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Chemical Structure of the Chromophore Biosynthesis of the Chromophore Critcial dehyrogenation reaction to juxtapose aromatic group with imidazlinone

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Chemical Structure of the Chromophore

Biosynthesis of the Chromophore Critcial dehyrogenation reaction to juxtapose aromatic group withimidazlinone

Excitation and Fluorescence Emission Spectra of GFP

Förster cycle

Hydroxyl formPhenol-OH

PhenolatePhenol-O-

Protonatedexcxited

Excited phenolate

wt

egfpS65t

t203i

YfpS65g, t203y

CfpF64l, s65t, y66w

BfpY66h, y145f

The Beta-Can Structure

Topology of Folding

Cysteins

The Environment of the Fluorophor

Light microscopy - fancy techniques.FLIM - fluorescence life-time imaging; used to study interactions between molecules in living cells. Used in conjunction with FRET.FLIM measures the fluorescent life-time of the FRET donor. Decay of the activated FRET donor depends on its environment and association with an acceptor. FLIM data can be used to calculate true FRET efficiencies and confirm molecular interaction in living cells.This requires a pulse laser and so is routinely performed on a multiphoton lm.

FCS - fluorescence correlation spectroscopy; monitors random motion of fluorescently labelled molecules in a defined cell volume, which is irradiated by a focussed laser beam. Monitored changes give information about rates of diffusion and so apparent mass. If mass is known - likely association with cell compartments can be implied. Hence this is versatile for dynamics studies in live cells.

Uses FCS module attached to routine confocal microscope