4ees
Crystal structure of iLOVCrystal structure of iLOV
Structural highlights
Publication Abstract from PubMedFluorescent proteins derived from Light, Oxygen or Voltage (LOV) domains offer advantages over Green Fluorescent Protein (GFP) from their small size and efficacy under anaerobic conditions. The flavoprotein iLOV was engineered from the blue-light receptor phototropin as a reporter of viral infection. To inform the molecular basis for the improved, photoreversible, fluorescent properties of iLOV, we employed directed evolution and determined five LOV crystallographic structures. Comparative structural analyses between iLOV and its progenitors reveal mutation-induced constraints in the environment of the flavin mononcleotide (FMN) chromophore: in iLOV, the methyl group of Thr 394 crowds the FMN isoalloxazine ring, Leu470 triggers side-chain flipping of Leu472, and the terminal FMN phosphate shows increased anchoring. We further engineered iLOV variants that are readily detectable in bacterial and mammalian cells, due to order-of-magnitude photostability increases. Structure determination of a resulting representative photostable iLOV (phiLOV) variant reveals additional constraints on the chromophore. Aromatic residues Tyr401 and Phe485 in phiLOV sandwich the FMN isoalloxazine ring from both sides, while Ser390 anchors the side chain of FMN-interacting Gln489 Our combined structural and mutational results reveal that constraining the FMN fluorophore yields improved photochemical properties for iLOV and its new photostable derivative. These findings provide a framework for structural fine-tuning of LOV-scaffold proteins to maximize their potential as oxygen-independent fluorescent reporters. Structural Tuning of the Fluorescent Protein iLOV for Improved Photostability.,Christie JM, Hitomi K, Arvai AS, Hartfield KA, Mettlen M, Pratt AJ, Tainer JA, Getzoff ED J Biol Chem. 2012 May 9. PMID:22573334[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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