This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Protein recognition of specific DNA sequences lies at the heart of many normal and disease-related processes, including gene expression and its regulation and genetic recombination. To understand the biophysical principles that govern recognition specificity in protein-DNA interactions, we study the interactions of three restriction endonucleases (EcoRI, BamHI and EcoRV) with their DNA recognition sites, using thermodynamics, kinetics, spectroscopy (NMR, EPR and fluorescence), genetics and computational simulations. Major attention is given to dynamic characteristics of protein-DNA complexes, including binding-dependent conformational transitions in proteins and DNA and conformational-vibrational fluctuations in the complexes. Our enormous accumulation of diverse information on these systems places us in position to pose sharply focused computational questions and to benchmark the computational results against experimental data. This proposal includes the following computational objectives: 1. To use computational simulations to study the interactions of a 'promiscuous' mutant EcoRI endonuclease with its DNA recognition site. We will first test if MD simulations (using the AMBER8 suite, with explicit solvent and treatment of long-range interactions by particle-mesh Ewald) can reproduce the relatively subtle differences between mutant and wild-type complexes as determined by x-ray crystallography, to pave the way for simulations of the mutants for which we lack experimental structures. We will then use MD simulations to study the dynamics of the mutant complex, with particular attention to the roles of interfacial water molecules, motions of the protein 'arms' that enfold the DNA, and possibly concerted motions in the complex. These studies will include calculation of Debye-Waller B-factors from the MD trajectories, and comparison with the experimental B-factors. Finally, we will do computational time-averaging crystallographic refinement (TACR) using the data on both wild-type and mutant complexes, to study molecular and solvent motions that are physically reasonable and constrained to the envelope defined by the experimental diffraction data. These studies will use the GROMOS package for the MD portion of the TACR. 2. To use MD simulations (with full explicit solvent) to study how conformational dynamics and molecular distortions in the EcoRI-DNA complex are affected by stereospecific methylphosphonate substitutions at two positions of the GAATTC recognition site, for which we have extensive biochemical data. We will use such simulations to study how these derivatives cause (a) disruption of the precise water relay required for catalysis; (b) subtle but crucial alterations in sidechain conformations; (c) prevention of Mg2+ cofactor binding to the active site. These studies should yield new insight into the role of active-site water structure and DNA distortion in the catalytic mechanism. 3. To use MD simulations to study the consequences of electrostatic repulsion (a form of molecular strain) in the active site of the BamHI-DNA complex. Experimental data indicate that the cluster of acidic sidechains in this active site have abnormally high pKa's, that the charge on the E111 sidechain is the critical central controller, and that the consequences of strain include increased molecular motion (broadened dynamic distribution) in the complex. We will first use in silico rebuilding and MD simulation (full explicit solvent) to produce an improved model of the wild-type BamHI-DNA complex, since our biochemical data show the crystal structure lacks essential DNA elements and suffers from distortions due to packing forces. We will then test the effect of protonation of E111 and D94 in the context of wild-type protein and mutant proteins E111A and D94A, examining the following issues: (a) Are the consequences of strain strictly local (e.g., rotamer relaxation) or extended over wider areas of the complex? (b) Are the increased conformational-vibrational motions in strained complexes primarily large motions in a small region or smaller motions over a larger region? (c) Do the protein and DNA undergo concerted motions? We will use a large explicit solvent shell, which allows us to treat protein flexibility and dielectric relaxation explicitly, Finally, we will use molecular dynamics free energy simulations to calculate the pKa shifts of E111 in the context of wild-type and D94A.
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