A physiological role for titin and nebulin in skeletal muscle

Abstract

Production of active force in skeletal muscle results from the interaction of myosin-containing thick filaments with actin-containing thin filaments. These muscles are also passively elastic, producing forces that resist stretch independently of ATP splitting or of interaction between the filaments. The mechanism of this passive elasticity is unknown; suggestions include gap filaments in the region between thick and thin filaments in muscles stretched beyond filament overlap, or intermediate filaments which connect successive Z-disks. Recently, the two exceptionally large proteins titin (also called connectin) and nebulin (originally called band 3) have been implicated in passive elasticity (for review see refs 7, 8). Here, we show that after these proteins are degraded by low doses of ionizing radiation, the ability of single skinned muscle cells to generate both passive tension in response to stretch and active tension in response to calcium is greatly reduced. These effects are accompanied by axial misalignment of thick filaments. Titin and/or nebulin apparently provide axial continuity for the production of resting tension on stretch and also tend to keep the thick filaments centred within the sarcomere during force generation.

Studies on titin PEVK peptides and their interaction

“…The IDP have a number of characteristic properties: (1) aberrant molecular weight as determined by SDS gel electrophoresis [39] or gel permeation [39,40]; (2) high net charge at neutral pH and low content of hydrophobic amino acids [41]; (3) CD spectra that are similar to denatured or random coil proteins (minima near 200 nm and lack of minima in the 210-230 nm range typical of alpha helix and beta structure) [38]; (4) amino acid compositional bias with higher contents of lysineglutamic acid -proline and lower amounts of aromatic residues [42,43]; (5) absence of cooperative folding transitions when the protein is heated [39]; (6) sensitivity to proteolytic degradation [39]. Conditions (1), (2), (3), and (4) are clearly met by the polyE peptides E115 and E137 since they have elevated SDS gel apparent molecular mass (35 KDa vs 18 KDa actual and 15 KDa vs 8 KDa actual respectively, Table 2 and Fig. 1), high negative charge due to the preponderance of glutamic acid residues, CD spectra that fit the typical disordered pattern, and amino acid compositional bias.…”

“…It has multiple functions including sarcomere assembly, sarcomere stability maintenance [1], passive tension generation [2,3] and serine/threonine kinase activity [4,5]. The over 3 million Dalton molecular weight makes it the largest protein in nature.…”

Calmodulin‐binding profiles for nebulin and dystrophin in human skeletal muscle

“…Until recently it was believed to play a purely structural role in stabilising the myofilament lattice. However direct evidence from radiation inactivation experiments suggests that both nebulin and titin (another highmolecular-mass myofibrillar protein) are associated with the maintenance of resting tension within the muscle fibre [16]. Titin and nebulin can be phosphorylated, although only 15% of total titin phosphates exchanged with cytosolic ATP within 3 days; it has been suggested that the majority of phosphorylation sites are therefore more likely to have a structural, rather than a regulatory role [17].…”

Global configuration of single titin molecules observed through chain-associated rhodamine dimers

“…Titin is a tandem array of Ig type C2 and fibronectin (FN) type III domains (Ϸ300 per molecule) interspersed with unique sequences, most notably the Pro-, Glu-, Val-, and Lys-rich PEVK segment, and the N2A and N2B segments (5). The I-band segment of titin acts as a molecular spring, whose elastic properties define the passive or restoring mechanical properties of striated muscle (6)(7)(8)(9). By contrast, titin's A-band segment is thought to function as a scaffold that defines structural regularity within the A band (10).…”