1H, 13C and 15N resonance assignments of S114A mutant of UVI31+ from Chlamydomonas reinhardtii

Almost complete sequence specific 1H, 13C and 15N resonance assignments of S114A mutant of UVI31+ from Chlamydomonas reinhardtii are reported. The cDNA of S114A mutant of UVI31+ was cloned from a eukaryotic green algae (C. reinhardtii) and overexpressed in E.coli from where the protein was purified to homogeneity. The point mutation S114A in UVI31+ reduces its DNA endonuclease activity substantially as compared with its wild type. As a prelude to the structural characterization of S114A mutant of UVI31+, we report here complete sequence-specific 1H, 13C and 15N NMR assignments.

genome analysis and found a protein, which was identified as UVI31?, one amongst several UV inducible transcripts/ proteins and a novel b-lactamase (Moharikar et al. 2006, Rout et al. 2010. UVI31? protein is also endowed with endonuclease function, in vitro. Cell biological regulation reveals interesting localization changes of UVI31? protein in Chlamydomonas reinhardtii chloroplast compartment (Shukla et al. 2012).
Chlamydomonas reinhardtii is about 10 lm in diameter that swims with two flagella. They have a cell wall made up of hydroxyproline-rich glycoproteins, a large cupshaped chloroplast, a large pyrenoid, and an ''eyespot'' that senses light (Moharikar et al. 2006). It undergoes apoptosis in response to UV-C irradiation (Moharikar et al. 2007). It shows classical hall-marks of animal cell apoptosis and hence can be used as a model system for studying its molecular mechanism in a plant-like environment. Certain candidate molecules were recently identified as to be either UV-regulated or involved in apoptosis. They include apoptosis protease activating factor (APAF), a caspase-3 like protein and a defender against apoptotic death (dad1). All of them exhibited a distinct activation pattern correlating with onset of death following UV irradiation (Moharikar et al. 2006(Moharikar et al. , 2007. One of the putative candidate molecules that is yet to be characterized is the aforementioned UV inducible gene, uvi31?, the focus of the present study. Uvi31? was originally isolated from Schizosaccharomyces pombe (Lee et al. 1994), whose expression was unaltered by other DNA damaging or cytotoxic agents. Interestingly, uvi31? showed no significant sequence homology to the known DNA repair genes. It was observed that uvi31? transcript increases during normal cell cycle in G1 phase before septation and also during diauxic shift (Kim et al. 1997). A null mutant of uvi31? from S. pombe showed sensitivity to UV-light, defects in septation and cytokinesis during the resumption of cell division following UV damage-induced cell cycle arrest (Kim et al. 2002).
The uvi31? protein has structural homology with the BolA protein of Escherichia coli, which was identified by its ability to induce round cell morphology when over-expressed in cells and also following a general stress response (Aldea et al. 1988;Santos et al. 1999;Huynen et al. 2005).
Recently, it has been found that uvi31? exhibited endonuclease activity (Shukla et al. 2012). However, uvi31? has no structural homology with the known endonucleases. The mutation at S114A site at uvi31? reduced its endonuclease activity as derived from biochemical analysis (yet to be published). And it has also been speculated that the Ser residue (S114) may be responsible for this activity. With this in the backdrop, we have mutated Ser114 to Ala114 and set out to structurally characterize the mutant of uvi31? (named as S114Auvi31?) by NMR spectroscopy, to find out whether S114 is involved in endonuclese activity. Towards this goal, we report almost complete sequence specific 1 H, 13 C and 15 N NMR assignments of S114A mutant of UVI31?.

