Studying biomolecules on surfaces is of great importance in the design of biosensors, advanced materials, and bioassays. Single molecule techniques, in particular, enable the understanding of properties and dynamic behavior that are usually obscured and inaccessible through ensemble (bulk) experiments. The nanomechanics of proteins has been studied via single molecule force spectroscopy on surfaces to identify energy landscapes and to reveal unique structural or temporal states of molecules under stress.(1-5) Atomic force microscopy (AFM)(6) is an ideal tool for imaging single molecules(7, 8) as well as studying molecular interactions and nanomechanics. Application of force spectroscopy(9, 10) to molecules such as titin,(1) myosin,(11, 12) and signaling proteins(13-17) has successfully demonstrated molecular rupture forces,(18) protein domain unfolding,(19-24) and receptor–ligand interactions.(5, 25-27)
Force spectroscopy of biopolymers requires the attachment of polymers of sufficient length between the tip and surface as well as the stretching of a single or a few polymers at a defined loading rate. Protein attachment has been the subject of intensive investigation. The commonly used physisorption on substrate allows polymers to be adsorbed and tethered by AFM cantilever but only at random, unspecified locations and yields hard-to-interpret force curves. The use of site-specific antibodies directed to protein sequences allows for tethering and stretching the protein between known sites.(28-30) Force curves of definitive single molecule events are even more challenging to achieve consistently.(31) Single molecule events are enhanced by reducing the surface density of attachment with either dilute solutions for physisorption or with limiting number of chemically reactive sites.(32) Tip–surface or adhesive interactions can obscure the first portion of the force spectrum,(32, 33) thus allowing only long polymers to yield any interpretable data. Hence, some of the first force spectra of single molecules were from long biopolymers such as polysaccharides,(34) DNA,(35) and titin(1) (the longest known protein). Refinement of protein immobilization techniques and surfaces have greatly reduced the obscuring effects of tip–surface adhesion, thereby allowing smaller proteins to be studied.(31, 32) For large modular proteins where there are significant conformational data on the fundamental domains, each of these domains can be studied by the use of engineered polyproteins of 8–12 identical domains.(36) Polymers of tandem repeats of identical domains render regularly spaced force events (e.g., the sawtooth pattern) and can be interpreted in fine detail by the unfolding of a single domain, e.g., from titin Ig/Fn3.(1, 37)
While these engineered polyproteins have been powerful in revealing the mechanism of mechanical unfolding of protein domains, many cytoskeletal proteins consist of numerous distinct modules that may interact intimately and fulfill collectively complicated mechanical roles in cells. Therefore, direct nanomechanical measurements of the full length, intact protein are required to understand biomechanics at the cellular and molecular levels. Such force curves are composites of many overlapping force events and are too complex to interpret with any degree of confidence. Dynamics stiffness measurement, taken simultaneously with the force curves, is a complementary technique that holds promise of resolving some of the complexity.(20, 38)
The giant modular protein nebulin (MW 700–800 kDa) plays a critical role in the structure and function of thin filaments and active contraction of skeletal muscles (Figure 1A).(39) Mutations in the nebulin gene have been linked to the muscle disease nemaline myopathy.(40) Nebulin associates with actin to form a 1 μm long composite thin filament in skeletal muscles and is thought to act as a protein ruler to regulate thin filament length,(41, 42) and perhaps as a Ca2+/calmodulin or S100-mediated regulatory protein.(43, 44) The protein (e.g., 6669 residues in the human adult nebulin isoform(45)) comprises highly homologous repeats of 35 amino acids that correlate with the number of actin monomers in the thin filament.(45, 46) Biochemical, structural, and developmental studies support the notion that nebulin, in conjunction with tropomodulin and other actin capping proteins, regulates thin filament length, but the architecture of nebulin-containing composite thin filaments remains speculative.(47, 48)
Nebulin is an attractive candidate for study by force spectroscopy because it plays a direct role in the force generating machinery of muscle and is likely to play a mechanical role in the stability and function of the muscle sarcomere. Force spectroscopy would help identify those structural features and transitions that are important in these mechanical roles. Because of its intimate association with actin, myosin, and its anchorage into the Z-bands of the muscle sarcomere, the isolation of native nebulin has remained elusive, and it is still much too large for recombinant expression in toto. Through the use of the mild detergent deoxycholate, we have successfully extracted and purified full-length, native nebulin from rabbit muscle for the first time. In the course of our study on the nanomechanical properties of nebulin, we have approached and resolved several key technical obstacles for investigating giant, modular proteins in general: (1) the use of pairs of antibodies to either termini or internal site to tether selected regions of the full-length nebulin; (2) the use of self-assembled monolayer surfaces (OEG-SAM) to isolate and covalently tether single proteins while avoiding tip–surface interactions and adsorption of protein to the surface;(32) (3) the analysis of the entire set of force curves without bias to establish empirical criteria for identifying single molecule events versus multiple molecule events; (4) the use of the adaptive, model independent HHT(49-51) method to search for periodicity of key mechanical events in the force spectra.