Folding of green fluorescent protein and the cycle3 mutant.
Although the correct folding of green fluorescent protein (GFP) is required for formation of the chromophore, it is known that wild-type GFP cannot mature efficiently in vivo in Escherichia coli at 37 degrees C or higher temperatures that the jellyfish in the Pacific Northwest have never experienced. Recently, by random mutagenesis by the polymerase chain reaction (PCR) method, a mutant called Cycle3 was constructed. This mutant had three mutations, F99S, M153T, and V163A, on or near the surface of the GFP molecule and was able to mature correctly even at 37 degrees C [Crameri et al. (1996) Nat. Biotechnol. 143, 315-319]. In the present study, we investigated the differences in their folding behavior in vitro. We observed the folding and unfolding reactions of both wild-type GFP and the Cycle3 mutant by using green fluorescence as an indicator of the formation of the native structure and examining hydrogen-exchange reactions by Fourier transform infrared spectroscopy. Both proteins showed unusually slow refolding and unfolding rates, and their refolding rates were almost identical under the native state at 25 and at 35 degrees C. On the other hand, aggregation studies in vitro showed that wild-type GFP had a strong tendency to aggregate, while the Cycle3 mutant did not. These results indicated that the ability to mature efficiently in vivo at 37 degrees C was not due to the improved folding and that reduced hydrophobicity on the surface of the Cycle3 mutant was a more critical factor for efficient maturation in vivo.
Fukuda
H
H
Department of Physics, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Arai
M
M
Kuwajima
K
K
D000818:Animals; D002942:Circular Dichroism; D017666:Deuterium Oxide / Q000737:chemistry; D049452:Green Fluorescent Proteins; D019791:Guanidine / Q000737:chemistry; D007202:Indicators and Reagents; D007700:Kinetics; D008027:Light; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D017666:Deuterium Oxide; D019791:Guanidine