Methods and results
Cloning, over-expression and purification of UVI31? were carried out as described earlier (Rout et al. 2010). 13 C 6glucose or/and 15 NH 4 Cl (Cambridge Isotopes Inc.) were used as the sole source of carbon or/and nitrogen. The purification of the protein was achieved by Ni ?? -NTA (Ni ?? -nitrilotriacetate) agarose (Qiagen, Hilgen, Germany). His6-tagged S114A mutant of UVI31? was eluted with 250 mM imidazole in 50 mM sodium phosphate (pH 7.6), 100 mM NaCl. The eluted fractions were dialyzed overnight against 50 mM sodium phosphate (pH 6.4), 100 mM NaCl.
For NMR experiments, protein samples were prepared in a mixed solvent of 90 % H 2 O and 10 % 2 H 2 O containing 50 mM sodium phosphate (pH 6.4) and 100 mM NaCl. The protein concentrations were 0.8 mM. NMR experiments were recorded on a Bruker Avance 800 MHz NMR spectrometer equipped with a 5 mm triple-resonance cryogenic probe. Experiments recorded at 298 K with uniformly 15 N-labled-S114A mutant of UVI31? included sensitivity-enhanced 2D [ 15 N-1 H]-HSQC using waterflipback for minimizing water saturation . The 3D experiments recorded with doubly ( 13 C and 15 N) labeled samples were, HNCO, HN (CA)CO, CBCANH, and CBCA(CO)NH ; Bax and Grzesiek 1993;Wuthrich et al. 1986), essentially for the assignment of backbone resonances. For the assignment of theThe side chain 1 H and 13 C resonances, we recordeds were assigned using 3D C(CO)NH and 3D H(CCO)NH (Montelione et al. 1992) and 13 C-and 15 N-edited NOESY spectra (Muhandiram et al. 1993). NMR data processing was carried out using Bruker Topspin 3.1 software and analyzed with TATAPRO (Atreya et al. 2000(Atreya et al. , 2002 and CARA (Keller 2002). 1 H chemical shifts were referenced with respect to the external standard 2,2-dimethyl-2-silapentene-5-sulfonates (DSS), while 15 N and 13 C chemical shifts were Fig. 1 2D [ 15 N-1 H]-HSQC of S114A mutant of UVI31? at pH 6.4 and 298 K with assignments. The spectrum was recorded with 256 and 1,024 complex points along t 1 and t 2 dimensions, respectively. The assignments are indicated by the one-letter amino acid code followed by the corresponding sequence number along the protein primary sequence. The peaks connected with horizontal lines are correlations from sidechain NH 2 spin pairs belonging to Asn and Gln residues calibrated indirectly (Edison et al. 1994; Chary and Govil 2008).

Extent of assignment and data deposition
Sequence specific resonance assignments of S114A mutant of UVI31?, which is free of Cys and Trp, could be carried out for nearly all 1 H, 13 C and 15 N spins using a suite of 3D NMR experiments mentioned in Methods. The 1 H N and 15 N assignments thus derived are shown in the 2D [ 15 N-1 H]-HSQC (Fig. 1). A total of 117( 1 H i , 13 C i-1 , 15 N i ) correlations are expected from non-proline residues in the 3D-HNCO spectrum of S114A mutant of UVI31? that has a His-tag of 6 residues. We could however observe 107 distinct correlations and assign almost all of them (97 %) under the experimental conditions described herein. In addition, the side chain 1 H and 13 C were assigned for most of the residues (88 % of aliphatic 1 H resonances and 92 % aliphatic 13 C resonances) with an exception of aromatic resonances. The peaks connected by horizontal lines in Fig. 1 belongs to the pairs of side chain NH 2 correlations belonging to Asn and Gln residues. The chemical shift data thus obtained has been deposited in the BioMagResBank (http://www.bmrbwisc.edu) under accession number 18567. Sharp [ 15 N-1 H] peaks of almost uniform intensity and large dispersion (of 4.4 ppm) of resonances along the 1 H N dimension (Fig. 1) revealed that the protein is in a well folded state. The secondary structure elements (Fig. 2) of the protein have been characterized using the well known empirical relations of 13 C chemical shifts (DC a -DC b ) ( Spera and Bax 1991;Wishart et al. 1995;Barnwal et al. 2008). The CSI plot (Fig. 2) reveals the presence of both ahelical and b-strand segments separated by short stretches of unstructured elements. Comparison of [ 15 N-1 H]-HSQC spectra of (S114A)-UVI31? with that of wild type-uvi31?, both recorded under similar experimental conditions reveal subtle spectral changes between them, though most of the 15 N-1 H peaks are unperturbed, suggesting that the overall structural topology of the protein may remain same with substantial conformational changes in the structure particularly near and around the S114A point mutation. Complete 3D structural characterization of the mutant UVI31? will throw more light on the intricate structural changes caused by the point mutation. This work is in progress. Fig. 2 The secondary structural characterization as derived from the difference between the DC a and DC b values (DC a -DC b ) of S114A mutant of UVI31?. The value DC a -DC b for a particular residue i represents the average over the three consecutive residues, i-1, i and i ? 1. A stretch, having negative values of DC a -DC b , indicates the presence of extended b-strand whereas positive values indicate a regular a-helical region