lecture 2 - craggs Flashcards
key points from the structure text
tertiary structure of all FPs similar
11 beta-strands form perfect cylinder
capped at each end by aloha helix
chromophore located in the central coaxial helix
size of cylinder in GFP
42 angstroms by 24 angstroms
why is chromophore encapsulated
shielded from collision quenchers
increased barrier to non-radiative relaxation
what happens in denaturation
chromophore still there but non-fluorescent
what allows for excited state proton transfer
tight packing around chromophore and network of hydrogen bonds
what are absorption bands of WT GFP
395 and 475 nm
what wavelength leads to ESPT stage 1
395 nm
can stage I convert to stage B easily
no rarely by a non radiative process (rotation of T203)
emission of state B
503 nm
absorption of state B
475 nm
emission of state I
508 nm
what disrupts state A
one point mutation disrupts hydrogen bonding network so no longer populated
what happens in the excited state
decarboxylation
which is the non-absorbed state
state B
how to get from state B to state A
excite to state I and that quickly converts to A which changes a bit so can’t go back to B
