This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The mechanical manipulation of DNA is central to all aspects of genetic regulation in cells [30]. Regulatory proteins mechanically manipulate DNA away from its equilibrium configuration by introducing bends, loops, twists and supercoils [31] against mechanical strain arising from the DNA. In some cases, this is done to prevent other proteins from binding DNA resulting in gene expression. Little is known about how regulatory proteins are able to handle and withstand the forces stemming from DNA. In many cases, changes in DNA structure are known to occur in protein-DNA interaction but the structure cannot be resolved experimentally when the changes are too large or when the DNA becomes disordered, e.g., when regulatory proteins force DNA into loops. The lac repressor (LacI) is a classic example of genetic regulation via DNA looping. It controls the function of the lac operon in E. coli, a set of genes involved in lactose catabolism [29]. The all-atom structure of LacI bound to its DNA binding sites has been determined, but with the DNA loop missing [33, 34]. It is therefore unclear what are the mechanisms that the protein uses to resist the strain from the connecting DNA loop, which likely changes the structure of LacI. The Resource has developed a multiscale method [37] for the simulation of protein- DNA complexes involving long segments of flexible DNA. This method combines conventional molecular dynamics simulation using NAMD with a mathematical model of DNA, the so-called elastic rod model. The elastic rod model accounts for the physical properties of DNA to build the structure of the missing DNA loop and computes the forces that this loop exerts on the protein [36, 38 40]. An all-atom molecular dynamics simulation of the protein incorporates these forces using the SMD method [60], revealing the structural dynamics of the protein-DNA interaction. The geometry of the elastic rod model of the DNA loop and the forces arising from it are updated and exchanged every 10 ps of MD simulation. The multiscale method was applied to the LacI in complex with a 76 base pair elastic loop [37]. The MD part of the simulation encompassed 320,000 atoms, simulated with the NAMD program on 256 processors. The results of our simulation corrected a long held view of the mechanism of LacI. While it was assumed previously that LacI opened its cleft in a hinge-like massive motion between the dimers to control the ability of LacI to enforce the DNA loop. The simulations revealed that the ability of LacI to maintain the DNA in a looped configuration is actually due to the extreme flexibility of its DNA-binding head groups connected to the rest of the protein through flexible linkers, while the dimers are essentially immobile with respect to each other. The computationally modeled behavior is in good agreement with experiment [32], and provides a new interpretation of existing data.
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