Titin is a large filamentous protein that forms a sarcomeric myofilament with a molecular
spring region that develops force in stretched sarcomeres. The molecular spring has a
complex make-up that includes the N2A element. This element largely consists of a 104-
residue unique sequence (N2A-Us) flanked by immunoglobulin domains (I80 and I81).
The N2A element is of interest because it assembles a signalosome with CARP (Cardiac
Ankyrin Repeat Protein) as an important component; CARP both interacts with the N2AUs
and I81 and is highly upregulated in response to mechanical stress. The mechanical
properties of the N2A element were studied using single-molecule force spectroscopy,
including how these properties are affected by CARP and phosphorylation. Three protein
constructs were made that consisted of 0, 1, or 2 N2A-Us elements with flanking I80
and I81 domains and with specific handles at their ends for study by atomic force
microscopy (AFM). The N2A-Us behaved as an entropic spring with a persistence
length (Lp) of 0.35 nm and contour length (Lc) of 39 nm. CARP increased the Lp
of the N2A-Us and the unfolding force of the Ig domains; force clamp experiments
showed that CARP reduced the Ig domain unfolding kinetics. These findings suggest
that CARP might function as a molecular chaperone that protects I81 from unfolding
when mechanical stress is high. The N2A-Us was found to be a PKA substrate, and
phosphorylation was blocked by CARP. Mass spectrometry revealed a PKA phosphosite
(Ser-9895 in NP_001254479.2) located at the border between the N2A-Us and I81.
AFM studies showed that phosphorylation affected neither the Lp of the N2A-Us nor
the Ig domain unfolding force (Funfold). Simulating the force-sarcomere length relation of
a single titin molecule containing all spring elements showed that the compliance of the
N2A-Us only slightly reduces passive force (1.4%) with an additional small reduction
by CARP (0.3%). Thus, it is improbable that the compliance of the N2A element
has a mechanical function per se. Instead, it is likely that this compliance has local
effects on binding of signaling molecules and that it contributes thereby to strain- and
phosphorylation- dependent mechano-signaling.
