Abstract
The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is >1 μm long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains. These are mostly arranged in long-range patterns or ‘super-repeats’. The large super-repeats each contain 11 domains and are repeated 11 times, thus forming nearly half the titin molecule. Through interactions with myosin and C-protein, they are involved in thick filament assembly. The importance of titin in muscle assembly is highlighted by the effect of mutations in the A-band portion, which are the commonest cause of dilated cardiomyopathy, affecting ~1 in 250 (Herman et al. in N Engl J Med 366:619–628, 2012). Here we report backbone 15N, 13C and 1H chemical shift and 13Cβ assignments for the A59–A60 domain tandem from the titin A59–A69 large super-repeat, completed using triple resonance NMR. Since, some regions of the backbone remained unassigned in A60 domain of the complete A59–A60 tandem, a construct containing a single A60 domain, A60sd, was also studied using the same methods. Considerably improved assignment coverage was achieved using A60sd due to its lower mass and improved molecular tumbling rate; these assignments also allowed the analysis of inter-domain interactions using chemical shift mapping against A59–A60.
Highlights
The giant protein titin is the third most abundant protein of vertebrate striated muscle
In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains
Through interactions with myosin and C-protein, they are involved in thick filament assembly
Summary
A major component of vertebrate striated muscle sarcomeres, is the largest known polypeptide, with isoforms up to nearly 4 MDa (Tskhovrebova and Trinick 2003) This is mostly folded into a string of about 300 immunoglobulin (Ig) and fibronectin (Fn, type III)-like domains, both of which have b-sandwich folds of 7–8 strands and about 100 amino acids. Our long-term aim is to determine the structure and dynamical properties of the A59–A69 large super-repeat unit using overlapping two and three domain constructs (Tskhovrebova et al 2010; Czajlik et al 2012) By inference, this should allow modeling of the entire large superrepeat region spanning 0.5 lm. Assignments of backbone resonances in these molecules provide a basis for understanding inter-domain flexibility, domain-domain interactions and the formation of higher oligomer in the system, based on homology models and chemical shift based backbone folds using residual dipolar couplings, paramagnetic relaxation enhancements, 1H–15N relaxation data and chemical shift perturbations, without the need for full sidechain assignment
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have