This Article Full Text Full Text (PDF) Supplement All Versions of this Article: biophysj.108.130237v1 95/3/1360    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Google Scholar Articles by Greene, D. N. Articles by Oberhauser, A. F. PubMed PubMed Citation Articles by Greene, D. N. Articles by Oberhauser, A. F.

Single-Molecule Force Spectroscopy Reveals a Stepwise Unfolding of Caenorhabditis elegans Giant Protein Kinase Domains

Dina N. Greene ^*, Tzintzuni Garcia , R. Bryan Sutton  ^¶, Kim M. Gernert , Guy M. Benian ^* and Andres F. Oberhauser   ^¶

^* Department of Pathology, and BimCore, Emory University School of Medicine, Atlanta, Georgia, 30322; Department of Neuroscience and Cell Biology, Department of Biochemistry and Molecular Biology, and ^¶ Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555

Correspondence: Address reprint requests to Andres F. Oberhauser, E-mail: afoberha{at}utmb.edu.

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 Caenorhabditis 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 β-sandwich domains (30–150 pN). Further, our atomic force microscopy 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.

This article has been cited by other articles:

				E. M. Puchner, A. Alexandrovich, A. L. Kho, U. Hensen, L. V. Schafer, B. Brandmeier, F. Grater, H. Grubmuller, H. E. Gaub, and M. Gautel Mechanoenzymatics of titin kinase PNAS, September 9, 2008; 105(36): 13385 - 13390. [Abstract] [Full Text] [PDF]