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Biophys. J. BioFAST: First Published October 28, 2005. doi:10.1529/biophysj.105.066571
© 2005 by the Biophysical Society.

A more recent version of this article appeared on January 15, 2006.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Frequency Modulation AFM Reveals Individual Intermediates Associated With Each Unfolded I27 Titin Domain

michael j Higgins 1*, john e sader 2 and suzanne p jarvis 1

1 Trinity College Dublin
2 University of Melbourne

* To whom correspondence should be addressed. E-mail: michael.higgins{at}tcd.ie.

Submitted on May 16, 2005
Revised on July 19, 2005
Accepted on 27 September 2005


  Abstract
In this study, we apply a dynamic Atomic Force Microscopy (AFM) technique, Frequency Modulation (FM) detection, to the mechanical unfolding of single titin I27 domains and make comparisons with measurements made using the AFM contact or static mode method. Static mode measurements revealed the well-known force transition occurring at 100-120 pN in the first unfolding peak which was less clear, or more often absent, in the subsequent unfolding peaks. In contrast, some FM-AFM curves clearly resolved a force transition associated with each of the unfolding peaks irrespective of the number of observed unfolded domains. As expected for FM-AFM, the frequency shift response of the main unfolding peaks and their intermediates could only be detected when the oscillation amplitudes used were smaller than the interaction lengths being measured. It was also shown that the forces measured for the dynamical interaction of the FM-AFM technique were significantly lower than those measured using the static mode. This study highlights the potential for using dynamic AFM for investigating biological interactions, including protein unfolding and the detection of novel unfolding intermediates.

Key Words: Atomic Force Microscopy, Frequency Modulation Detection, Protein Unfolding, Single Molecule AFM, Titin Protein




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Copyright © 2005 by the Biophysical Society.