Abstract
The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria has attracted widespread interest since the discovery that its chromophore is generated by the autocatalytic, posttranslational cyclization and oxidation of a hexapeptide unit. This permits fusion of the DNA sequence of GFP with that of any protein whose expression or transport can then be readily monitored by sensitive fluorescence methods without the need to add exogenous fluorescent dyes. The excited state dynamics of GFP were studied following photo-excitation of each of its two strong absorption bands in the visible using fluorescence upconversion spectroscopy (about 100 fs time resolution). It is shown that excitation of the higher energy feature leads very rapidly to a form of the lower energy species, and that the excited state interconversion rate can be markedly slowed by replacing exchangeable protons with deuterons. This observation and others lead to a model in which the two visible absorption bands correspond to GFP in two ground-state conformations. These conformations can be slowly interconverted in the ground state, but the process is much faster in the excited state. The observed isotope effect suggests that the initial excited state process involves a proton transfer reaction that is followed by additional structural changes. These observations may help to rationalize and motivate mutations that alter the absorption properties and improve the photo stability of GFP.
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bokman S. H., Ward W. W. Renaturation of Aequorea gree-fluorescent protein. Biochem Biophys Res Commun. 1981 Aug 31;101(4):1372–1380. doi: 10.1016/0006-291x(81)91599-0. [DOI] [PubMed] [Google Scholar]
- Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802–805. doi: 10.1126/science.8303295. [DOI] [PubMed] [Google Scholar]
- Cody C. W., Prasher D. C., Westler W. M., Prendergast F. G., Ward W. W. Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein. Biochemistry. 1993 Feb 9;32(5):1212–1218. doi: 10.1021/bi00056a003. [DOI] [PubMed] [Google Scholar]
- Delagrave S., Hawtin R. E., Silva C. M., Yang M. M., Youvan D. C. Red-shifted excitation mutants of the green fluorescent protein. Biotechnology (N Y) 1995 Feb;13(2):151–154. doi: 10.1038/nbt0295-151. [DOI] [PubMed] [Google Scholar]
- Heim R., Cubitt A. B., Tsien R. Y. Improved green fluorescence. Nature. 1995 Feb 23;373(6516):663–664. doi: 10.1038/373663b0. [DOI] [PubMed] [Google Scholar]
- Heim R., Prasher D. C., Tsien R. Y. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12501–12504. doi: 10.1073/pnas.91.26.12501. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Perozzo M. A., Ward K. B., Thompson R. B., Ward W. W. X-ray diffraction and time-resolved fluorescence analyses of Aequorea green fluorescent protein crystals. J Biol Chem. 1988 Jun 5;263(16):7713–7716. [PubMed] [Google Scholar]
- Rao B. D., Kemple M. D., Prendergast F. G. Proton nuclear magnetic resonance and fluorescence spectroscopic studies of segmental mobility in aequorin and a green fluorescent protein from aequorea forskalea. Biophys J. 1980 Oct;32(1):630–632. doi: 10.1016/S0006-3495(80)84999-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ward W. W., Bokman S. H. Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein. Biochemistry. 1982 Sep 14;21(19):4535–4540. doi: 10.1021/bi00262a003. [DOI] [PubMed] [Google Scholar]
- al-Taylor C., Ashraf el-Bayoumi M., Kasha M. Excited-state two-proton tautomerism in hydrogen-bonded n-heterocyclic base pairs. Proc Natl Acad Sci U S A. 1969 Jun;63(2):253–260. doi: 10.1073/pnas.63.2.253. [DOI] [PMC free article] [PubMed] [Google Scholar]