Structural basis of light dependent modulation of phytochrome.

PhDThis thesis presents structural and biochemical studies of a phytochrome, Agp1, a bilin-binding red-light receptor protein. Crystallographic studies were undertaken in order to obtain structural insight into the mechanism of photoconversion and signal transduction from the sensor domain to the signalling domain, the latter hypothesised to be universally conserved among two-component histidine kinases. Using Agp1 from a non-photosynthetic plant pathogen, Agrobacterium tumefaciens as a model phytochrome, structural determination of both Pr and Pfr-forms was attempted, in order to permit unbiased structural comparison of the two. Application of the surface entropy reduction strategy led to determination of the structure of Agp1 in its Pr-form and crystals of Agp1 in the Pfr-like form were obtained for the first time. Biochemical studies were undertaken to probe the conformational differences between Agp1 as apoprotein, Pr-form, and Pfr-like form. Limited differences in secondary structure exist between the forms of Agp1. Conformational differences between the Pr and the Pfr-like form seem to underlie the fact that the space group of Agp1 crystals in the Pfr-like form is different from that of Agp1 in the Pr-form. The ability of the Agp1 apoprotein to form a dimer via a disulphide bond at the N-terminal chromophore-binding cysteine residue implies flexibility of the N-terminal region which allows for the initial bilin incorporation during holoprotein formation.Queen Mary University of London college studentship. University of London central research fun

Improved fluorescent phytochromes for in situ imaging

Modern biology investigations on phytochromes as near-infrared fluorescent pigments pave the way for the development of new biosensors, as well as for optogenetics and in vivo imaging tools. Recently, near-infrared fluorescent proteins (NIR-FPs) engineered from biliverdin-binding bacteriophytochromes and cyanobacteriochromes, and from phycocyanobilin-binding cyanobacterial phytochromes have become promising probes for fluorescence microscopy and in vivo imaging. However, current NIR-FPs typically suffer from low fluorescence quantum yields and short fluorescence lifetimes. Here, we applied the rational approach of combining mutations known to enhance fluorescence in the cyanobacterial phytochrome Cph1 to derive a series of highly fluorescent variants with fluorescence quantum yield exceeding 15%. These variants were characterised by biochemical and spectroscopic methods, including time-resolved fluorescence spectroscopy. We show that these new NIR-FPs exhibit high fluorescence quantum yields and long fluorescence lifetimes, contributing to their bright fluorescence, and provide fluorescence lifetime imaging measurements in E.coli cells

Functional comparison of the WD-repeat domains of SPA1 and COP1 in suppression of photomorphogenesis

The Arabidopsis COP1/SPA complex acts as a cullin4-based E3 ubiquitin ligase to suppress photomorphogenesis in darkness. It is a tetrameric complex of two COP1 and two SPA proteins. Both COP1 and SPA are essential for the activity of this complex, and they both contain a C-terminal WD-repeat domain responsible for substrate recruitment and binding of DDB1. Here, we used a WD domain swap-approach to address the cooperativity of COP1 and SPA proteins. We found that expression of a chimeric COP1 carrying the WD-repeat domain of SPA1 mostly complemented the cop1-4-mutant phenotype in darkness, indicating that the WD repeat of SPA1 can replace the WD repeat of COP1. In the light, SPA1-WD partially substituted for COP1-WD. In contrast, expression of a chimeric SPA1 protein carrying the WD repeat of COP1 did not rescue the spa-mutant phenotype. Together, our findings demonstrate that a SPA1-type WD repeat is essential for COP1/SPA activity, while a COP1-type WD is in part dispensible. Moreover, a complex with four SPA1-WDs is more active than a complex with only two SPA1-WDs. A homology model of SPA1-WD based on the crystal structure of COP1-WD uncovered two insertions and several amino acid substitutions at the predicted substrate-binding pocket of SPA1-WD

Investigating the roles of the C-terminal domain of Plasmodium falciparum GyrA

Malaria remains as one of the most deadly diseases in developing countries. The Plasmodium causative agents of human malaria such as Plasmodium falciparum possess an organelle, the apicoplast, which is the result of secondary endosymbiosis and retains its own circular DNA. A type II topoisomerase, DNA gyrase, is present in the apicoplast. In prokaryotes this enzyme is a proven, effective target for antibacterial agents, and its discovery in P. falciparum opens up the prospect of exploiting it as a drug target. Basic characterisation of P. falciparum gyrase is important because there are significant sequence differences between it and the prokaryotic enzyme. However, it has proved difficult to obtain soluble protein. Here we have predicted a new domain boundary in P. falciparum GyrA that corresponds to the C-terminal domain of prokaryotic GyrA and successfully purified it in a soluble form. Biochemical analyses revealed many similarities between the C-terminal domains of GyrA from E. coli and P. falciparum, suggesting that despite its considerably larger size, the malarial protein carries out a similar DNA wrapping function. Removal of a unique Asn-rich region in the P. falciparum protein did not result in a significant change, suggesting it is dispensable for DNA wrapping

Proportion of secondary structures calculated from CD-spectra using the Dichroweb server [32].

