Dwell-time distribution analysis of polyprotein unfolding using force-clamp spectroscopy.

Using the recently developed single molecule force-clamp technique we quantitatively measure the kinetics of conformational changes of polyprotein molecules at a constant force. In response to an applied force of 110 pN, we measure the dwell times of 1647 unfolding events of individual ubiquitin modules within each protein chain. We then establish a rigorous method for analyzing force-clamp data using order statistics. This allows us to test the success of a history-independent, two-state model in describing the kinetics of the unfolding process. We find that the average unfolding trajectory is independent of the number of protein modules N in each trajectory, which varies between 3 and 12 (the engineered protein length), suggesting that the unfolding events in each chain are uncorrelated. We then derive a binomial distribution of dwell times to describe the stochastic dynamics of protein unfolding. This distribution successfully describes 81% of the data with a single rate constant of alpha = 0.6 s(-1) for all N. The remainder of the data that cannot be accounted for suggests alternative unfolding barriers in the energy landscape of the protein. This method investigates the statistical features of unfolding beyond the average measurement of a single rate constant, thus providing an attractive alternative for measuring kinetics by force-clamp spectroscopy.

[1]  Robin S. Dothager,et al.  Differences in the folding transition state of ubiquitin indicated by φ and ψ analyses , 2004 .

[2]  Hongbin Li,et al.  The unfolding kinetics of ubiquitin captured with single-molecule force-clamp techniques. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Julio M Fernandez,et al.  Single-molecule force spectroscopy reveals signatures of glassy dynamics in the energy landscape of ubiquitin , 2006 .

[4]  A. Oberhauser,et al.  Single protein misfolding events captured by atomic force microscopy , 1999, Nature Structural Biology.

[5]  A. Fersht.  Structure and mechanism in protein science , 1998 .

[6]  X. Xie,et al.  Protein Conformational Dynamics Probed by Single-Molecule Electron Transfer , 2003, Science.

[7]  C. Stevens,et al.  Presynaptic mechanism for long-term potentiation in the hippocampus , 1990, Nature.

[8]  Stephen A. Wainwright,et al.  Mechanical Design in Organisms , 2020 .

[9]  K. Lindorff-Larsen,et al.  Parallel protein-unfolding pathways revealed and mapped , 2003, Nature Structural Biology.

[10]  William Stafford Noble,et al.  Large-scale identification of yeast integral membrane protein interactions. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[11]  R. Keynes.  The ionic channels in excitable membranes. , 1975, Ciba Foundation symposium.

[12]  Everett A Lipman,et al.  Single-Molecule Measurement of Protein Folding Kinetics , 2003, Science.

[13]  K. Schulten,et al.  Multiple probes reveal a native-like intermediate during low-temperature refolding of ubiquitin. , 2004, Journal of Molecular Biology.

[14]  G. I. Bell.  Models for the specific adhesion of cells to cells. , 1978, Science.

[15]  Matthias Rief,et al.  Sensing specific molecular interactions with the atomic force microscope , 1995 .

[16]  Robin S. Dothager,et al.  Differences in the folding transition state of ubiquitin indicated by phi and psi analyses. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[17]  T. Sosnick,et al.  Distinguishing between two-state and three-state models for ubiquitin folding. , 2000, Biochemistry.

[18]  D. Leeson,et al.  Protein folding and unfolding on a complex energy landscape. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[19]  S. Khorasanizadeh,et al.  Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues , 1996, Nature Structural Biology.

[20]  Christopher M. Dobson,et al.  Molecular recycling within amyloid fibrils , 2005, Nature.

[21]  Carlos Bustamante,et al.  Direct Observation of the Three-State Folding of a Single Protein Molecule , 2005, Science.

[22]  Aaron Ciechanover,et al.  The ubiquitin–proteasome pathway: on protein death and cell life , 1998, The EMBO journal.

[23]  Wolfgang A. Linke,et al.  Reverse engineering of the giant muscle protein titin , 2002, Nature.

[24]  A G Hawkes,et al.  The quality of maximum likelihood estimates of ion channel rate constants , 2003, The Journal of physiology.

[25]  L. Itzhaki,et al.  The unfolding story of three-dimensional domain swapping. , 2003, Structure.

[26]  N. Oppenheimer,et al.  Structure and mechanism , 1989 .

[27]  B. Sakmann,et al.  Fluctuations in the microsecond time range of the current through single acetylcholine receptor ion channels , 1981, Nature.

[28]  R. Morris.  Foundations of cellular neurophysiology , 1996 .

[29]  E. Katayama,et al.  Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity , 2003, The EMBO journal.

[30]  Michael D. Stone,et al.  Structural transitions and elasticity from torque measurements on DNA , 2003, Nature.

[31]  Joshua W Shaevitz,et al.  Statistical kinetics of macromolecular dynamics. , 2005, Biophysical journal.

[32]  R. Milne,et al.  Hidden Markov analysis of mechanosensitive ion channel gating. , 2005, Mathematical biosciences.

[33]  Julio M Fernandez,et al.  Force-Clamp Spectroscopy Monitors the Folding Trajectory of a Single Protein , 2004, Science.

[34]  Ignacio Tinoco,et al.  Probing the mechanical folding kinetics of TAR RNA by hopping, force-jump, and force-ramp methods. , 2006, Biophysical journal.