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Mutagenesis of solvent-exposed hydrophobic residues in firefly luciferase Laurence C. Tisi*; James A.H. Murray; Chris Lowe; Tennis court road, cambridge, cb2 1qt, england      *(lct11@mole.bio.cam.ac.uk) The crystal structure of firefly luciferase (FFL) demonstrates that the enzyme has a number of solvent-exposed hydrophobic residues on its surface. This reflects a number of previous observations regarding the hydrophobic nature of FFL. Solvent exposed hydrophobic residues in protein structures can be expected to i) increase protein aggregation and ii) compromise the protein's stability. In an attempt to improve the performance of FFL in assay systems, a protein engineering approach was taken to systemically decrease the hydrophobicicty of FFL. Amino acids were identified in the structure of FFL which were i) large hydrophobic residues (FLIVW), ii) solvent exposed and iii) non-conserved. These amino acids (eight in total) were individually mutated to alanines, so reducing the hydrophobicity without introducing any functional groups. Analysis of the resulting point mutations showed that whilst one of the mutations was delerterious, four of the mutations demonstrated significantly increased thermostability. None of the mutations effect the wavelength of bioluminescent emission, however a number of subtle effects on the 'flash-kinetics' of FFL were observed. [Poster: tisi.lauren.13132]

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