Single-molecule force spectroscopy (SMFS) is by now well established as a standard technique in biophysics and mechanobiology. In recent years, the technique has benefitted greatly from new approaches to bioconjugation of proteins to surfaces. Indeed, optimized immobilization strategies for biomolecules and refined purification schemes are being steadily adapted and improved, which in turn has enhanced data quality. In many previously reported SMFS studies, poly(ethylene glycol) (PEG) was used to anchor molecules of interest to surfaces and/or cantilever tips. The limitation, however, is that PEG exhibits a well-known trans-trans-gauche to all-trans transition, which results in marked deviation from standard polymer elasticity models such as the worm-like chain, particularly at elevated forces. As a result, the assignment of unfolding events to protein domains based on their corresponding amino acid chain lengths is significantly obscured. Here, we provide a solution to this problem by implementing unstructured elastin-like polypeptides as linkers to replace PEG. We investigate the suitability of tailored elastin-like polypeptides linkers and perform direct comparisons to PEG, focusing on attributes that are critical for single-molecule force experiments such as linker length, monodispersity, and bioorthogonal conjugation tags. Our results demonstrate that by avoiding the ambiguous elastic response of mixed PEG/peptide systems and instead building the molecular mechanical systems with only a single bond type with uniform elastic properties, we improve data quality and facilitate data analysis and interpretation in force spectroscopy experiments. The use of all-peptide linkers allows alternative approaches for precisely defining elastic properties of proteins linked to surfaces.

Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal adhesion complexes formed at the cytoplasmic side of cell attachment to the ECM and is activated after force generation via actomyosin fibers attached to this complex. The mechanism of translating mechanical force into a biochemical signal is not understood, and it is not clear whether FAK is activated directly by force or downstream to the force signal. We use experimental and computational single-molecule force spectroscopy to probe the mechanical properties of FAK and examine whether force can trigger activation by inducing conformational changes in FAK. By comparison with an open and active mutant of FAK, we are able to assign mechanoactivation to an initial rupture event in the lowforce range. This activation event occurs before FAK unfolding at forces within the native range in focal adhesions. We are also able to assign all subsequent peaks in the force landscape to partial unfolding of FAK modules. We show that binding of ATP stabilizes the kinase domain, thereby altering the unfolding hierarchy. Using all-atom molecular dynamics simulations, we identify intermediates along the unfolding pathway, which provide buffering to allow extension of FAK in focal adhesions without compromising functionality. Our findings strongly support that forces in focal adhesions applied to FAK via known interactions can induce conformational changes, which in turn, trigger focal adhesion signaling.atomic force microscopy | mechanobiology | focal adhesion signaling | protein kinase regulation | single-molecule force spectroscopy