Titin extensibility in situ: entropic elasticity of permanently folded and permanently unfolded molecular segments

Abstract

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 sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science. 270:293-296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff 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 following 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 (approximately 2.0 micron) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from approximately 2.0 to 2.7 micron, the "contracted" tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond approximately 2.7 micron, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112-1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain folded and (b) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force-SL relation that is not achievable with a single elastic segment.

Figure 1

Electron micrographs of sarcomeres labeled with antititin antibodies T12, N2A, 514, and Ti102. (A1) sequence of I-band segment of human soleus muscle titin (Labeit and Kolmerer, 1995). (A2) Control. (A3–A6) Labeled with N2A. (B1) Labeled with T12 and Ti102. (B2–B5) Labeled with 514. (A1, red and white) Ig-like and fibronectin-like domains, respectively. (Blue) Unique sequence. (Yellow) PEVK segment. Domain numbering according to that of cardiac titin with extra domains and amino acid residues of human soleus titin indicated (Labeit and Kolmerer, 1995). Bar, 1.0 μm.

Figure 1

Electron micrographs of sarcomeres labeled with antititin antibodies T12, N2A, 514, and Ti102. (A1) sequence of I-band segment of human soleus muscle titin (Labeit and Kolmerer, 1995). (A2) Control. (A3–A6) Labeled with N2A. (B1) Labeled with T12 and Ti102. (B2–B5) Labeled with 514. (A1, red and white) Ig-like and fibronectin-like domains, respectively. (Blue) Unique sequence. (Yellow) PEVK segment. Domain numbering according to that of cardiac titin with extra domains and amino acid residues of human soleus titin indicated (Labeit and Kolmerer, 1995). Bar, 1.0 μm.

Figure 1

Electron micrographs of sarcomeres labeled with antititin antibodies T12, N2A, 514, and Ti102. (A1) sequence of I-band segment of human soleus muscle titin (Labeit and Kolmerer, 1995). (A2) Control. (A3–A6) Labeled with N2A. (B1) Labeled with T12 and Ti102. (B2–B5) Labeled with 514. (A1, red and white) Ig-like and fibronectin-like domains, respectively. (Blue) Unique sequence. (Yellow) PEVK segment. Domain numbering according to that of cardiac titin with extra domains and amino acid residues of human soleus titin indicated (Labeit and Kolmerer, 1995). Bar, 1.0 μm.

Figure 3

SL dependence of the end-to-end length of tandem Ig and PEVK segments. (Solid lines) Predicted extensions assuming that tandem Ig and PEVK segments behave as wormlike entropic elasticities in series. Predictions based on Kellermayer et al. (1997) assume persistence lengths of tandem Ig and PEVK segments of 15 and 2 nm, respectively. Lines indicated by 0, 1, 2, and 4 assume that 0, 1, 2, and 4 Ig domains of the first-tandem Ig segment were permanently unfolded (A, 2 nm; L, 38 nm). Predictions based on Tskhovrebova et al. (1997) assume persistence lengths of tandem Ig and PEVK segments of 4.8 and 0.15 nm, respectively.

Figure 2

Z-line to epitope distances as a function of SL. The T12 epitope maintains a fixed distance from the Z-line. Other epitopes are merged with T12 at a SL of ∼1.8 μm, but depart from T12 as sarcomeres are stretched.

Figure 4

Entropic force–SL relation of elastic region of titin modeled as two serially linked WLCs with different persistence length (tandem Ig and PEVK segments). Force increases modestly at lengths where tandem Ig extension predominates and steeply at lengths where PEVK extension predominates. Inset: relation between predicted entropic force and extension ratio (z/L) of PEVK segment and tandem Ig segment.