Crystal structure of Venus, a yellow fluorescent protein with improved maturation and reduced environmental sensitivity

被引:155
作者
Rekas, A
Alattia, JR
Nagai, T
Miyawaki, A
Ikura, M [1 ]
机构
[1] Univ Toronto, Div Mol & Struct Biol, Ontario Canc Inst, Toronto, ON M5G 2M9, Canada
[2] Univ Toronto, Dept Med Biophys, Toronto, ON M5G 2M9, Canada
[3] JST, PRESTO, Nakagyo Ku, Kyoto 6040847, Japan
[4] RIKEN, Brain Sci Inst, Adv Technol Dev Ctr, Lab Cell Funct & Dynam, Wako, Saitama 3510198, Japan
关键词
D O I
10.1074/jbc.M209524200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Yellow emission variants of green fluorescent protein (GFP) have been found useful in a variety of applications in biological systems due to their red-shifted emission spectrum and sensitivity to environmental parameters, such as pH and ionic strength. However, slow maturation properties and new requirements for more intense fluorescence necessitated further mutagenesis studies of these proteins. Venus, a new variant with improved maturation and brightness, as well as reduced environmental dependence, was recently developed by introducing five mutations into the well characterized variant, enhanced yellow fluorescent protein (EYFP). In this paper, we present the crystal structure of Venus at 2.2 Angstrom resolution, which enabled us to correlate its novel features with these mutation points. The rearrangement of several side chains near the chromophore, initiated by the F46L mutation, was found to improve maturation at 37degreesC by removing steric and energetic constraints, which may hinder folding of the polypeptide chain, and by accelerating the oxidation of the Calpha-Cbeta bond of Tyr(66) during chromophore formation. M153T, V163A, and S175G were also found to improve the rate of maturation by creating regions of greater flexibility. F64L induced large conformational changes in the molecule, leading to the removal of halide sensitivity by preventing ion access to the binding site.
引用
收藏
页码:50573 / 50578
页数:6
相关论文
共 25 条
[1]   THE CCP4 SUITE - PROGRAMS FOR PROTEIN CRYSTALLOGRAPHY [J].
BAILEY, S .
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 1994, 50 :760-763
[2]  
BASTIANUTTA R, 2000, PROTEINS, V41, P429
[3]   Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed) [J].
Bevis, BJ ;
Glick, BS .
NATURE BIOTECHNOLOGY, 2002, 20 (01) :83-87
[4]   Crystallography & NMR system:: A new software suite for macromolecular structure determination [J].
Brunger, AT ;
Adams, PD ;
Clore, GM ;
DeLano, WL ;
Gros, P ;
Grosse-Kunstleve, RW ;
Jiang, JS ;
Kuszewski, J ;
Nilges, M ;
Pannu, NS ;
Read, RJ ;
Rice, LM ;
Simonson, T ;
Warren, GL .
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 1998, 54 :905-921
[5]   Structural and spectral response of green fluorescent protein variants to changes in pH [J].
Elsliger, MA ;
Wachter, RM ;
Hanson, GT ;
Kallio, K ;
Remington, SJ .
BIOCHEMISTRY, 1999, 38 (17) :5296-5301
[6]   Reducing the environmental sensitivity of yellow fluorescent protein - Mechanism and applications [J].
Griesbeck, O ;
Baird, GS ;
Campbell, RE ;
Zacharias, DA ;
Tsien, RY .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (31) :29188-29194
[7]   WAVELENGTH MUTATIONS AND POSTTRANSLATIONAL AUTOXIDATION OF GREEN FLUORESCENT PROTEIN [J].
HEIM, R ;
PRASHER, DC ;
TSIEN, RY .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (26) :12501-12504
[8]   IMPROVED GREEN FLUORESCENCE [J].
HEIM, R ;
CUBITT, AB ;
TSIEN, RY .
NATURE, 1995, 373 (6516) :663-664
[9]   Mechanism and cellular applications of a green fluorescent protein-based halide sensor [J].
Jayaraman, S ;
Haggie, P ;
Wachter, RM ;
Remington, SJ ;
Verkman, AS .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (09) :6047-6050
[10]   IMPROVED METHODS FOR BUILDING PROTEIN MODELS IN ELECTRON-DENSITY MAPS AND THE LOCATION OF ERRORS IN THESE MODELS [J].
JONES, TA ;
ZOU, JY ;
COWAN, SW ;
KJELDGAARD, M .
ACTA CRYSTALLOGRAPHICA SECTION A, 1991, 47 :110-119