1H, 15N and 13C backbone chemical shift assignment of titin domains A59–A60 and A60 alone

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.

Abstract The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is [1 lm 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 15 N, 13 C and 1 H chemical shift and 13 Cb 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.
Keywords Muscle protein Á Titin A-band Á Large superrepeat unit Á Fibronectin type III domain tandem

Biological and medical context
Titin, 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. The filamentous molecule spans from the Z-discs at the ends of the sarcomere (N-terminus) to the M-line in its middle (C-terminus), a distance of *1 lm. In the A-band titin is integral with thick filaments and is thought to control exact filament assembly (Whiting et al. 1989) via interactions with myosin, C-protein and other filament components. In the C-zone of the A-band the Ig and Fn domains are arranged in repeating patterns called large super-repeats: Ig-Fn-Fn-Fn-Ig-Fn-Fn-Fn-Ig-Fn-Fn (Labeit et al. 1992). Thus domains at the same positions in different super-repeats are most similar. This pattern is repeated 11 times, making 121 domains in total and forming nearly half the molecule. Next generation sequencing has recently shown that A-band titin is of great importance in heart disease. Mutations throughout this region are the commonest cause A. Czajlik of the important disease dilated cardiomyopathy, which affects 1 in 250 in the population and is a major cause of death (Herman et al. 2012).
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. Here we report chemical shift assignments of the backbones of the A59-A60 fibronectin type III (Fn3) double and A60sd single domain constructs using triple resonance NMR. 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, 1 H-15 N relaxation data and chemical shift perturbations, without the need for full sidechain assignment.

Methods and experiments
The A59-A60 domain tandem and A60sd single domain were cloned into the pET 15b_A170 and PET19b vectors, respectively. They were over-expressed as a N-terminal His6-tagged fusion protein in Escherichia coli strain BL21(DE3). A59-A60 was uniformly 15 N, 13 C and 2 H-labeled and A60sd was uniformly enriched in 15 N and 13 C. Cells were grown at 37°C in D 2 O-M9 minimal medium containing 15 NH 4 Cl (1 g/L) and either [100 %-D7 and 100 %-13 C] glucose (4 g/L) or [100 %-13 C] glucose (4 g/L) as the sole nitrogen and carbon sources. For induction of expression, 1 mM IPTG was added when the cells reached an OD 600 of 0.7, after which they were left shaking at 37°C for an additional 15 h. The cells were then disrupted by sonication in a 50 mM TRIS buffer (pH 7.9) containing 6 M guanidinium chloride, 0.5 M NaCl and 15 mM DTT and harvested by centrifugation. Finally the proteins were purified from the supernatant using Ni-NTA resin and eluted with 50 mM EDTA (pH 6.2). Typically 5 mg protein was purified from 1 l of A59-A60 culture. For A60sd yields were much higher, *15 mg/l of culture. Purity and isotope enrichment were checked by SDS-PAGE and mass spectrometry.

Assignments and data deposition
In order to prove that the secondary structure of the C-terminal domain of A59-A60 and A60sd are the same, their Ca chemical shifts were compared. The Ca chemical shifts for A60 from the A59-A60 tandem and A60sd were highly correlated (R 2 = 0.99) and, excluding changes at the termini where different flanking residues were present, only small shift changes were observed (Fig. 3A), with the largest being 0.57 ppm indicating very little change in overall secondary structure. However, consistent differences in chemical shifts (dCa C 0.3 ppm) are observed for the loops regions between b-strands A60-b2 and A60-b3, as well as between b-strands A60-b6 and A60-b7. This suggests that there is an inter-domain interaction. In order to examine the interaction between A59 and A60 further, the changes in chemical shift between the 1 H-15 N HSQC spectra of the C-terminal domain of A59-A60 and A60sd were calculated (Fig. 3B) using the chemical shift distance metric D = [(d15 N) 2 ? (5 9 d1H) 2 ] 0.5 . Aside from the termini, only the loop regions between b-strands A60-b2 and A60-b3, A60-b4 and A60-b5, as well as a number of residues in bsheets A60-b6 and b7, show a D [ 0.5 ppm, indicating that their environment has changed significantly. As these regions are localized to the N-terminal face of A60 (assuming a Fn3 fold) this suggests that there are distinct interactions between A59 and A60. Three further strands of evidence support a lack of flexibility between the two domains. Firstly from the observed secondary structure the linker between the b-sheets A59-b6 and A60-b1 is extremely short (residues 95-100). Secondly the observed correlation time of 21 ns for A59-A60 which was measured using a TRACT experiment is consistent with that expected for a rigid double domain construct with small amounts of a higher oligomer present (Tskhovrebova and Trinick 2003). Finally { 1 H}-15 N heteronuclear nOe measurements show that the linker region has nOes intensities covering a range from 0.77 ± 0.03 to 0.78 ± 0.03, which are in agreement with the observed nOes for the secondary structure elements in the same system which have an average value of 0.76 ± 0.05 over secondary structure elements. Overall these results clearly point to the conclusion that the A59-A60 double domain has an inflexible linker and a structured domain-domain interface. This observation therefore has important implications for the overall flexibility, structure and biological role of the large super repeat in titin and its role in myosin assembly. Backbone 1 H, 13 C and 15 N resonance assignments of titin A59-A60 tandem and A60sd single domain have been submitted to the BMRB (http://www.bmrb.wisc.edu) under accession numbers 19010 and 19011, respectively.