Titin and nebulin are unprecedented giant proteins discovered and named in this laboratory. Our recent work has focused on the roles of titin and nebulin in the structure, regulation, mechanics and assembly of sarcomeres in striated muscles. We have employed a multidisciplinary approach that draws on methodologies and concepts of biochemistry, biophysics, cell and molecular biology, and physiology. (A) Titin: A primary theme of our research is the understanding of the structural and physiological roles of titin, a family of giant structural proteins that constitute an elastic matrix in the striated muscle sarcomeres and some nonmuscle cells. Titin and the elastic matrix may play major physiological roles, including the genesis of long range elasticity, the maintenance of sarcomere stability, the assembly of nascent sarcomeres in developing muscle cells, and the assembly of myosin in the microfilaments in nonmuscle cells. The following questions of central importance are being addressed: 1. How do titin filaments respond to stretch and release? 2. What is the molecular basis of titin elasticity? 3. Do thin filaments modulate titin elasticity? 4. Does titin participate in the regulation of actin-myosin interactions? 5. How do the signaling capacity of titin and titin kinase unction in muscle? We are testing the hypothesis that reversible unfolding of ordered domains such as titin kinase and reversible unraveling of intrinsically disordered segments are two distinct and complementary mechanisms of titin elasticity and function. The elasticity of isolated full length titin and engineered fragments and analogous elastic molecules from skeletal and cardiac and flight muscles are being measured directly, as single molecules and as gel networks, with laser trap techniques, atomic force microscopy (AFM) and rheometric techniques. The conformation of titin domains and synthetic peptides are being studied by high resolution NMR and X-ray crystallography. Titin elasticity is being explained with theories of biopolymers combined with realistic and experimental structural features of titin domains. In the past two years, we have succeeded in revealing that the elastic PEVK segment of titin is modular in construction, display repeating proline II helix /coil conformational motifs and most importantly, interact with thin filaments in a calcium/S100 sensitive manner. Our observations established the conformational basis of titin PEVK elasticity and identified PEVK as a site of interfilament interaction and that S100 or calcium sensor proteins regulate interaction and titin elasticity. Moreover, we discovered that the titin PEVK segment is a giant force sensing molecule of the SH3 signaling pathways. High resolution structures of a homopolymer of a PEVK module in solution and in gel phase has been determined by NMR techniques in collaboration with Prof. Wittebort at University of Louisville. The extensive differential splicing of titin PEVK exons in various human heart muscles was also elucidated. The PEVK domain is an intrinsically disordered protein with short-range structure. AFM measurements on PEVK suggested that its elasticity was driven in part by salt bridges. NMR data demonstrated the existence of specific salt bridges. Using the NIH Biowulf cluster, we applied molecular modeling techniques to understand dynamic interactions at the atomic level. TheMD and SMD simulations have shown further that the manifold of ionic interactions constantly evolves throughout the stretching. These new insights into the behavior of salt bridges in titin PEVK may provide insights in designing elastic polyampholytes as biomaterials for nanotechnology and tissue engineering. These simulations also help us to understand how ligand binding can be regulated by force and only bind when the protein is stretched to unmask SH3 binding sites. We are also designing a new type of instrrument to measure ligand binding and protein-protein interactions as proteins are being stretched. This new instrument will allow us to investigate directly how signaling protein interactions respond to mechanical stress. (B) Nebulin: Nebulin is the second largest protein ( 0.7 MDa) in the skeletal muscle sarcomere. It acts as a ruler for thin filament length and may act as a calcium-linked regulatory protein that is distinct from the classic troponin/tropomyosin system. In muscle development, nebulin is thought to expedite the maturation process of the thin filament assembly. Mutation in the nebulin gene leads to the disease nemalin myopathy, which causes muscle weakness and early death. The following questions are being addressed: 1. How does nebulin regulate the length of thin filaments? 2. How does nebulin regulate actin-myosin interaction? 3. What role does the nanomechanics of nebulin play in muscle function The length, mass and sequence of nebulin size isoforms have been compared with thin filament lengths of a wide range of skeletal muscles in developing and adult muscles. Together with the effect of over expression of nebulin domains on myofibrillogenesis in cultured skeletal muscle cells, we concluded that both the number of nebulin repeats and the specific N-terminal and C-terminal termination sequences are necessary to give rise to uniform thin filament length distribution within each sarcomere. In the past two years, we have successfully purified native full length nebulin from skeletal muscles and tested its effect on the length distribution of polymerizing or performed actin filaments. The measurement of actin filment length unfortunately was complicated by the gelling of actin filaments by the multivalent actin binding sites on each nebulin molecule. To alleviate this roblem, we are designing a new nanotechnology-based platform to measure the effect of nebulin on actin filament length distribution on a nanopatterned substrate that allows parallel actin filaments to elongate in the same direction and polarity without gelation. The kinetics of nebulin on ATPase activities is being studied by stop flow methods to define plausible molecular mechanisms of action. The influence of nebulin and calcium/calmodulin on the sliding velocity of actin over myosin is being investigated to reveal the effect of nebulin on mechanical coupling of myosin motors. We are searching for other potential calcium mediators by affinity chromatography and blotting. We are particularly interested in the possible involvement of S100 proteins as well as the kinase/phosphotase that phosphorylate nebulin as physiological effectors of actin/myosin interaction. We measured the nanomechncial properties of full-length native nebulin with the AFM. These required the implementation of the use of high affinity sitespecific antibodies pairwise to stretch nebulin between the nebulin;the use nof protein resistant self-assembled monolayers to prevent non-specific tip-surface interaction, a new method of sorting out single vs. multiple molecular stretching and the adaption of a powerful empirical mode decomposition (HHT) method to identify force-generating events. The most unique and unexpected finding is that nebulin must be stretched long enough to wrap around and match the peroriodicity of F-actin. We believe that these studies of full length nebulin and nebulin fragments will lead to a deeper understanding of how nebulin interacts with actin, myosin and other cellular components in the multiple roles it plays in muscle function. These results and the technology platform can also be applied to other members of the nebulin family, such as NRAP and cortactin which plays a role in muscle assemblyin muscle assemblyin muscle assembly, cell motility and hin muscle assembly, cell motility and has been implicated in muscle diseases and metastasis of cancer.