Green fluorescent protein variants as ratiometric dual emission pH sensors. 2. Excited-state dynamics.
In the preceding paper [Hanson, G. T., McAnaney, T. B., Park, E. S., Rendell, M. E. P., Yarbrough, D. K., Chu, S., Xi, L., Boxer, S. G., Montrose, M. H., and Remington, S. J. (2002) Biochemistry 41, 15477-15488], novel mutants of the green fluorescent protein (GFP) that exhibit dual steady-state emission properties were characterized structurally and discussed as potential intracellular pH probes. In this work, the excited-state dynamics of one of these new dual emission GFP variants, deGFP4 (C48S/S65T/H148C/T203C), is studied by ultrafast fluorescence upconversion spectroscopy. Following excitation of the high-energy absorption band centered at 398 nm and assigned to the neutral form of the chromophore, time-resolved emission was monitored from the excited state of both the neutral and intermediate anionic chromophores at both high and low pH and upon deuteration of exchangeable protons. The time-resolved emission dynamics and isotope effect appear to be very different from those of wild-type GFP [Chattoraj, M., King, B. A., Bublitz, G. U., and Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 8362-8367]; however, due to overlapping emission bands, the apparent difference can be analyzed quantitatively within the same framework used to describe GFP excited-state dynamics. The results indicate that the pH-sensitive steady-state emission characteristics of deGFP4 are a result of a pH-dependent modulation of the rate of excited-state proton transfer. At high pH, a rapid interconversion from the excited state of the higher energy neutral chromophore to the lower energy intermediate anionic chromophore is achieved by proton transfer. At low pH, excited-state proton transfer is slowed to the point where it is no longer rate limiting.
McAnaney
Tim B
TB
Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.
Park
Eun Sun
ES
Hanson
George T
GT
Remington
S James
SJ
Boxer
Steven G
SG
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D003903:Deuterium; D014644:Genetic Variation; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008954:Models, Biological; D008956:Models, Chemical; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D011522:Protons; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D008164:Luminescent Proteins; D020033:Protein Isoforms; D011522:Protons; C409504:blue fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins; D003903:Deuterium