TITIN EXTENSIBILITY IN-SITU - ENTROPIC ELASTICITY OF PERMANENTLY FOLDED AND PERMANENTLY UNFOLDED MOLECULAR SEGMENTS

Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarc omere is stretched, developing what is known as passive force. The I-b and region of titin contains tandem Ig segments (consisting of seriall y linked immunoglobuln-like domains) with the unique PEVK segment in b etween (Labeit, S., and B. Kolmerer. 1995. Science. 270:293-296), Alth ough the tandem Ig and PEVK segments have been proposed to behave as s tiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By fol lowing the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established, In slack sarcomeres (similar to 2.0 mu m) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from sim ilar to 2.0 to 2.7 mu m, the ''contracted'' tandem Ig segments straigh tened while their individual Ig domains remained folded. When sarcomer es were stretched beyond similar to 2.7 mu m, the tandem Ig segments d id not further extend, instead PEVK extension was now dominant. Modeli ng tandem Ig and PEVK segments as entropic springs with different bend ing rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustama nte. 1997. Science. 276:1112-1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain fol ded and (b) the PEVK segment behaves as a permanently unfolded polypep tide. Our model provides a molecular basis for the sequential extensio n of titin's different segments. Initially, the tandem Ig segments ext end at low forces due to their high bending rigidity. Subsequently, ex tension of the PEVK segment occurs only upon reaching sufficiently hig h external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provi des a unique passive force-SL relation that is not achievable with a s ingle elastic segment.