The arrangement of I-band titin in the sarcomere and the existence of titin spring elements in a monomeric state. (A) Schematic of a half sarcomere and the I-band domains of the three main human titin isoforms. The recombinant titin constructs generated for the various in vitro assays of this study are shown. (B–E) Continuous sedimentation-coefficient distributions, c(s), calculated from sedimentation-velocity experiments for purified recombinant titin constructs in medium ionic-strength buffer at 20°C. (B) Titin N2-Bus (13 µmol/l) at 52,000 rpm; (C) N2-B (5 µmol/l) at 45,000 rpm; (D) N2-A (9.5 µmol/l) at 47,000 rpm; and (E) PEVK (63 µmol/l) at 50,000 rpm. Absorbances were measured at 280 nm (220 nm for PEVK) and analyzed using SedFit. (F and G) The behavior of two representative I-band titin segments in size-exclusion chromatography experiments performed at room temperature. (F) The four-Ig domain fragment I9-12 (41.3 kD) and (G) PEVK (19.9 kD). Note that the peak heights are not comparable because of the different protein concentrations used. BSA (66 kD) was used as the elution marker.