Titin, which has a molecular weight of about 3 × 10⁶, is the largest polypeptide yet described. Titin molecules extend from muscle thick filaments to the Z disc; they are thought to act as springs to keep the thick filaments centered in the sarcomere. Titin is composed of a large number of repeated immunoglobulin (Ig) sequences of 89 amino acids, each of which is folded into a domain about 4 nm in length (Figure Q3–2A).

You suspect that the springlike behavior of titin is caused by the sequential unfolding (and refolding) of individual Ig domains. You test this hypothesis using the atomic force microscope, which allows you to pick up one end of a protein molecule and pull with an accurately measured force. For a fragment of titin containing seven repeats of the Ig domain, this experiment gives the sawtooth force-versus- extension curve shown in Figure Q3–2B. If the experiment is repeated in a solution of 8 M urea (a protein denaturant), the peaks disappear and the measured extension becomes much longer for a given force. If the experiment is repeated after the protein has been cross-linked by treatment with glutaraldehyde, once again the peaks disappear but the extension becomes much smaller for a given force.

Figure Q3–2A

A. Are the data consistent with your hypothesis that titin’s springlike behavior is due to the sequential unfolding of individual Ig domains? Explain your reasoning.

B. Is the extension for each putative domain-unfolding event the magnitude you would expect? (In an extended polypeptide chain, amino acids are spaced at intervals of 0.34 nm.)

C. Why is each successive peak in Figure Q3–2B a little higher than the one before?

D. Why does the force collapse so abruptly after each peak?

Transcribed Image Text:

(A) (B) 400 300 200 100 50 100 150 200 extension (nm) force (pN)