The molecular elasticity of the insect flight muscle proteins projectin and kettin

doi:10.1073/pnas.0509016103

The molecular elasticity of the insect flight muscle proteins projectin and kettin

*European Molecular Biology Laboratory, D-69012 Heidelberg, Germany;
^‡Department of Neuroscience and Cell Biology and Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX 77555;
^§Institute of Physiology, University of Heidelberg, D-69120 Heidelberg, Germany;
^¶Clarendon Laboratory, University of Oxford, Oxford OX1 3PU, United Kingdom; and
^‖Physiology and Biophysics Laboratory, University of Muenster, D-48149 Muenster, Germany

Edited by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved December 28, 2005 (received for review October 14, 2005)

Abstract

Projectin and kettin are titin-like proteins mainly responsible for the high passive stiffness of insect indirect flight muscles, which is needed to generate oscillatory work during flight. Here we report the mechanical properties of kettin and projectin by single-molecule force spectroscopy. Force–extension and force-clamp curves obtained from Lethocerus projectin and Drosophila recombinant projectin or kettin fragments revealed that fibronectin type III domains in projectin are mechanically weaker (unfolding force, F_u ≈ 50–150 pN) than Ig-domains (F_u ≈ 150–250 pN). Among Ig domains in Sls/kettin, the domains near the N terminus are less stable than those near the C terminus. Projectin domains refolded very fast [85% at 15 s⁻¹ (25°C)] and even under high forces (15–30 pN). Temperature affected the unfolding forces with a Q₁₀ of 1.3, whereas the refolding speed had a Q₁₀ of 2–3, probably reflecting the cooperative nature of the folding mechanism. High bending rigidities of projectin and kettin indicated that straightening the proteins requires low forces. Our results suggest that titin-like proteins in indirect flight muscles could function according to a folding-based-spring mechanism.

Footnotes

**To whom correspondence should be addressed. E-mail: afoberha{at}utmb.edu
↵ ^†Present address: Department of Biology, University of York, York YO10 5DD, United Kingdom.
Author contributions: W.A.L. and A.F.O. designed research; T.G., M.C.L., W.A.L., and A.F.O. performed research; B.B., V.B., and A.F.O. contributed new reagents/analytic tools; A.F.O. analyzed data; and M.C.L. and A.F.O. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

AFM,

atomic force microscopy;

FnIII,

fibronectin type III;

IFM,

indirect flight muscle.