This Article Full Text Full Text (PDF) 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 Puchner, E. M. Articles by Gaub, H. E. PubMed PubMed Citation Articles by Puchner, E. M. Articles by Gaub, H. E.

Comparing Proteins by Their Unfolding Pattern

Elias M. Puchner ^*, Gereon Franzen , Mathias Gautel  and Hermann E. Gaub ^*

^* Lehrstuhl für Angewandte Physik and Center for Nanoscience, LMU München, Munich, Germany; and Cardiovascular Research Division and The Randall Division, King's College London, London, Great Britain

Correspondence: Address reprint requests to Elias M. Puchner, Tel.: 49-89-2180-2306; Fax: 49-89-2180-2050; E-mail: elias.puchner{at}physik.lmu.de.

Single molecule force spectroscopy has evolved into an important and extremely powerful technique for investigating the folding potentials of biomolecules. Mechanical tension is applied to individual molecules, and the subsequent, often stepwise unfolding is recorded in force extension traces. However, because the energy barriers of the folding potentials are often close to the thermal energy, both the extensions and the forces at which these barriers are overcome are subject to marked fluctuations. Therefore, force extension traces are an inadequate representation despite widespread use particularly when large populations of proteins need to be compared and analyzed. We show in this article that contour length, which is independent of fluctuations and alterable experimental parameters, is a more appropriate variable than extension. By transforming force extension traces into contour length space, histograms are obtained that directly represent the energy barriers. In contrast to force extension traces, such barrier position histograms can be averaged to investigate details of the unfolding potential. The cross-superposition of barrier position histograms allows us to detect and visualize the order of unfolding events. We show with this approach that in contrast to the sequential unfolding of bacteriorhodopsin, two main steps in the unfolding of the enzyme titin kinase are independent of each other. The potential of this new method for accurate and automated analysis of force spectroscopy data and for novel automated screening techniques is shown with bacteriorhodopsin and with protein constructs containing GFP and titin kinase.

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]