Graciela Sala-Newby

Engineering the Ca2+-activated photoprotein aequorin with reduced affinity for calcium

Two stage PCR has been used to introduce single amino acid substitutions into the EF hand structures of the Ca(2+)-activated photoprotein aequorin. Transcription of PCR products, followed by cell free translation of the mRNA, allowed characterisation of recombinant proteins in vitro. Substitution of D to A at position 119 produced an active photoprotein with a Ca2+ affinity reduced by a factor of 20 compared to the wild type recombinant aequorin. This recombinant protein will be suitable for measuring Ca2+ inside the endoplasmic reticulum, the mitochondria, endosomes and the outside of live cells.

Engineering firefly luciferase as an indicator of cyclic AMP-dependent protein kinase in living cells

A bioluminescent indicator for protein kinase A has been developed by mutating V217 in firefly (Photinus pyralis) luciferase to R, and the C‐terminal peroxisomal signal removed by PCR. The cDNA for normal and the RRFS mutant luciferase were inserted into pSV7d and expressed in COS‐7 cells. Transient expression in approximately 5% of cells was confirmed by extraction of active luciferase, light emission from cells in the presence of luciferin, and immuno‐localisation. The cyclic‐AMP analogue, 8‐(4‐chlorophenylthio)‐cyclic AMP caused a 5–10% decrease in light emission within 4 min in COS cells expressing the RRFS mutant, but not in cells expressing normal luciferase. This provides for the first time an indicator for detecting and quantifying protein kinase A activation in living cells.

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.

Stepwise removal of the C-terminal 12 amino acids of firefly luciferase results in graded loss of activity.

The C-terminus of the firefly luciferase (550 amino acids) was engineered using PCR followed by in vitro transcription-translation in order to investigate the role of the last 12 amino acids in the bioluminescence. Coding sequences were removed stepwise and the decapeptide MRSAMSGLHL, a putative AMP-activated protein kinase phosphorylation site, was used to replace the last 8-12 amino acids in order to test for amino acid specificity at the C-terminus. Removal of up to seven of the C-terminal amino acids resulted in no detectable loss of bioluminescent activity. However, the luciferase activity decreased stepwise from 50 to 0.1% when 8-12 amino acids were removed. Replacement of amino acids 539-550 and 543-550 by MRSAMSGLHL generated luciferases that retained 22 and 35% of catalytic activity respectively. These results have important implications for the further development of engineered luciferases as intracellular indicators and the understanding of the active centre of beetle luciferases.

Agonist-stimulated free calcium in subcellular compartments - Delivery of recombinant aequorin to organelles using a replication deficient adenovirus vector

Changes in the concentration of calcium ions ([Ca2+]) within cellular organelles play a central role in controlling cellular function. We have engineered the Ca2+ sensitive photoprotein aequorin to monitor selectively [Ca2+] within defined subcellular compartments, namely the cytosol, nucleus and endoplasmic reticulum. DNA encoding the engineered aequorins have been inserted into a replication deficient adenovirus (Ad) type 5 E1-vector, under control of the cytomegalovirus (CMV) major immediate early promoter. The Ad vector provides a simple and efficient method to express the photoproteins in a wide variety of mammalian cell types. Efficient targeting of the photoproteins to the appropriate cellular compartment was established immunocytochemically in COS7 cells, where it was expressed in up to 100% of the target population. Levels of expression could be controlled by virus dose and chemical agents which affect the activity of the CMV promoter. In HeLa cells expressing nuclear targeted aequorin or cytosolic aequorin, ATP or histamine induced immediate biphasic elevations of both nuclear and cytosolic [Ca2+]; subsequent challenge with agonist evoked similar responses. In addition to epithelial type adherent cell lines (COS7 and HeLa), aequorin expression was also readily detected in non-adherent cells of myeloid lineage (K562 and HL60) and non-adherent primary cells polymorphonuclear leucocytes (neutrophils). The Ad vectors can, therefore, be used to express targeted aequorin in a range of different cell types and represents a novel method to monitor changes in free [Ca2+] in cellular organelles.

Imaging bioluminescent indicators shows Ca2+ and ATP permeability thresholds in live cells attacked by complement.

A series of permeability thresholds to Ca2+ metabolites and macromolecules, occurring at different times when cells are attacked by complement, has been established by imaging HeLa cells transiently expressing a recombinant cytosolic fusion protein of firefly luciferase and aequorin (luciferase–aequorin) to measure changes in ATP and cytosolic free Ca2+. Nuclear fluorescence of propidium was used as a measure of permeability to small molecules, and luciferase activity imaged to assess lysis. The rise in cytosolic free Ca2+ observed after C9 attack preceded by at least 60 s both the increase in propidium fluorescence, measured in single cells, and the decrease in ATP monitored by luciferase light emission. These effects were dependent on the concentration of C9. At concentrations of C9 up to 4 μg/ml no loss of luciferase–aequorin protein was detected at the end of the experiment. Thus the membrane integrity of the cells remained intact, even though the cells were permeable to propidium. These results confirmed our earlier observations that propidium permeability in cells attacked by complement was not a reliable measure of cell death. They also show that it is vital to take account of cellular heterogeneity if the mechanisms by which cells respond to membrane pore former attack are to be correctly interpreted.

Removal of twelve C-terminal amino acids from firefly luciferase abolishes activity

cDNA coding for the luciferase in the firefly Photinus pyralis was cloned using pcDV1 primer and Honjo linker containing SP6 RNA polymerase promoter. This enabled conditions to be established to produce mRNA, capped with m7 GpppG, in vitro and then translated to form light emitting protein. Full length recombinant luciferase produced by in vitro translation, was fully active, had the same isoelectric focusing point as the native enzyme and produced a similar, yellow emission. Removal of the coding sequence for the last 12 amino acids at the C terminus, containing the peroxisome signal peptide, by polymerase chain reaction resulted in greater than or equal to 99% loss in activity of the protein formed from mRNA in vitro. This has important implications for using this luciferase as an indicator or reporter gene in eukaryotic cells, and for identifying the active centre of the enzyme.