A more flexible nucleotide building block for the synthesis of new DNA based porphyrin-zipper arrays is described. Changing the rigid acetylene linker between the porphyrin substituent and the 2'-deoxyuridine to a more flexible propargyl amide containing linkage leads in part to an increased duplex stability. The CD spectra reveal different electronic interactions between the porphyrins depending on the type of linker used. Molecular modelling suggests large variation of the relative orientation of the porphyrins within the major groove of the DNA. The porphyrins can be metallated post-synthetically with different metals as shown with zinc, cobalt and copper. The spectroscopic features do not alter drastically upon metallation apart from the CD spectra, and the stability of the metal complex is highly dependent on the nature of the metal. As shown by CD spectroscopy, the zinc porphyrin is rapidly demetallated at high temperatures. Globular structure determination using SAXS indicates that a molecular assembly comprised of a two to four helical bundle dominates in solution at higher concentrations (≥50 μM) which is not observed by spectroscopy at lower concentrations (≤1 μM).

RG42 6ET. 5-Aminolaevulinic acid dehydratase (ALAD; also called porphobilinogen synthase; EC 4.2.1.24) catalyses a key step in the tetrapyrrole biosynthetic pathway; the Knorr type condensation reaction between two molecules of 5-aminolaevulinic acid ( A M ) to form the monopyrrole, porphobilinogen. At present, the three-dimensional structure of this enzyme has not been determined although studies using electron microscopy and low angle X ray scattering have been reported [l, 21. This work describes the analysis of the structure of ALAD from yeast using circular dichmism (CD).Yeast ALAD was isolated and purified to homogeneity from E. coli JM109 cells overexpressing the yeast HEM2 gene as described in [3]. Protein characterisation revealed an octameric, zinc dependent, magnesium binding ALAD. Using JASCO spectrophotometers, CD spectra of the backbone and aromatic regions were obtained and analysis revealed yeast ALAD secondary structure to consist of approximately 33% a-helix and 21% p-pleated sheet.Yeast ALAD requires zinc for catalytic activity [3,4].However, when metal chelation was carried out using 1 ,lo-phenanthroline [5] and spectra were measured again, no changes in enzyme backbone or aromatic environment were observed, suggesting that metal removal from yeast ALAD does not grossly affect its structure. This is interesting as it has been suggested that one function of the metals in ALADs is to maintain structural integrity in the octameric enzymes [6J. The role of metals was investigated further using native PAGE of metal bound yeast and E. coli ALADs chelated with 1,lO phenanthroline or EDTA. Results revealed predominantly a single band for both metalbound enzymes but on metal chelation, a laddering of bands was observed for E. coli L A D but not that from yeast. It therefore appears that metals in yeast ALAD are unlikely to fill a major structural role although such a function is suggested for E. coli ALAD [6]. CD studies with the denaturants urea and sodium dodecyl sulphate suggest that the yeast ALAD structure is remarkably stable to disruption.Similarly,CD was used to study yeast ALAD structure in the presence of increasing stoichiometric ratios of ALA. No changes in either spectral region were noted. suggesting that conformational changes required during catalysis are small. This is in accordance with conclusions drawn from the study of the enzyme's pH dependent kinetics [3].Thermal denaturation of yeast ALAD was followed in the backbone region. The melting curve showed a temperature sensitive enzyme with two stage irreversible denaturation. The first stage was quick q,I, = 49.2OC) whereas the second stage was slower (Tm = 60.2oC). For comparison, the experiment was repeated with E. coli ALAD, and this also showed two stage melting with a slower first phase qm=61.60C) and fast second stage vm=64.50C). Differences in the arrangement of structural motifs are therefore anticipated between the two enzymes.Towards the goal of a three-dimensional structure for the enzyme, crystallisation of both native an...

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