The unfolding kinetics of ubiquitin captured with single-molecule force-clamp techniques
Publikation: Beitrag in Fachzeitschrift › Forschungsartikel › Beigetragen › Begutachtung
Beitragende
Abstract
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 memory-less 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.
Details
Originalsprache | Englisch |
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Seiten (von - bis) | 7299-7304 |
Seitenumfang | 6 |
Fachzeitschrift | Proceedings of the National Academy of Sciences of the United States of America : PNAS |
Jahrgang | 101 |
Ausgabenummer | 19 |
Publikationsstatus | Veröffentlicht - 11 Mai 2004 |
Peer-Review-Status | Ja |
Externe IDs
Scopus | 2442448427 |
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ORCID | /0000-0002-6209-2364/work/142237659 |
Schlagworte
Schlagwörter
- IMMUNOGLOBULIN DOMAINS, PROTEIN, TITIN, SPECTROSCOPY, ADHESION, FIBRONECTIN, MICROSCOPY, STABILITY, DYNAMICS, LIFETIME