It has only recently become possible to study the dynamics of large time scale biological processes computationally in explicit solvent and atomic detail. This required a combination of advances in computer hardware, utilization of parallel and special purpose hardware as well as numerical and theoretical approaches. In this work we report advances in these areas contributing to the feasibility of a work of this scope in a reasonable time. We then make use of them to study an interesting model system, the action of the DNA bending protein 1IHF and demonstrate such an effort can now be performed on GPU equipped PC workstations. Many cellular processes require DNA bending. In the crowded compartment of the cell, DNA must be efficiently stored but this is just one example where bending is observed. Other examples include the effects of DNA structural features involved in transcription, gene regulation and recombination. 1IHF is a bacterial protein that binds and kinks DNA at sequence specific sites. The 1IHF binding to DNA is the cause or effect of bending of the double helix by almost 180 degrees. Most sequence specific DNA binding proteins bind in the major groove of the DNA and sequence specificity results from direct readout. 1IHF is an exception; it binds in the minor groove. The final structure of the binding/bending reaction was crystallized and shows the protein arm like features "latched" in place wrapping the DNA in the minor grooves and intercalating the tips between base pairs at the kink sites. This sequence specific, mostly indirect readout protein-DNA binding/bending interaction is therefore an interesting test case to study the mechanism of protein DNA binding and bending in general. Kinetic schemes have been proposed and numerous experimental studies have been carried out to validate these schemes. Experiments have included rapid kinetics laser T jump studies providing unprecedented temporal resolution and time resolved (quench flow) DNA foot-printing. Here we complement and add to those studies by investigating the mechanism and dynamics of the final latching/initial unlatching at an atomic level. This is accomplished with the computational tools of molecular dynamics and the theory of Milestoning. Our investigation begins by generating a reaction coordinate from the crystal structure of the DNA-protein complex and other images generated through modelling based on biochemical intuition. The initial path is generated by steepest descent minimization providing us with over 100 anchor images along the Steepest Descent Path (SDP) reaction coordinate. We then use the tools of Milestoning to sample hypersurfaces (milestones) between reaction coordinate anchors. Launching multiple trajectories from each milestone allowed us to accumulate average passage times to adjacent milestones and obtain transition probabilities. A complete set of rates was obtained this way allowing us to draw important conclusions about the mechanism of DNA bending. We uncover two possible metastable intermediates in the dissociation unkinking process. The first is an unexpected stable intermediate formed by initial unlatching of the IHF arms accompanied by a complete "psi-0" to "psi+140" conformational change of the IHF arm tip prolines. This unlatching (de-intercalation of the IHF tips from the kink sites) is required for any unkinking to occur. The second intermediate is formed by the IHF protein arms sliding over the DNA phosphate backbone and refolding in the next groove. The formation of this intermediate occurs on the millisecond timescale which is within experimental unkinking rate results. We show that our code optimization and parallelization enhancements allow the entire computational process of these millisecond timescale events in about one month on 10 or less GPU equipped workstations/cluster nodes bringing these studies within reach of researchers that do not have access to supercomputer clusters. / text

Protein mechanical responses play a critical role in a wide variety of biological phenomena, impacting events as diverse as muscle contraction and stem cell differentiation. Recent advances in both experimental and computational techniques have provided the opportunity to explore protein constitutive properties at the molecular level. However, despite these advances many questions remain about how proteins respond to applied mechanical forces, particularly as a function of load magnitude. In order to address these questions, relatively simple helical structures were computationally tested to determine the mechanisms and kinetics of unfolding at a range of physiologically relevant load magnitudes. Atomically detailed constant force molecular dynamics simulations combined with the Milestoning kinetic analysis framework revealed that the mean first passage time (MFPT) of the initiation of unfolding of long (~16nm) isolated helical domains was a non-monotonic function of the magnitude of applied tensile load. The unfolding kinetics followed a profile ranging from 2.5ns (0pN) to a peak of 3.75ns (20pN) with a decreasing MFPT beyond 40pN reflected by an MFPT of 1ns for 100pN. The application of the Milestoning framework with a coarse-grained network analysis approach revealed that intermediate loads (15pN-25pN) retarded unfolding by opening additional, slower unfolding pathways through non-native [pi]-helical conformations. Analysis of coiled-coil helical pairs revealed that the presence of the second neighboring helix delayed unfolding initiation by a factor of 20, with calculated MFPTs ranging from 55ns (0pN) to 85ns (25pN per helix) to 20ns (100pN per helix). The stability of the coiled-coil domains relative to the isolated helix was shown to reflect a decreased propensity to break flexibility restraining intra-helix hydrogen bonds, thereby delaying [psi] backbone dihedral angle rotation and unfolding. These results show for the first time a statistically determined profile of unfolding kinetics for an atomically detailed protein that is non-monotonic with respect to load caused by a change in the unfolding mechanism with load. Together, the methods introduced for analyzing the mechanical response of proteins as well as the timescales determined for the initiation of unfolding provide a framework for the determination of the constitutive properties of proteins and non-biological polymers with more complicated geometries. / text