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<div> <p> <span>Sample Preparation.</span> Slr1694-DNA encoding the full-length SLR protein (amino acids 1–160; GenBank accession no. <span><a href="http://www.pubmedcentral.nih.gov/redirect3.cgi?&&auth=0hCTkuRzDNjYkmu4AqIXeSfBJR9AqEqFXWx4MT5Ta&reftype=extlink-entrez-nucleotide&artid=1544145&iid=133948&jid=2&FROM=Article%7CBody&TO=Entrez%7CTerm%7CNucleotide&article-id=1544145&journal-id=2&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=1653348"><span></span></a><a href="http://www.pubmedcentral.nih.gov/redirect3.cgi?&&auth=08CBHw_7vESLpQgpf0UEcCTnMed49ou6l77HxFAat&reftype=extlink-entrez-nucleotide&artid=1544145&iid=133948&jid=2&FROM=Article%7CBody&TO=Entrez%7CTerm%7CNucleotide&article-id=1544145&journal-id=2&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=1653348">D90913</a></span>, <span><a href="http://www.pubmedcentral.nih.gov/redirect3.cgi?&&auth=0-3mMEbVdEDfDFsEGBv3kb85jX_g1pRKcY1k7-AMP&reftype=extlink-entrez-protein&artid=1544145&iid=133948&jid=2&FROM=Article%7CBody&TO=Entrez%7CTerm%7CProtein&article-id=1544145&journal-id=2&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=protein&dopt=GenPept&list_uids=16330981"><span></span></a><a href="http://www.pubmedcentral.nih.gov/redirect3.cgi?&&auth=0b3U4omUMO0TJjnPRoaSLmDXdrdT2PXu4AdycKkyb&reftype=extlink-entrez-protein&artid=1544145&iid=133948&jid=2&FROM=Article%7CBody&TO=Entrez%7CTerm%7CProtein&article-id=1544145&journal-id=2&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=protein&dopt=GenPept&list_uids=16330981">NP_441709</a></span>), was amplified by PCR from <em>Synechocystis</em> 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 <em>Escherichia coli</em> 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 D<sub>2</sub>O at least three times. </p> <p> <span>Spectroscopy.</span> Femtosecond transient absorption spectroscopy was carried out with a Ti:sapphire-based regenerative amplification system as described (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1544145#b38">38</a>). 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. </p> <p> <span>Data Analysis.</span> 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 <em>Supporting Text</em> 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. <a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1544145#b39">39</a>. </p> </div>
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