Quaternary ammonium oxidative demethylation: X-ray crystallographic, resonance Raman, and UV-visible spectroscopic analysis of a Rieske-type demethylase.

Herein, the structure resulting from in situ turnover in a chemically challenging quaternary ammonium oxidative demethylation reaction was captured via crystallographic analysis and analyzed via single-crystal spectroscopy. Crystal structures were determined for the Rieske-type monooxygenase, stachydrine demethylase, in the unliganded state (at 1.6 Å resolution) and in the product complex (at 2.

Correlated single-crystal electronic absorption spectroscopy and X-ray crystallography at NSLS beamline X26-C.

The research philosophy and new capabilities installed at NSLS beamline X26-C to support electronic absorption and Raman spectroscopies coupled with X-ray diffraction are reviewed. This beamline is dedicated full time to multidisciplinary studies with goals that include revealing the relationship between the electronic and atomic structures in macromolecules. The beamline instrumentation has been fully integrated such that optical absorption spectra and X-ray diffraction images are interlaced.

Single-crystal Raman spectroscopy and X-ray crystallography at beamline X26-C of the NSLS.

Three-dimensional structures derived from X-ray diffraction of protein crystals provide a wealth of information. Features and interactions important for the function of macromolecules can be deduced and catalytic mechanisms postulated. Still, many questions can remain, for example regarding metal oxidation states and the interpretation of `mystery density', i.

Ultrafast dynamics of protein proton transfer on short hydrogen bond potential energy surfaces: S65T/H148D GFP.

Ultrafast proton transfer dynamics on a short H-bond in a protein were studied through the time-resolved fluorescence of the S65T/H148D green fluorescent protein (GFP) mutant. In response to the change in chromophore pK(a) upon excitation, the donor-proton-acceptor structure evolves on a sub-100 fs time scale, followed by picosecond time scale vibrational cooling and host structure reorganization.

Ultrafast Electronic and Vibrational Dynamics of Stabilized A State Mutants of the Green Fluorescent Protein (GFP): Snipping the Proton Wire.

Two blue absorbing and emitting mutants (S65G/T203V/E222Q and S65T at pH 5.5) of the green fluorescent protein (GFP) have been investigated through ultrafast time resolved infra-red (TRIR) and fluorescence spectroscopy. In these mutants, in which the excited state proton transfer reaction observed in wild type GFP has been blocked, the photophysics are dominated by the neutral A state.

An alternate proton acceptor for excited-state proton transfer in green fluorescent protein: rewiring GFP.

The neutral form of the chromophore in wild-type green fluorescent protein (wtGFP) undergoes excited-state proton transfer (ESPT) upon excitation, resulting in characteristic green (508 nm) fluorescence. This ESPT reaction involves a proton relay from the phenol hydroxyl of the chromophore to the ionized side chain of E222, and results in formation of the anionic chromophore in a protein environment optimized for the neutral species (the I* state). Reorientation or replacement of E222, as occurs in the S65T and E222Q GFP mutants, disables the ESPT reaction and results in loss of green emission following excitation of the neutral chromophore.

Proton relay reaction in green fluorescent protein (GFP): Polarization-resolved ultrafast vibrational spectroscopy of isotopically edited GFP.

The complex transient vibrational spectra of wild type (wt) GFP have been assigned through polarization anisotropy measurements on isotopically edited proteins. Protein chromophore interactions modify considerably the vibrational structure, compared to the model chromophore in solution. An excited-state vibrational mode yields information on excited-state electronic structure.

Observation of excited-state proton transfer in green fluorescent protein using ultrafast vibrational spectroscopy.

The photodynamics of wtGFP have been studied by ultrafast time-resolved infrared spectroscopy (TIR). In addition to the expected bleaching and transient infrared absorption of bands associated with the chromophore, we observe the dynamics of the proton relay reaction in the protein. Protonation of a protein carboxylate group occurs on the tens of picoseconds time scale following photoexcitation.

Light-driven decarboxylation of wild-type green fluorescent protein.

The response of wild-type GFP to UV and visible light was investigated using steady state absorption, fluorescence, and Raman spectroscopies. As reported previously [van Thor, Nat. Struct.