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BLUF (blue light sensing using FAD) domains constitute a recently discovered class …


Biology Articles » Biophysics » Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor » Materials and methods

Materials and methods
- Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor

Sample Preparation. Slr1694-DNA encoding the full-length SLR protein (amino acids 1–160; GenBank accession no. D90913, NP_441709), was amplified by PCR from Synechocystis sp. PCC6803 DNA (kindly provided by I. Maldener, University of Regensburg, Regensburg, Germany) and cloned between EcoRI and HindIII sites of the pet28a(+) vector (Invitrogen). Protein was expressed in Escherichia coli strain BL21(DE3) at 18°C in 0.7 mM IPTG overnight. Slr1964 was purified on Ni-nitrilotriacetic acid resin (Qiagen, Hilden, Germany) according to the supplier’s instruction. The eluate was dialyzed 2× again 200 volumes of 10 mM NaPi/10 mM NaCl (pH 8.0) and concentrated by ultrafiltration (PM30; Millipore). For isotopic replacement, the protein was concentrated and subsequently diluted with buffered D2O at least three times.

Spectroscopy. Femtosecond transient absorption spectroscopy was carried out with a Ti:sapphire-based regenerative amplification system as described (38). A 400-nm pump beam was obtained by frequency-doubling the output from the amplifier and attenuated to 300 nJ. Femtosecond time delays up to 5 ns between pump and probe were controlled by a delay line, and time-gated spectra at 109 delay times were recorded. The polarizations of pump and probe beams were set at the magic angle (54.7°). The instrument response function was fit to a Gaussian of 120 fs full-width at half-maximum (FWHM). The samples were loaded in a flow system of 4-ml volume, including a flow cuvette of 1-mm path length, and flowed by a peristaltic pump.

Data Analysis. The transient absorption spectra were globally analyzed in terms of a kinetic model with sequentially interconverting EADS, i.e., 1 → 2 → 3 → …. The arrows indicate successive monoexponential decays with increasing time constants, which can be regarded as the lifetime of each species. The first EADS corresponds to the time 0 difference spectrum. The EADS are not necessarily associated with pure molecular species and often reflect mixtures of molecular species. To further unravel the pathways for product-state formation, a target analysis was applied, in which a specific kinetic scheme was tested (see Supporting Text and Figs. 6 and 7). With this procedure, the SADS of the pure molecular states are estimated. The global and target analysis procedures have been extensively reviewed in ref. 39.


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