Direct Observation of Markovian Behavior of the Mechanical Unfolding of Individual Proteins
Yi Cao 1, Rachel Kuske 2 and Hongbin Li 2*
1 The University of British Columbia
2 University of British Columbia
* To whom correspondence should be addressed. E-mail: hongbin{at}chem.ubc.ca.
Submitted on December 26, 2007
Revised on February 21, 2008
Accepted on 10 March 2008
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Abstract |
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Single molecule force-clamp spectroscopy is a valuable tool to analyze unfolding kinetics of proteins. Previous force-clamp spectroscopy experiments have demonstrated that the mechanical unfolding of ubiquitin deviates from the generally assumed Markovian behavior and involves the features of glassy dynamics. Here we use single molecule force-clamp spectroscopy to study the unfolding kinetics of a computationally designed fast-folding mutant of the small protein GB1, which shares a similar -grasp fold as ubiquitin. By treating the mechanical unfolding of polyproteins as the superposition of multiple identical Poisson processes, we developed a simple stochastic analysis approach to analyze the dwell time distribution of individual unfolding event in polyprotein unfolding trajectories. Our results unambiguously demonstrate that the mechanical unfolding of NuG2 fulfills all criteria of a memoryless Markovian process. This result, in contrasts with the complex mechanical unfolding behaviors observed for ubiquitin, serves as a direct experimental demonstration of the Markovian behavior for the mechanical unfolding of a protein and reveals the complexity of the unfolding dynamics amongst structurally similar proteins. Furthermore, we extended our method into a robust and efficient pseudo-dwell time analysis method, which allows one to make full use of all the unfolding events obtained in force-clamp experiments without categorizing the unfolding events. This method enabled us to measure the key parameters characterizing the mechanical unfolding energy landscape of NuG2 with improved precision. We anticipate that the methods demonstrated here will find broad applications in single molecule force-clamp spectroscopy studies for a wide range of proteins.
Key Words:
Atomic force microscopy, Force-clamp spectroscopy, Markovian process, Polyprotein, Protein unfolding, Stochastic analysis
