a °C scale) in thermal stability for XMod­(XL2V)-Doc-TFL is in contrast to the increase (7%) in mechano-stability of XMod that we observed under TFL incorporation (Figure J) and confirms that non-equilibrium mechanical stability does not necessarily need to be correlated with enhanced thermal stability. − …”

“…Mechanical stability describes how much tension a folded domain can withstand prior to unfolding, or how much force is required to dissociate a receptor-ligand complex. As prior work has shown, − mechanical stability is typically independent from thermostability, and mutant proteins with higher thermal stability are not necessarily more mechanostable. Single-molecule force spectroscopy (SMFS) − with the atomic force microscope (AFM) can be used to stretch single protein molecules, quantify intermediate folding states, , and elucidate [un]­folding energy landscapes while accounting for differences in loading geometry − or the presence of dual modes of ligand recognition. , The goal of this work was therefore to investigate the role of protein fluorination on non-equilibrium mechanostability, specifically investigating any discrepancies in trends between equilibrium thermodynamic stability and non-equilibrium mechanostability.…”

The hierarchical emergence of worm-like chain behaviour from globular domain polymer chains

“…For general SMFS experiments on polyproteins, it is often the case that the WLC model is used simply as a fitting model to validate the measurement of single protein properties, such as the peak forces and the peak-to-peak distances of the SMFS unfolding curves. Nevertheless, many SMFS experiments observe persistence lengths of around the length of a single amino acid: in titin 27,28,32,33 , general polyproteins [34][35][36] , and other structural motifs [37][38][39][40] , and this magnitude of persistence length corresponds well to that of individual, flexible amino acid chains 41,42 . Yet we have seen from the work of Li et al that in the absence of applied tensile force, the persistence length can be significantly larger.…”

Conformational entropy of a single peptide controlled under force governs protease recognition and catalysis

“…Design of a Single-Molecule Enzymatic Assay. We engineered a substrate consisting of a polyprotein (I27ΔTevSite) 8 that contains eight repeated I27 Ig domains from human titin (14)(15)(16).…”