Mutational analysis of highly conserved aspartate residues essential to the catalytic core of the piggyBac transposase

<p>Abstract</p> <p>Background</p> <p>The <it>piggyBac </it>mobile element is quickly gaining popularity as a tool for the transgenesis of many eukaryotic organisms. By studying the transposase which catalyzes the movement of <it>piggyBac</it>, we may be able to modify this vector system to make it a more effective transgenesis tool. In a previous publication, Sarkar A, Sim C, Hong YS, Hogan JR, Fraser MJ, Robertson HM, and Collins FH have proposed the presence of the widespread 'DDE/DDD' motif for <it>piggyBac </it>at amino acid positions D268, D346, and D447.</p> <p>Results</p> <p>This study utilizes directed mutagenesis and plasmid-based mobility assays to assess the importance of these residues as the catalytic core of the <it>piggyBac </it>transposase. We have functionally analyzed individual point-mutations with respect to charge and physical size in all three proposed residues of the 'DDD' motif as well as another nearby, highly conserved aspartate at D450. All of our mutations had a significant effect on excision frequency in S2 cell cultures. We have also aligned the <it>piggyBac </it>transposase to other close family members, both functional and non-functional, in an attempt to identify the most highly conserved regions and position a number of interesting features.</p> <p>Conclusion</p> <p>We found all the designated DDD aspartates reside in clusters of amino acids that conserved among <it>piggyBac </it>family transposase members. Our results indicate that all four aspartates are necessary, to one degree or another, for excision to occur in a cellular environment, but D450 seems to have a tolerance for a glutamate substitution. All mutants tested significantly decreased excision frequency in cell cultures when compared with the wild-type transposase.</p

A FUNCTIONAL ANALOGY BETWEEN CROWN ETHERS AND METALLACROWNS

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DEVELOPMENT OF METALLOCROWN ETHERS

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Harnessing natures ability to control metal ion coordination geometry using de novo designed peptides

Advances in protein chemistry and molecular and structural biology have empowered modern chemists to build complex biological architectures using a “first principles” approach, which is known as de novo protein design. In this Perspective we demonstrate how simple three-stranded α-helical constructs can be prepared by the sole consideration of the primary amino acid sequence of a peptide. With these well defined systems, we then demonstrate that metal binding cavities can be carved out of the hydrophobic cores of these aggregates in order to bind metal ions such as cadmium with well defined coordination geometries. Examples will be given of homoleptic CdS(3) complexes, CdS(3)O sites and proteins which contain equilibrium mixtures of these two species. We will provide a description of a strategy that allows us to build heterochromic peptides (small proteins that complex two metals in nearly identical environments but which result in different physical properties and allow for metal site selectivity). We conclude with a new class of designed peptides, diastereopeptides, which can exploit changes in amino acid chirality to control metal ion coordination number and lead to an alternative path towards heterochromic systems. The constructs described herein represent the initial steps of preparing protein structures that may simultaneous contain structural and catalytic metal binding centers. These studies inform the community on a developing field, which promises new opportunities for the study of bioinorganic chemistry