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

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. •I27Y9P mechanostability is similar to the wild-type and different from that published•NMR can be fully applied to the high-resolution structural analysis of polyproteins•NMR studies show that polyproteins may induce structural changes in the monomer•Structural data from the I27Y9P polyprotein fully explain its nanomechanics The polyprotein strategy can alter the structure of the protein domain under study. Oroz et al. prove that NMR spectroscopy is a good method to test this possibility. Using this approach, they show that the titin I27Y9P variant has similar mechanical stability to the wild-type.