<p>Proportion of secondary structures calculated from CD-spectra using the Dichroweb server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref032" target="_blank">32</a>].</p

Gyrase β-pinwheel blade motif alignments.

<p>Aligned sequences of the region corresponding to the first (<b>A</b>), the second (<b>B</b>) and the sixth (<b>C</b>) β-pinwheel blade motifs of prokaryotic gyrases. Red, yellow and green elements above the sequences represent α-helices, β-strands and coils in the structure of <i>X</i>. <i>campestris</i> GyrA CTD [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref038" target="_blank">38</a>], respectively (PDB code: 3L6V chain A, as viewed by PyMOL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref039" target="_blank">39</a>]). Grey-filled triangle indicates the trypsinolysis site for EcGyrA (Arg571) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref040" target="_blank">40</a>] whereas unfilled triangles indicate those for PfGyrA (Arg854, Lys856 and Lys862). The putative coiled-coil of PfGyrA predicted by the SMART server is shown in red below the sequences in A. Annotations and smaller filled arrows underneath the sequences in A and B indicate the elements found in PfGyrA that are conserved with prokaryotic GyrA. <b>D.</b> X-ray crystal structure of <i>X</i>. <i>campestris</i> GyrA (PDB code: 3L6V [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref038" target="_blank">38</a>]) shown in cartoon format with secondary structure elements coloured as described for A-C. Each of the six blades are numbered. The hydrophobic region of blade 1 is coloured orange and shown in stick format. The conserved S/T residue of blade 1 is shown in blue, in stick format and the conserved G of blade 1 is shown in cyan, in stick format.</p

Molecular weights of GyrA-CTD proteins calculated from their amino acid sequences and elution volumes from analytical size exclusion chromatography.

<p>Molecular weights of GyrA-CTD proteins calculated from their amino acid sequences and elution volumes from analytical size exclusion chromatography.</p

Topology footprinting assay for GyrA-CTD proteins.

<p>Ability of GyrA-CTD proteins to wrap DNA was visualised by incubating approx. 2.7 nM nicked (topologically free) DNA with the indicated molar excess of protein; subsequently the nick was sealed by T4 ligase, allowing the topological changes introduced as a result of protein-DNA interaction to be observed. EcGyrA-CTD(ΔT) and EcGyrA-CTD(WT) behaved as previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref031" target="_blank">31</a>]. Similar to EcGyrA-CTD(ΔT), MBP-PfGyrA-CTD and PfGyrA-CTD introduced writhe into topologically free nicked plasmid DNA, and the effect increases in a concentration dependent manner. “R” and “S” represent the positions of relaxed and supercoiled DNA respectively and apply to all gels in the figure.</p

Limited trypsinolysis of PfGyrA-CTD.

<p>Protein digests were visualised as a function of time. Time elapsed in minutes after initiation of reaction is indicated along the top of the gel. Decreases in band intensities of the original proteins (black triangles) are associated with reciprocal increases in band intensities of the fragments (white triangles). Edman degradation has identified the following amino acid sequences from the denoted fragments. Ec1, 572-IKEED-576; Ec2 1-<u>GSH</u>ME-2 [underlined Gly-Ser-His residues derived from pET28a(+)]; Pf1, 855-LKFND-859 (also 857-FNDLQ-861 and 863-GNEQE-867 at lower intensities); Pf2, MKDHL (derived from the N-terminal His-tag).</p

Analyses of the MBP-PfGyrA-CTD variant without the Asn-rich region.

<p>MBP-PfGyrA-CTD without the Asn-rich region (887–902) was subjected to <b>A.</b> EMSA in the presence of 1 nM substrate 140 bp DNA and the indicated excess of protein and <b>B</b>. topology footprinting assay performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.g005" target="_blank">Fig 5</a>. MBP-PfGyrA-CTD(ΔN) exhibits similar affinity for DNA when compared to MBP-PfGyrA-CTD. No difference could be observed in terms of ability to wrap DNA.</p