SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Single-molecule force spectroscopy reveals a stepwise unfolding of C. elegans giant protein kinase domains
Dina Greene 1, Tzintzuni Garcia 2, Bryan Sutton 2, Kim M Gernert 1, Guy M Benian 1 and Andres F Oberhauser 2*
1 Emory University School of Medicine
2 UTMB
* To whom correspondence should be addressed. E-mail: afoberha{at}utmb.edu.
Submitted on January 24, 2008
Revised on February 28, 2008
Accepted on 18 March 2008
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Abstract |
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Myofibril assembly and disassembly are complex processes that regulate overall muscle mass. Titin kinase has been implicated as an initiating catalyst in signaling pathways that ultimately result in myofibril growth. In titin, the kinase domain is in an ideal position to sense mechanical strain that occurs during muscle activity. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and autoinhibitory/regulatory region. Molecular dynamics simulations suggest that human titin kinase acts as a force sensor. However, the precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Here we measured the mechanical properties of the kinase domain and flanking Ig/Fn domains of the C. elegans titin-like proteins twitchin and TTN-1 using single-molecule atomic force microscopy. Our results show that these kinase domains have significant mechanical resistance, unfolding at forces similar to those for Ig/Fn beta-sandwich domains (30-150pN). Further, our AFM data is consistent with molecular dynamic simulations, which show that these kinases unfold in a stepwise fashion, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data support the hypothesis that titin kinase may function as an effective force sensor.
Key Words:
atomic force microscopy, enzyme activation, kinase, nanomechanics, titin, twitchin
