SMFS experiments can measure the forces required for the mechanical unfolding of protein molecules and can resolve the changes in length with single amino acid resolution (Carrion-Vazquez et al. 1999b). However, at present they cannot provide a detailed structural resolution of the process. To this end computer simulations have proven to be very important for the atomic analysis of this process. Simulations allow us to follow how mechanical forces change the structure of the proteins under stress at the atomic level, ttese simulations are easy to implement since the denaturing agent (force) and the reaction coordinate (N-C distance) are simple to simulate. Since these techniques also deal with single molecules they are especially suitable for direct comparison with SMFS results, which constitutes one of the main appeals of the SMFS field. Two main groups of methods have been used to this end: all-atom molecular dynamics (with and without explicit water solvent) and on- and off-lattice simulations (Isralewitz et al. 2001).
Simulation techniques have much higher time resolution than the single-molecule techniques (which can only capture slow conformational motions), ttey also use shorter computational times in the range of nanoseconds or tenths of nanoseconds, which is achieved by increasing the pulling speed in the case of constant-velocity simulations (or by increasing the force in the case of constant-force simulations). In most of the cases, however, a direct comparison is not possible because these techniques simulate the unfolding process over a very short period of time (picosecond to nanosecond range), whereas SMFS experimental data are obtained over a much longer period of time (millisecond to second range), tte simulations are generally performed at pulling speeds many orders of magnitude faster than the experiment (eight or more, typically 106-107 vs. 0.1-1 nm ms_1), As a consequence of this time reduction, much irreversible work is done to stretch the molecule, which typically results in force peaks 1 order of magnitude higher than those observed in the AFM experiments. Hence, it is not clear whether they should precisely model the real process in these short periods of time and they may not fully reproduce the experimental conditions.
In spite of these difficulties, the synergy between experiment and simulation is very powerful. It is expected that this will be central to the future development of the field in obtaining the necessary high-resolution picture of the mechanical unfolding of proteins. A good example of this will be shown in Sect. 8.4.1.
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