期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2004
卷号：101
期号：19
页码：7299-7304
DOI：10.1073/pnas.0400033101
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We use single-molecule force spectroscopy to study the kinetics of unfolding of the small protein ubiquitin. Upon a step increase in the stretching force, a ubiquitin polyprotein extends in discrete steps of 20.3 {+/-} 0.9 nm marking each unfolding event. An average of the time course of these unfolding events was well described by a single exponential, which is a necessary condition for a memoryless Markovian process. Similar ensemble averages done at different forces showed that the unfolding rate was exponentially dependent on the stretching force. Stretching a ubiquitin polyprotein with a force that increased at a constant rate (force-ramp) directly measured the distribution of unfolding forces. This distribution was accurately reproduced by the simple kinetics of an all-or-none unfolding process. Our force-clamp experiments directly demonstrate that an ensemble average of ubiquitin unfolding events is well described by a two-state Markovian process that obeys the Arrhenius equation. However, at the single-molecule level, deviant behavior that is not well represented in the ensemble average is readily observed. Our experiments make an important addition to protein spectroscopy by demonstrating an unambiguous method of analysis of the kinetics of protein unfolding by a stretching force.
