Understanding Optical Behaviour of Green Fluorescent Protein through It's Chromophore Analogues

Chatterjee, Tanmay (2016) Understanding Optical Behaviour of Green Fluorescent Protein through It's Chromophore Analogues. PhD thesis, Indian Institute of Science Education and Research Kolkata.

PDF (PhD thesis of Tanmay Chatterjee (10RS004)) Thesis_Tanmay.pdf - Submitted Version Restricted to Repository staff only Download (6MB)

Abstract

The green fluorescent protein (GFP) from jellyfish Aequorea victoria has attracted great interest as a biological fluorescence marker. The chromophore responsible for bright fluorescence of GFP is p-hydroxybenzilidineimidazolinone (p-HBDI). In our present effort, it is observed that suitable three step chemical tweaking of the parent chromophore (p-HBDI) produces highly bright coumarinic fluorophore. Utilising this ability of three step chemical tweaking of p-HBDI to produce highly bright fluorophore, a photoactivable analogue of p-HBDI viz; ONBYOHBO has been prepared. When excited at 375 nm, this nonfluorescent chromophore produces the same coumarinic fluorophore (cOHBO) which fluoresce brightly. Successful use of cOHBO towards live stem cell imaging has also been performed. In the present effort, to bridge between the emission properties of p-HBDI and o-HBDI, a number of model analogues have been synthesized. Interestingly, dual fluorescence of GFP chromophore analogues appearing from both the normal and tautomer species has been observed. One of the derivatives, CHBDI exhibiy hitherto unobserved dual emission with equal intensity proton transfer and charge transfer band. Out of other two derivatives OHIM exhibit distinctly only CT band and MHBDI exhibit proton transfer band. It could be shown that differential optical behaviour of o-HBDI and CHBDI is not due to conformational variation in solid or in solution phase. Rather control of excited state acidity/basicity of donor and acceptor site and excited state electronic energy level by functional group modification could be shown to be responsible for differential optical behaviour. Chemical modification induced electronic control over relative intensity of charge transfer and proton transfer bands could thus be evidenced. Support from single crystal X-ray structure, NMR, femtosecond fluorescence decay analysis provided important information and thus helped us understand the photophysics better. Excited state electronic energy level diagrams as well as acidity constant have been provided which could successfully explain differential chemical structure as well as solvent polarity dependent steady state and fluorescence dynamics of all these derivatives. It could be shown that for OHIM, PT is unfavoured both kinetically and thermodynamically. For CHBDI PT is favoured kinetically but not thermodynamically and for MHBDI, PT is favoured both kinetically and thermodynamically.

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