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
four processes to form chromophore
folding
cyclisation
oxidation
dehydration
why is folding required
maturation of chromophore
what is involved in cyclisation
nucleophilic attack of the carbonyl to form high energy intermediate 1
how does the protein act like an enzyme
aligning the nucleophile N67 and electrophile carbonyl-C65
what angle of distorted helix for correct alignment
80 degrees
how is no explicit enthalpic cost
reduced number of hydrogens before and after cyclisation
other important residues
G67 and some proline residues
key roles in folding and pre-organisation
R96 E222 and Y66
roles of R96 and E222
catalyse key steps in the mechanism
