Spectroscopically Validated pH-dependent MSOX Movies Provide Detailed Mechanism of Copper Nitrite Reductases.

Copper nitrite reductases (CuNiRs) exhibit a strong pH dependence of their catalytic activity. Structural movies can be obtained by serially recording multiple structures (frames) from the same spot of a crystal using the MSOX serial crystallography approach. This method has been combined with on-line single crystal optical spectroscopy to capture the pH-dependent structural changes that accompany during turnover of CuNiRs from two Rhizobia species. The structural movies, initiated by the redox activation of a type-1 copper site (T1Cu) via X-ray generated photoelectrons, have been obtained for the substrate-free and substrate-bound states at low (high enzymatic activity) and high (low enzymatic activity) pH. At low pH, formation of the product nitric oxide (NO) is complete at the catalytic type-2 copper site (T2Cu) after a dose of 3 MGy (frame 5) with full bleaching of the T1Cu ligand-to-metal charge transfer (LMCT) 455 nm band (S(σ)Cys â†’ T1Cu2+) which in itself indicates the electronic route of proton-coupled electron transfer (PCET) from T1Cu to T2Cu. In contrast at high pH, the changes in optical spectra are relatively small and the formation of NO is only observed in later frames (frame 15 in Br2DNiR, 10 MGy), consistent with the loss of PCET required for catalysis. This is accompanied by decarboxylation of the catalytic AspCAT residue, with CO2 trapped in the catalytic pocket.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies.

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (<1 µL) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

A microfluidic device for both on-chip dialysis protein crystallization and in situ X-ray diffraction.

This paper reports a versatile microfluidic chip developed for on-chip crystallization of proteins through the dialysis method and in situ X-ray diffraction experiments. A microfabrication process enabling the integration of regenerated cellulose dialysis membranes between two layers of the microchip is thoroughly described. We also describe a rational approach for optimizing on-chip protein crystallization via chemical composition and temperature control, allowing the crystal size, number and quality to be tailored. Combining optically transparent microfluidics and dialysis provides both precise control over the experiment and reversible exploration of the crystallization conditions. In addition, the materials composing the microfluidic chip were tested for their transparency to X-rays in order to assess their compatibility for in situ diffraction data collection. Background scattering was evaluated using a synchrotron X-ray source and the background noise generated by our microfluidic device was compared to that produced by commercial crystallization plates used for diffraction experiments at room temperature. Once crystals of 3 model proteins (lysozyme, IspE, and insulin) were grown on-chip, the microchip was mounted onto the beamline and partial diffraction data sets were collected in situ from several isomorphous crystals and were merged to a complete data set for structure determination. We therefore propose a robust and inexpensive way to fabricate microchips that cover the whole pipeline from crystal growth to the beam and does not require any handling of the protein crystals prior to the diffraction experiment, allowing the collection of crystallographic data at room temperature for solving the three-dimensional structure of the proteins under study. The results presented here allow serial crystallography experiments on synchrotrons and X-ray lasers under dynamically controllable sample conditions to be observed using the developed microchips.