W0284

High Resolution X-ray and Neutron Diffraction Studies of Dihydrofolate Reductase from Escherichia coli. Brad C. Bennett, Chris G. Dealwis, and Elizabeth E. Howell, The Center of Excellence for Structural Biology and Dept. of Biochemistry, Cellular & Molecular Biology, Univ. of Tennessee- Knoxville, Knoxville, TN, USA 37996.

High-resolution x-ray and neutron diffraction techniques may help solve disputed mechanistic questions in enzymes. The use of neutrons in protein diffraction studies facilitates the determination of hydrogen atom positions in the molecule. Therefore, catalytically relevant protons along a reaction pathway may be visualized. This approach will be applied to dihydrofolate reductase (DHFR), a monomeric enzyme that catalyzes a critical step in the de novo synthesis of thymidylate and is involved in the production of other purine nucleosides and methionine. Although E. coli DHFR has been exhaustively studied both kinetically and structurally, the protonation states of the catalytically important aspartate 27 (D27) residue, dihydrofolate (DHF) and the inhibitor methotrexate (MTX) remain unresolved. Despite numerous x-ray crystallographic and computational studies, there is no consensus on the origin and path of proton donation.

We have purified E. coli DHFR to homogeneity and co-crystallized it with different ligands for x-ray and neutron diffraction studies. We have performed in-house x-ray diffraction on two different complexes (DHFR·MTX, and DHFR·folate·NADP⁺), each of which have crystallized into several different space groups (for example, the DHFR·MTX complex: C2 and P6₁; and, the DHFR·folate·NADP⁺ complex: P2₁, C2, and P2₁2₁2₁). Using molecular replacement, we have completely solved the structure of the P6₁ form of the DHFR·MTX binary complex to 1.8 Å resolution. Preliminary characterization indicates all crystals diffract to ≤ 2.3 Å resolution. We have expressed DHFR in minimal media in preparation for growth in the presence of deuterated metabolites. We have also expressed DHFR from D₂0-“adapted” E. coli in fully deuterated rich media source. Work is ongoing to determine deuteration level in the protein, to crystallize this perdeuterated form in anticipation of neutron diffraction experiments, and to take protonated crystals to a synchrotron for high-resolution analysis. The neutron scattering behavior of deuterium is similar to that of much larger atoms found in protein crystals, namely C, O, N, and S. The exchange of hydrogen for deuterium, either by vapor diffusion or soaking the crystals in a D₂0-based buffer or by cell growth in the D₂0-based media, will reduce the incoherent background scattering of hydrogen, replace a negative scatterer of the neutron beam with a positive scatterer, and allow crystals <1.0mm³ to be used for diffraction.