ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2015, Vol. 41, No. 3, pp. 266–270. © Pleiades Publishing, Ltd., 2015. Original Russian Text © O.A. Zlobovskaya, K.S. Sarkisyan, K.A. Lukyanov, 2015, published in Bioorganicheskaya Khimiya, 2015, Vol. 41, No. 3, pp. 299–304. 266 1 INTRODUCTION Fluorescent proteins of the Green Fluorescent Protein family (GFP) are widely used in biological studies as genetically encoded tags due to the ability for autocatalytic formation of the chromophore group [1]. A panel of improved fluorescent proteins covering almost entire visible spectrum was created based on the natural GFPlike proteins using directed molecu lar evolution. The proteins fluorescing in the far red and near infrared range are of special interest for basic and biomedical sciences due to the relatively low autofluorescence and relatively high transparency of the tissues of vertebrates in this region [2]. Farred fluores cent proteins with excitation maxima at 590–610 nm and emission maxima at 630–670 nm were obtained on the basis of GFPlike proteins [3–8]. The further move in the red region was possible to achieve via cre ating bacteriophytochromebased fluorescent pro teins [9]. These proteins covalently bind biliverdin IXa as a prosthetic group. With the help of directed molec ular evolution it was possible to obtain variants of bac teriophytochromes fluorescing in the near infrared region of the spectrum (excitation at 660–690 nm, emission at 680–720 nm) [9–12]. Spectral variety of fluorescent proteins is used for multicolor labeling allowing visualization of different Abbreviations: GFP, green fluorescent protein; FRET, Forster (or fluorescence) resonance energy transfer; iRFP, infrared fluo rescent protein. 1 Corresponding author: phone: + 7 (499) 7428122; email: kluk@ibch.ru. structures in the same cells. Moreover, the suitable pairs of fluorescent proteins are used for analysis of protein–protein interactions and for developing of fluorescent sensors based on the effect of Förster reso nance energy transfer (FRET) [13, 14]. The FRET is based on the fact that the excited fluorophore donor, instead of emitting light, transfers its energy to the flu orophore–acceptor in a resonance manner, which in turn emits a quantum of light or dissipates the energy in the form of heat. Several key conditions should be met for the suc cessful FRET between two fluorophores [13, 14]. Firstly, the emission spectrum of the donor fluoro phore must significantly overlap with the absorption spectrum of the acceptor. Secondly, the donor and acceptor molecules must be located close to each other (usually no farther than 5–8 nm) because the efficiency of FRET decreases proportionally to the sixth power of the distance between the fluorophores. Furthermore, the efficiency of energy transfer depends on the relative orientation of the dipole moment of the donor emission and the dipole moment of the acceptor absorption. The maximum efficiency is achieved when the dipoles are oriented parallel to each other and the minimal, when perpendicular. The FRET efficiency can be estimated from the decrease of the donor fluorescence intensity, decrease of the lifetime of the donor excited state, increase of the acceptor fluorescence intensity, and from some other spectral characteristics. Infrared Fluorescent Protein iRFP as an Acceptor for Resonance Excitation Energy Transfer O. A. Zlobovskaya a , K. S. Sarkisyan a , and K. A. Lukyanov a, b, 1 a Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. MiklukhoMaklaya 16/10, Moscow, 117997 Russia b ScientificResearch Institute of Biomedical Technologies, Nizhni Novgorod State Medical Academy, pl. Minina 10/1, Nizhni Novgorod, 603005 Russia Received November 24, 2014; in final form, December 8, 2014 Abstract—The possibility of bacteriochromebased infrared fluorescent protein iRFP use as an acceptor for the Förster resonance energy transfer (FRET) was investigated. GFPlike farred fluorescent proteins mKate2, eqFP650, and eqFP670 were used as energy donors. Bacterial expression vectors encoding donor and acceptor proteins joined by a heptadecapeptide linker were constructed with the goal to test FRET. The efficiency of FRET was estimated in vitro for the isolated proteins from the increase of the donor emission following cleavage of the linker. Among the three tested constructs the eqFP650iRFP pair demonstrated the most efficient energy transfer (approximately 30%). Keywords: fluorescent protein, GFP, iRFP, genetically encoded sensor, FRET DOI: 10.1134/S1068162015030139