Metal-enhanced fluorescence of single green fluorescent protein (GFP)

ABSTRACT The green fluorescent protein (GFP) gene offers many advantages as a viability reporter for high-throughput antimicrobial drug screening. However, screening for antituberculosis compounds by using GFP driven by the heat shock promoter, hsp60, has been of limited utility due to the low signal-to-noise ratio. Therefore, an alternative promoter was evaluated for its enhanced fluorescence during microplate-based culture and its response to 18 established antimicrobial agents by using a green fluorescent protein microplate assay (GFPMA). Mycobacterium tuberculosis strains H37Rv, H37Ra, and Erdman were transformed with pFPCA1, which contains a red-shifted gfp gene driven by the acetamidase promoter of M. smegmatis mc2155. The pFPCA1 transformants achieved higher levels of GFP-mediated fluorescence than those carrying the hsp60 construct, with signal-to-noise ratios of 20.6 to 27.8 and 3.8 to 4.5, respectively. The MICs of 18 established antimicrobial agents for all strains carrying pFPCA1 in the GFPMA were within 1 to 2 twofold dilutions of those determined by either the fluorometric or the visual microplate Alamar Blue assay (MABA). No significant differences in MICs were observed between wild-type and pFPCA1 transformants by MABA. The optimized GFPMA is sufficiently simple, robust, and inexpensive (no reagent costs) to be used for routine high-throughput screening for antituberculosis compounds.

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ON MECHANISM OF ENHANCED FLUORESCENCE IN GREEN FLUORESCENT PROTEIN

Biophysical Reviews and Letters ◽

10.1142/s1793048007000568 ◽

2007 ◽

Vol 02 (03n04) ◽

pp. 221-227 ◽

Cited By ~ 1

Author(s):

EVGENI B. STARIKOV ◽

ITAI PANAS ◽

YUJI MOCHIZUKI ◽

SHIGENORI TANAKA ◽

YI LUO ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Degrees Of Freedom ◽

Fluorescent Protein ◽

Fluorescence Emission ◽

Enhanced Fluorescence ◽

Additional Energy ◽

Definitive Answer ◽

Green Fluorescent ◽

Experimental And Theoretical Studies ◽

Intrinsic Strain

In spite of the numerous experimental and theoretical studies on green fluorescent protein and its modifications, there is still no definitive answer to the central question: why such systems exhibit enhanced fluorescence. Based upon detailed quantum-chemical estimations, we advocate the following hypothesis. In the green fluorescent protein ground electronic state, the protein surrounding strains the chromophore with respect to its native intramolecular conformational preference in vacuo or in solution. Absorbing a photon of the proper wavelength not only causes a joint proton–electron transfer in and around the chromophore, but also increases the intrinsic strain of the latter. Since conformational relaxation of such a structure will not require any additional energy input, the energy gained by the chromophore cannot be dissipated into the chromophore's internal non-radiative degrees of freedom, and thus it returns as a fluorescence emission.

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