1H, 13C, and 15N resonance assignment of photoactive yellow protein

Photoactive yellow protein (PYP) is involved in the negative phototactic response towards blue light of the bacterium Halorhodospira halophila. Here, we report nearly complete backbone and side chain 1H, 13C and 15N resonance assignments at pH 5.8 and 20 °C of PYP in its electronic ground state.

Photoactive yellow protein (PYP) is a 125 amino acid (14 kDa) water-soluble, blue-light sensor protein, first found in the halophilic bacterium Halorhodospira halophila (Meyer 1985). PYP is a photoreceptor, believed to be responsible for the negative phototactic response of its host organism (Sprenger et al. 1993). This kind of response is required for organisms to evade potentially harmful short-wavelength light. Based on this observation, PYP has become a suitable model to understand the signal-transduction mechanism in Per-Arnt-Sim (PAS) domain signaling (Crosthwaite et al. 1997;Nambu et al. 1991). Several PYP-like proteins have meanwhile been found in other organisms, where they are also thought to act as light sensors. In addition, PYP-like proteins found in purple bacteria are involved in cell buoyancy or sensing bacteriophytochromes (Jiang et al. 1999;Kyndt et al. 2004).
Understanding of light transduction in PYP requires structural information in atomic detail. A 1.4 Å crystallographic structure was determined in 1995 by Borgstahl et al. and in 1998 Düx and coworkers revealed the solution structure and backbone dynamics of PYP by NMR spectroscopy. The reaction center of PYP is protected from solvent by R52, which is believed to function as a gateway in the photocycle (Borgstahl et al. 1995;Genick et al. 1997). The chromophore, para-coumaric acid (pCA), is covalenty bound to C69 with a thioester bond and pCA participates in two short hydrogen bonds with E46 and Y42 to stabilize the negative charge of pCA in the electronic ground state, pG. Upon blue-light capture, the chromophore undergoes transcis isomerisation and the intermediate pR is formed, which subsequently relaxes to the proposed signaling state, pB. In the latter state, the reaction center is exposed and the two short hydrogen bonds are broken (Borgstahl et al. 1995;Sigala et al. 2009;Yamaguchi et al. 2009).
In this paper, we present the nearly complete assignment of the backbone and side chain resonances of the pG state of PYP.
All spectra were processed using NMRPipe (Delaglio et al. 1995) and they were analyzed with Sparky (Goddard and Kneller 2008).

Completeness of assignments and data deposition
The obtained backbone assignment is over 96 % complete (see Fig. 1). Missing assignments are the amide backbone 15 N frequencies of the four Pro residues, and the amide nitrogen and proton frequencies of M1 and G7. At this pH no backbone assignment was possible for E12 and the 13 C' frequencies of F6, D10 and I11 were not found.
The entire 1 H, 13 C and 15 N side chain resonance assignment is over 85 % complete. Unassigned side chain resonances mainly pertain to labile protons and their attached heteroatoms, such as N g /H g of the two Arg residues, protons connected to oxygen (i.e. Tyr, Ser and Thr OH), protons bound to nitrogen and their corresponding nitrogens in the imidazole moiety of His residues, H f of Lys, and also resonances due to several non-labile groups, such as methyl C e /H e of Met residues, and a number of signals due to Phe and Trp ring systems.
The C f frequency of the tyrosine residues were found in the 1 H-13 C HSQC CParo experiment (run overnight) and could be assigned by using a combination of CB(CGCD)HD and CB(CGCDCE)HE spectra. However, Y76 and Y118 have too similar H e frequencies to be able to determine which H e -C f peak corresponds to which residue. Therefore this information was obtained from pH-titration experiments, using the fact that tyrosine C c and C f chemical shifts change simultaneously by large amounts during this titration. As the Y76 signals start moving at a lower pH than those of Y118, it was possible to assign the C f nuclei in this fashion. We also can detect signals due to two short hydrogen bonds, which belong to Y42 and E46, using a 1D proton water flip-back sequence that suppresses the water signal without saturating it (see Fig. 2). These NMR signals have been assigned by Sigala et al. (2009).
Previously, 1 H and 15 N chemical shift assignments have been made at 37°C by Düx et al. (1998), but these have not been submitted to the BMRB. Our results agree well with these, with a few exceptions. 1 H, 13 C and 15 N assignments for the N-terminal deletion variant D25-PYP at 20°C are available under BMRB accession number 6321 (Bernard et al. 2005), and these show significant differences as a result of the removal of part of the native protein structure. The