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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Halliwell, Lisa Marie
Languages: English
Types: Doctoral thesis
Subjects: QR
The bioluminescence reaction is catalysed by firefly luciferase, converting the substrates D-luciferin, ATP and molecular oxygen with Mg2+ to produce light and this reaction has had wide ranging implications within a number of fields from industry to academia. The discovery of luciferase has been revolutionary in the real time in vivo study of cells given that it requires no energy for excitation, delivering a high signal to background ratio providing a highly sensitive assay. This protein, to date, has been utilised in molecular cell biology, cellular imaging, microbiology and numerous other fields. The extensive application of this protein has paved the way for the generation of toolbox of variants with altered properties. Protein engineering involving substitution mutations made within Photinus pyralis (Ppy) FLuc has led to the discovery of a number of novel variants however there is a bank of growing evidence displaying the power of deletions as an alternative for the development of proteins with altered properties since deletions can sample structural diversity not accessible to substitutions alone.\ud A novel mutagenic strategy was implemented to incorporate single amino acid deletions within thermostable firefly luciferase (x11FLuc) targeting loop structures (M1-G10, L172- T191, T352-F368, D375-R387, D520-L526, K543-L550). Of 43 deletion mutants obtained, 41 retained bioluminescent activity and other characteristics such as resistance to thermal inactivation. Surprisingly, only 2 variants, ΔV365 and ΔV366, exhibited a complete loss of activity showing that the luciferase protein is largely tolerant to single amino acid deletions.\ud \ud In order to identify useful mutants in the extensive library, a 96-well format luminometric cell lysate assay was developed which indicated the effect of deletions was largely region specific, for example, N- terminal deletions did not alter the activity of x11FLuc, whilst deletions within L172- T191, D375-R387, D520-L526 and the C- terminal loop reduced overall activity. On the other hand, deletions within T352-F368 enhanced overall bioluminescent activity and remarkably exhibited other important characteristics such as increases in specific activity and a reduced KM for luciferin. Therefore, a novel motif (omega loop) was identified as important for FLuc activity after full characterization of mutants.\ud \ud Characterisation of the deletion mutants originating from the omega loop (T352-F368), ΔP359 and ΔG360 both presented a reduced KM for luciferin, whilst ΔA361, ΔV362, ΔG363 presented an increase in KM towards ATP as compared to x11FLuc. Thus, deletions in the omega loop, in the main, improved activity and altered reaction kinetics, in particular ΔG363 retained 53% of initial activity after 250s. As such, it is considered that the field of protein engineering should not only overlook the utility of single amino acid deletions, since such mutagenic strategies may sample structural space not achieved by substitutions alone and mutations within less popularized secondary structures such as omega loops are can act as a useful tool in the improvement of proteins.
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