Titin (also known as connectin) is a giant protein with a wide range of cellular functions, including providing muscle cells with elasticity. Its physiological extension is largely derived from the PEVK segment, rich in proline (P), glutamate (E), valine (V), and lysine (K) residues. We studied recombinant PEVK molecules containing the two conserved elements: Ϸ28-residue PEVK repeats and E-rich motifs. Single molecule experiments revealed that calcium-induced conformational changes reduce the bending rigidity of the PEVK fragments, and site-directed mutagenesis identified four glutamate residues in the E-rich motif that was studied (exon 129), as critical for this process. Experiments with muscle fibers showed that titin-based tension is calcium responsive. We propose that the PEVK segment contains E-rich motifs that render titin a calciumdependent molecular spring that adapts to the physiological state of the cell.T itin comprises a multifunctional myofilament system in muscle, with a single molecule spanning the half-sarcomere (1-5). A large portion of the molecule functions as a molecular spring that, when extended, develops force. This force underlies the passive muscle force, which maintains the structural integrity of the contracting sarcomere and influences the filling behavior of the heart (3). Physiological force levels are largely determined by extension of the proline-glutamate-valine-lysine (PEVK) rich segment (6, 7). PEVK-like sequences are found in many titin-like proteins among evolutionary divergent organisms (8-11). The PEVK region of the human titin gene contains 114 exons; most code for conserved Ϸ28-residue PEVK repeats and 10 are more complex and encode E-rich motifs (12, 13). Here, we studied recombinant proteins that contain both PEVK repeats and glutamate (E)-rich motifs. Previous work suggests that the PEVK segment binds calcium with high affinity, raising the possibility that the extensibility of the PEVK segment may be calcium regulated (14). Thus, an important goal was to examine the effect of calcium on the mechanical behavior of the PEVK segment.
MethodsProteins. We engineered only PEVK sequences (''naked'' PEVK fragment) or PEVK sequences flanked by Ig-like domains (PEVK-Ig fusion fragment). Fragments were cloned, expressed, and purified by using routine methods (12, 15). All fragments were His-tagged at their amino terminus and, except when intended for structural analysis, had two Cys residues at the carboxyl terminus for covalent attachment to gold-coated substrates. Purified proteins were dialyzed into AB buffer (in mM; 25 Mops, pH 7.4͞150 KCl͞1 EGTA͞1 DTT), quick frozen, and stored at Ϫ80°C. For additional details, see Supporting Text, which is published as supporting information on the PNAS web site.Single Molecule Mechanics. Molecules were stretched by using an atomic force microscope specialized for stretching molecules (15, 16). Proteins (Ϸ100 g͞ml) were allowed to bind for Ϸ10 min to gold-coated microscope slides (precleaned glass slides and freshly cleaved mica were also...