Comment on ‘‘Force-Clamp Spectroscopy Monitors the Folding Trajectory of a Single Protein’’ Taking advantage of major improvements in their atomic force microscopy (AFM) appa- ratus, Fernandez and Li were able to follow single-molecule refolding trajectories of the ubiquitin protein (1). They observed a rich variety of kinetic behavior. Using a poly- cistronic version of ubiquitin, lengths of three to eight tethered proteins were picked up at random locations and unfolded using a pull- ing force. Upon relaxation of the force, refolding occurred in continuous stages. The results were interpreted in terms of a folding scenario with no defined kinetic barrier between the unfolded and folded states. Monomeric ubiquitin free in solution has been demonstrated to fold in a barrier-limited process (2–5), often in a two-state manner (6–15) without the multiple early collapse phases (12) seen in the AFM studies. Two- state behavior persists even when there is transition-state heterogeneity (11). The dis- crepancy between the ensemble and AFM measurements cannot be solely attributed to the measurement of single molecules; other single-molecule measurements, in which the proteins were monomeric and free in solu- tion, were fully consistent with analogous solution results that show two-state folding and discrete transitions (16, 17). One suspects that the nondiscrete folding behavior observed for tethered proteins in the AFM studies was due to the intimacy of the multiple ubiquitin chains. In free solution, detectable aggregation of refolding ubiquitin occurs at 2 6M concentration (15), which is resolved on the millisecond-to-second time scale (3, 6, 13). In the AFM measurements, the tethered ubiquitins are at relative concen- tration above the mM range. Therefore, the still-unfolded ubiquitin chains might be ex- pected to associate when the pulling force is reduced, which would produce the kinds of results observed by Fernandez and Li (1). The small number of single-protein fold- ing events observed by Fernandez and Li appear to be barrier-limited. The trajectories [see figure 5 in (1)] have a quiescent period followed by a sudden collapse to the native state, the hallmark of a nucleation process. Furthermore, a histogram of the dwell times results in a zero-force extrapolated rate that is within a factor of two of the value observed for barrier-limited folding in solution. For the single-protein events, the collapse process itself takes 0.1 s. This time scale is orders of magnitude slower than what is an- ticipated from the solution studies. In solution, post–transition state species do not accumu- late. Hence, their lifetimes must be less than a millisecond, the approximate time constant of the entire two-state reaction. Hopefully, fur- ther studies will clarify the nature of the slow collapse phase observed in the AFM studies. T. R. Sosnick Department of Biochemistry and Molecular Biology University of Chicago Chicago, IL 60637, USA References 1. J. M. Fernandez, H. Li, Science 303, 1674 (2004). 2. S. Khorasanizadeh, I. D. Peters, T. R. Butt, H. Roder, Biochemistry 32, 7054 (1993). 3. S. Khorasanizadeh, I. D. Peters, H. Roder, Nature Struct. Biol. 3, 193 (1996). 4. M. S. Briggs, H. Roder, Proc. Natl. Acad. Sci. U.S.A. 89, 2017 (1992). 5. J. Sabelko, J. Ervin, M. Gruebele, Proc. Natl. Acad. Sci. U.S.A. 96, 6031 (1999). 6. B. A. Krantz, L. Mayne, J. Rumbley, S. W. Englander, T. R. Sosnick, J. Mol. Biol. 324, 359 (2002). 7. G. W. Platt, S. A. Simpson, R. Layfield, M. S. Searle, Biochemistry 42, 13762 (2003). 8. C. G. Benitez-Cardoza et al., Biochemistry 43, 5195 (2004). 9. S. T. Gladwin, P. A. Evans, Fold. Des. 1, 407 (1996). 10. T. Sivaraman, C. B. Arrington, A. D. Robertson, Nature Struct. Biol. 8, 331 (2001). 11. B. A. Krantz, R. S. Dothager, T. R. Sosnick, J. Mol. Biol. 337, 463 (2004). 12. J. Jacob, B. Krantz, R. S. Dothager, P. Thiyagarajan, T. R. Sosnick, J. Mol. Biol. 338, 369 (2004). 13. B. A. Krantz, T. R. Sosnick, Biochemistry 39, 11696 (2000). 14. B. A. Krantz, L. B. Moran, A. Kentsis, T. R. Sosnick, Nature Struct. Biol. 7, 62 (2000). 15. H. M. Went, C. G. Benitez-Cardoza, S. E. Jackson, FEBS Lett. 567, 333 (2004). 16. E. A. Lipman, B. Schuler, O. Bakajin, W. A. Eaton, Science 301, 1233 (2003). 17. A. A. Deniz et al., Proc. Natl. Acad. Sci. U.S.A. 97, 5179 (2000). 1 June 2004; accepted 26 August 2004 TECHNICAL COMMENT www.sciencemag.org SCIENCE VOL 306 15 OCTOBER 2004 411b on May 20, 2020 http://science.sciencemag.org/ Downloaded from