chemistry of zs yellow fluorophorezs yellow protein ¥ sequence substantially different than gfp but...
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Chemistry of ZS Yellow fluorophore
Chem184
Green Fluorescent Protein (GFP)
• protein makes its own fluorophore out of amino acids
GFP
fluorophore
cyclization
dehydration
-ser65-tyr66-gly67-
oxidation
N
N
OO
NH
HO
NH
O
HO
HN
HN
OO
NH
HONH
O
HO O
Green Fluorescent Mouse
ZS Yellow Protein
• sequence substantially different than GFP
but same fold
The chromophore
• conjugated pi-system
• more orbitals involved than GFP
N
N
O
R
HO
N
N
N
O
R
- O
NH+
N
N
O
R
O
NH
Mechanism for formation
• self-catalyzed reactions (no other enzymes / reagents (exc. O2) involved)
HNHN
OO
NH
NH
O
HO O
H2N
NN
OO
NH
H2N
O
HO
N
lys66
tyr67
gly68
Mechanism for formation
HNHN
OO
NH
NH
O
HO O
H2N
HNN
OO
NH
NH
O
HO OH
H2Nlys66
tyr67
gly68cyclization
NN
OO
NH
NH
O
HO
H2N
NN
OO
NH
NH
O
HO
H2N
OH
O2 (oxid)elimination
(-H2O)
Mechanism for formation
N
N
OO
NH
NH
O
HO
H2N
OH
NN
OO
NH
NH
O
HO
H2N
elimination
(-H2O)
NN
OO
NH
NH
O
HO
H2N
OH
O2 (oxid)N
N
OO
NH
N
O
HO
H2N
elimination
(-H2O)
Mechanism for formation
N
N
OO
NH
N
O
HO
H2N
NN
OO
NH
NH
O
HO
NH
cyclization
NN
OO
NH
H2N
O
HO
N
chain cleavage
mature chromophore
Similarity to green fluorescent protein (GFP)
• additional orbitals in ZSY chromophore structure (red-shifted)
• protein surroundings can make a difference also
N
N
OO
NH
HO
NH
O
HON
N
OO
NH
HO
N
GFP ZSY
The Zsyellow protein environment around the chromophore
• different sidechains can make a difference sterically, electronically
• changes in amino acids can indirectly affect chromophore also
Why do conjugated molecules absorb visible light?
- light can excite electrons from lower energy molecular orbitals to higher
benzene
h!
benzene*
(excited state)
!E
Why do conjugated molecules absorb visible light?
The closer together the energy levels,
the less energy it takes to excite the electrons
!E smaller!
The more orbitals involved, the smaller the HOMO-LUMO gap is
Why do conjugated molecules absorb visible light?
The closer together the energy levels,
the less energy it takes to excite the electrons
E = h! ! = C/"
fromWe see that the longer the wavelength,
the lower the energy of the light
Therefore, small organic molecules absorb in the UV,
large conjugated molecules absorb in the visible
and
(v. large ones in the infrared)
!E smaller!
Some notes on what makes a molecule fluorescent
• light absorption, loss of small amount of energy (as heat)
• re-emission of photon, shifted to lower energy (redder)
absorption relaxation emission relaxation
(excitation) (fluorescence)
Some notes on what makes a molecule fluorescent
• if interested in visible fluorescence (~400-700nm),
then need several conjugated bonds
UV
UV/violet
blue-green
yellow
Some notes on what makes a molecule fluorescent
• planarity important for efficient conjugation
O
N
N
O
POPOP
Some notes on what makes a molecule fluorescent
• rigid enforcement of planarity
(avoid radiationless decay via internal rotations)
low quantum yield higher quantum yield
Some notes on what makes a molecule fluorescent
• for efficient fluorescence (high quantum yield), need
avoidance of strong electron acceptors / donors
or heavy atoms
N
High quantum yield
low quantum yield
Some notes on what makes a molecule fluorescent
• strong dipoles in fluorescent molecules can lead to
environmental sensitivity (e.g. changes with solvent polarity)
CN
N
environmentally insensitivesensitive to local environment
Some notes on what makes a molecule fluorescent
• strong dipoles in fluorescent molecules can lead to
environmental sensitivity (e.g. changes with solvent polarity)
CN
N
CN -
N+
The Zsyellow protein environment around the chromophore
• different sidechains can make a difference sterically, electronically
• changes in amino acids can indirectly affect chromophore also