Biophysical and Functional Characterization of asFP504,
a Novel Fluorescent Protein from the Philippines
Neil Andrew D. Bascos1,*, Francine Lianne C. Emralino1, Franco Carlos Liu1,
Carla P. Concepcion2, Marvin Altamia2, Yen-Chieh Huang3, Yin-Cheng Hsieh3,
Chun-Jung Chen3,4,5,*, and Cynthia Palmes-Saloma2,*
1Protein Structure and Immunology Laboratory, National Institute of Molecular Biology and Biotechnology,
University of the Philippines Diliman, Quezon City, Philippines
2Laboratory of Molecular and Cellular Biology, National Institute of
Molecular Biology and Biotechnology, University of the Philippines Diliman, Quezon City, Philippines
3Life Science Group, Scientific Research Division,
National Synchrotron Radiation Research Center, Hsinchu, Taiwan
4Department of Physics, National Tsing Hua University, Hsinchu, Taiwan
5Institute of Biotechnology, University Center for Bioscience and
Biotechnology, National Cheng Kung University, Tainan, Taiwan
*Corresponding authors: This email address is being protected from spambots. You need JavaScript enabled to view it.
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ABSTRACT
Fluorescent proteins have proven to be invaluable for a myriad of applications in scientific research. The discovery and characterization of novel fluorescent proteins promises to expand this range even further. This report focuses on the biophysical and functional characterization of a novel green fluorescent protein cloned from a Philippine soft coral species. The asFP504 protein showed peak excitation at 471 nm and at 494 nm (λE1= 471 nm; λE2=494 nm), its emission maximum from 471 nm excitation was observed at 504 nm. The fluorescence was observed to be related to its oligomeric state. Both fluorescence and oligomerization were robustly maintained for a range of temperatures, pH conditions, treatment with chaotropic agents, and proteolysis. X-ray crystallography documented a molecular packing of three dimers within each asymmetric unit for the asFP504 protein. The observed absorbance and fluorescence properties are comparable to that of commercially available fluorescence proteins. Despite its lower absorbance, asFP504 has higher quantum yield than mCitrine. In addition, the stability of asFP504 in the presence of multiple denaturants presents the potential of this protein – the first fluorescent protein from the Philippines – for use in many different research applications.
Key words: crystal structure, fluorescent protein, FRET, GFP, mutagenesis
INTRODUCTION
The green fluorescent proteins or GFPs (Tsien 1998) comprise a small class of chromoproteins found in bioluminescent hydrozoan and anthozoan coelenterates. Among the first of these to be discovered are members from the Aequorea victoria (avGFP) and Renilla reniformis (Renilla GFP) species. The GFP family of proteins has now expanded to include a number of GFP-like fluorescent and non-fluorescent proteins that have become important tools in molecular and cell biology. These proteins are used as protein labels, reporter genes, selection markers, fusion tags, and biosensors (Lippincott-Schwartz & Patterson 2003). Most of the current information on GFPs is based on the extensive research on the avGFP and Renilla GFP variants. Continued research on the properties of the expanding repertoire of GFPs promises the discovery of new ways for which these proteins may be utilized. . . . read more
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