Photoreduction by GFP: what does it mean biologically?

In this post, I want to make two technical points about the new report from Bogdanov et al which I mentioned previously.

The first point is that the further-oxidized, red fluorescent state of GFP is quite similar to the known red fluorescent protein dsRed. This point isn’t really mine; in fact Bogdanov and his co-authors suggest it in their paper (when they cite reference 14). But, possibly due to space constraints, they were unable to explain the similarities in detail. Let me do so here.

The part of GFP responsible for its fluorescence is its chromophore, which is formed by post-translational modification of three consecutive amino acid residues of the GFP protein. The backbone amide nitrogen of the 68th residue, a glycine (G), is believed to attack the backbone carbonyl carbon of residue 66 (often a serine (S) or a threonine (T)). The resulting cyclic intermediate loses water to form an imidazalone ring. The Cα and Cβ carbons of residue 67, a tyrosine (Y), are then oxidized by molecular oxygen, forming a Cα-Cβ double bond and thereby creating a ~12 atom conjugated system extending from tyrosine 66’s aromatic ring to the newly formed imidazalone ring. This conjugated system is the mature, green fluorescent chromophore.

Bogdanov and others found that this chromophore could be further oxidized to a red fluorescent state in an apparent two-electron process. This trait is shared by GFP and widely known red fluorescent protein dsRed. dsRed’s chromophore passes through a green fluorescent state, very similar to GFP’s, during oxidative maturation to its final red fluorescent state. Roger Tsien’s laboratory found that the additional oxidation step in dsRed further extends the conjugation in the chromophore by oxidizing the single Cα-N bond of residue 65 to a double bond.

The key differences between GFP and dsRed, however, are that the oxidant for both steps of dsRed maturation is apparently molecular oxygen, and neither step of dsRed maturation has been reported to require light. For GFP, the first oxidation, necessary for forming its green chromophore, uses molecular oxygen, and needs no light, but the newly reported, “redding”, second oxidation apparently requires light, and is vastly accelerated by biological redox agents like quinones or cytochrome c.

My second point concerns what this all may mean for the biology of GFP-possessing organisms. Bogdanov et al. conclude their paper by saying “an active role of GFPs in light-induced electron transfer should be kept in mind when one considers the biology of GFPs and potential applications”. That statement is true enough, but in my view it is worthwhile to point out important caveats that likely apply to this statement, especially since these caveats arise from data we already know.

The new properties of GFP may well come to be exploited in a variety of exciting new biotechnology applications, but I personally think it is unlikely that light capture by GFP and of other cellular redox agents is an energetically important process in jellyfish metabolism. I base this fact on the low quantum yield of GFP photobleaching or photoredding. Bogdanov did not give explicit estimates of the quantum yield of photobleaching or photoredding of the GFPs they studied, but I made order-of-magnitude estimates from their data, especially from their Supplemental Figure 1. The maximum quantum yield for GFP photoredding is likely to be around 6×10-4. This low efficiency combined with the low intensity of photoredding-active radiation from sunlight (as compared to what Bogdanov et al. used in their lab) means that the maximum energy a jellyfish can derive from photoredding is miniscule. Even if jellyfishes’ entire bodies were filled with GFP, and they spent an entire year at the equator in noontime-strength sunlight, GFP-powered photoreduction of NAD+ would be able to supply only about 2% of its energy needs. So the photoredding of GFP and concomitant reduction of biological redox molecules is unlikely to be significant energy source.

My tables below detail my calculations.

ESTIMATION OF GFP CONCENTRATION
Value Units Description Reference

55000

M-1 cm-1 GFP extinction coefficient Suppl. Info pg. 7

0.055

AU Height of GFP absorbance peak
before photoredding experiment
Suppl. Info Fig. 3c

1

cm Path length through cuvette Assumed by CF

1

mM GFP concentration used in experiments calculated
ESTIMATION OF PHOTON QUANTITY
Value Units Description Reference

3.91E-19

J / g energy per photon E = h c / l, l= 488nm

2.35E+05

J / mol g energy per einstein

0.1

W / cm2 Irradiance at cuvette surface Suppl. Info pg. 7

1

cm2 Cuvette surface area Assumed by CF

4.25E-01

mmol g / s inflow of redding-active photons into cuvette estimated
CALCULATION OF CONVERSION EFFICIENCY
Value Units Description Reference

3.00E-02

s-1 maximum observed rate of fractional depletion per second of green GFP fluorescence
in presence of 2.6 mM cytochrome c
Suppl. Info Fig. 1c

3.00E-05

mol / s mmol of gfp lost per second calculated

7.06E-05

mmol gfp / mmol g yield of bleached/redded gfp per incident photon calculated

88.10%

transmittance of gfp in cuvette T = 10-absorbance

5.94E-04

mmol gfp / mmol g yield of bleached/redded gfp per absorbed photon
COMPARISON OF GFP CONVERSION TO SEA LIGHT INTENSITY
Value Units Description Reference

1.4

W / m2 / nm approximate solar irradation at 488 nm, sea level,
noon at equator
http://en.wikipedia.org/wiki/File:Solar_Spectrum.png

60

nm gfp green absorbance peak full width at half max. Suppl. Info Fig. 3c

84

W / m2 gfp-redding active solar irradiation at sea surface calculated

0.0084

W / cm2 gfp-redding active solar irradiation at sea surface

3.57E-08

mol g / cm2 / s gfp-redding active solar irradiation at sea surface calculated

6.68

mol gfp / m2 / y maximum amount of photobleachable gfp per year, if sunlight (not gfp protein production) was limiting calcuated
THERMODYNAMICS OF NADH oxidation by O2
Value Units Description Reference

0.315

V standard half-reaction potential
for NADH oxidation to NAD+

0.82

V standard half-reaction potential
for 1/2 O2 reduction to H2O

49

kJ / mol free energy per mol
from O2-oxidized NADH
at standard conditions
calculated

0.10

m assumed jellyfish radius assumed

30

m2 / m3 surface area to volume ratio for a 0.1 m sphere

1

mmol O2 / g / h representative jellyfish O2 consumption rate http://www.biolbull.org/cgi/content/abstract/187/1/84

8760

mol O2 / m3 / y jellyfish O2 consumption rate assuming
jellyfish have density of water
assumed

30

m2 / m3 surface area to volume ratio for a 0.1 m sphere

292

mol O2 / m2 / y yearly jellyfish O2 consumption rate on a per surface area basis calculated

2%

ratio between maximum gfp-redding rate from sunlight and jellyfish energy needs calculated
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