Structure and characteristics of reassembled fluorescent protein, a new insight into the reassembly mechanisms

Bioorganic and Medicinal Chemistry Letters

Volumn 21, Issue 10, 2011, Pages 3021-3024

  Isogai, Masami ^a   Kawamoto, Yoshihiro ^a   Inahata, Kazuto ^a   Fukada, Harumi ^a   Sugimoto, Kenji ^a   Tada, Toshiji ^a  

^a Osaka Prefecture University   (Japan)

Author keywords

BiFC; Crystal structure; Fluorescent protein; Thermal stability; Venus

Indexed keywords

FLUORESCENT PROTEIN; GREEN FLUORESCENT PROTEIN; MONOMER; OLIGOMER; PEPTIDES AND PROTEINS; PROTEIN VENUS; UNCLASSIFIED DRUG;

ANION EXCHANGE CHROMATOGRAPHY; ARTICLE; CRYSTAL STRUCTURE; MUTATIONAL ANALYSIS; PROTEIN ASSEMBLY; PROTEIN FOLDING; PROTEIN PROTEIN INTERACTION; PROTEIN STRUCTURE; THERMOSTABILITY; TIME OF FLIGHT MASS SPECTROMETRY; X RAY ANALYSIS;

CRYSTALLOGRAPHY, X-RAY; FLUORESCENT DYES; LUMINESCENT PROTEINS; MODELS, MOLECULAR;

EID: 79955553606     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2011.03.039     Document Type: Article

References (19)

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10. The anion exchange chromatography was performed using Mono Q™ 4.6/100 PE column (GE Healthcare) and the conditions are described below. Flow rate: 0.5 mL/min at 4 °C, gradient: 0-80% elution buffer in 25 column volumes, start buffer: 20 mM Tris-HCl, pH 8.0 + 20 mM NaCl, elution buffer: 20 mM Tris-HCl + 1.0 M NaCl.
11. Whole Venus, reassembled Venus and their mutants were diluted to 0.1 mg/mL with 20 mM sodium phosphate buffer, pH 7.0 for fluorescence spectral analysis. Fluorescence spectra were measured with a FP-175 fluorescence spectrophotometer (JASCO). Fluorescent proteins were excited at 485 nm, and emission spectra were recorded at a wavelength range from 500 to 600 nm at 20 °C.
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14. The molecular mass of each component was measured on an autoflex II MALDI-TOF analyzer (Bruker Daltonics) using saturated α-cyano-4- hydroxycinnamic acid in 50% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid as a matrix.
15. Samples were diluted to 0.1 mg/mL with 20 mM sodium phosphate buffer, pH 7.0 for CD spectral analysis. CD experiments were performed on a J-820 spectropolarimeter (JASCO) with a Peltier PTC-423L thermo-unit (JASCO). The far-UV CD spectra (260-190 nm) were recorded using a 0.1 cm path length cell under constant nitrogen flush with a step size of 0.2 nm, bandwidth of 1 nm, and an averaging time of 2 s at 20 °C. The final spectra reported were an average of 10 scans.
16. Coordinates have been deposited with the Protein Data Bank with the following accession code: 3AKO.
17. Three mutants of VN155 (Y143F, Y145F, H148G) were constructed by site-directed mutagenesis of plasmids carrying VN155 (pET-16b) by PCR using Pfu Turbo (Stratagene). The sequence of mutants were verified by DNA sequencing with a dye terminator cycle sequencing kit (Beckman Coulter) and a CEQ2000 fragment analysis system (Beckman Coulter). Reassembled Venus consisting of VN155 mutants and VC155 (denoted as rV-Y143F, rV-Y145F, rV-H148G) were coexpressed and purified by the same procedures as those used for reassembled Venus.
18. Calorimetric experiments were carried out with a nanoDSC (TA instruments). Samples were prepared in concentrations of 0.5 and 1.0 mg/mL. The buffer used for the sample was 20 mM sodium phosphate, pH 7.0. Experiments were performed over a temperature range of 25-95 °C at a scan rate of 1 °C/min and excess pressure of 2.8 atm.
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