Identification of a Mechanical Rheostat in the Hydrophobic Core of Protein L

Abstract

The ability of proteins and their complexes to withstand or respond to mechanical stimuli is vital for cells to maintain their structural organisation, to relay external signals and to facilitate unfolding and remodelling. Force spectroscopy using the atomic force microscope allows the behaviour of single protein molecules under an applied extension to be investigated and their mechanical strength to be quantified. protein L, a simple model protein, displays moderate mechanical strength and is thought to unfold by the shearing of two mechanical sub-domains. Here, we investigate the importance of side-chain packing for the mechanical strength of protein L by measuring the mechanical strength of a series of protein L variants containing single conservative hydrophobic volume deletion mutants. Of the five thermodynamically destabilised variants characterised, only one residue (I60V) close to the interface between two mechanical sub-domains was found to differ in mechanical properties to wild type (ΔFI60V–WT = − 36 pN at 447 nm s− 1, ΔxuI60V–WT = 0.2 nm). Φ-value analysis of the unfolding data revealed a highly native transition state. To test whether the number of hydrophobic contacts across the mechanical interface does affect the mechanical strength of protein L, we measured the mechanical properties of two further variants. protein L L10F, which increases core packing but does not enhance interfacial contacts, increased mechanical strength by 13 ± 11 pN at 447 nm s− 1. By contrast, protein L I60F, which increases both core and cross-interface contacts, increased mechanical strength by 72 ± 13 pN at 447 nm s− 1. These data suggest a method by which nature can evolve a varied mechanical response from a limited number of topologies and demonstrate a generic but facile method by which the mechanical strength of proteins can be rationally modified.

Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments

“…3(b), it is clear that the probability of seeing two unfolding events in a force-extension trace is significantly higher than the probability of seeing five unfolding events. This is in contrast to the MC simulations typically used to fit the data from these experiments, where an equal probability of measuring between two and five unfolding events is assumed [32]. The unequal unfolding probability measured in Fig.…”

“…4(b), empty symbols] [32]. These simulations use the assumption that there is an equal probability of picking up and unfolding any length of polyprotein chain, from two domains up to five, through a nonspecific interaction between the tip and the polyprotein.…”

“…Monte Carlo (MC) simulations were performed in Igor Pro (Version 6, Wavemetrics, Lake Oswego, OR) using modifications to code previously developed to obtain estimates for the coarse parameters of the mechanical unfolding energy landscape for polyproteins containing two different protein domains [32]. Each protein domain is assumed to unfold via a two-state, all-or-none process, given by the following adaptation of the Zhurkov-Bell model:…”

“…1(d)]. As unfolding is a kinetically controlled process, measuring the speed dependence of unfolding can yield information on the basic parameters of the one-dimensional energy landscape of the protein, namely the unfolding rate constant [k U (0)] and the distance from the native state of the protein to the mechanical unfolding transition state ( x U ) [32] [ Fig. 1(e)].…”

Towards design principles for determining the mechanical stability of proteins

“…Mechanical clamps have since been identified in many 75 other proteins 9, [71][72][73][74] . Further studies have examined side chain packing and long-range interactions in topologically similar proteins 52 , hydrophobic packing in the hydrophobic core of a protein 75 , solvent accessibility of hydrogen bonds 76 , non-native interactions 71 and bond patterns as well as the identification of 80 "strong" and "weak" sequence motifs in protein families 24,25,[76][77][78][79] . …”