Engineering the C-terminus of firefly luciferase as an indicator of covalent modification of proteins.
Protein kinase recognition sequences and proteinase sites were engineered into the cDNA encoding firefly luciferase from Photinus pyralis in order to establish whether these modified proteins could be developed as bioluminescent indicators of covalent modification of proteins. Two key domains of the luciferase were modified in order to identify regions of the protein in which peptide sequences may be engineered whilst retaining bioluminescent activity; one between amino acids 209 and 227 and the other at the C-terminus, between amino acids 537 and 550. Mutation of amino acids between residues 209 and 227 reduced bioluminescent activity to less than 1% of wild-type recombinant. In contrast engineering peptide sequences at the C-terminus resulted in specific activities ranging from 0.06-120% of the wild-type recombinant. Addition of cyclic AMP dependent protein kinase catalytic subunit, to a variant luciferase incorporating the kinase recognition sequence, LRRASLG, with a serine at amino-acid position 543 resulted in a 30% reduction in activity. Alkaline phosphatase treatment restored activity. The bioluminescent activity of a variant luciferase containing a thrombin recognition sequence, LVPRES, with the cleavage site positioned between amino acid 542 and 543, decreased by 50% when incubated in the presence of thrombin. The results indicate regions within luciferase where peptide sequences may be engineered while retaining bioluminescent activity and have shown changes in bioluminescent activity when these sites are subjected to covalent modification. Changes in secondary structure, charge and length at the C-terminus of luciferase disrupt the microenvironment of the active site, leading to alterations in light emission. This has important implications both in understanding the evolution of beetle bioluminescence and also in development of bioluminescent indicators of the covalent modification of proteins.
Waud
J P
JP
Department of Medical Biochemistry, Llandough Hospital, Llandough, Penarth, Cardiff, UK.
Sala-Newby
G B
GB
Matthews
S B
SB
Campbell
A K
AK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001517:Coleoptera / Q000201:enzymology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism / Q000494:pharmacology; D017931:DNA Primers; D004795:Enzyme Stability; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology
D017931:DNA Primers; D011994:Recombinant Proteins; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; D013917:Thrombin