Three photoconvertible forms of green fluorescent protein identified by spectral hole-burning.
Several studies have led to the conclusion that, in the green fluorescent protein (GFP) of the jellyfish Aequorea victoria, a photoconversion involving excited-state proton transfer occurs from an A- to a B-form, while an intermediate I-form was held responsible for the green fluorescence. Here we have identified the I-form of wild-type GFP in absorption, located the 0-0 transitions of all three forms A, B and I, and determined vibrational frequencies of the ground and excited states. The intrinsically narrow 0-0 transitions are revealed by the wavelengths at which holes can be burnt. The pathways of photointerconversion are unraveled by excitation, emission and hole-burning spectroscopy. We present an energy-level scheme that has significant implications for GFP-mutants, which likewise can occur in the three photo-interconvertible forms.
Creemers
T M
TM
Center for the Study of the Excited States of Molecules, Huygens and Gorlaeus Laboratories, University of Leiden, The Netherlands.
Lock
A J
AJ
Subramaniam
V
V
Jovin
T M
TM
Völker
S
S
D000042:Absorption; D000818:Animals; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D007834:Lasers; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011522:Protons; D037401:Scyphozoa; D013057:Spectrum Analysis; D013696:Temperature
D008164:Luminescent Proteins; D011522:Protons; D049452:Green Fluorescent Proteins