LASER PHYSICS LETTERS
www.lphys.org
EDITORIAL BOARD W. Becker, Berlin D. Chorvat, Bratislava S. DeSilvestri, Milan M. V. Fedorov, Moscow A. Gaeta, Ithaca S. A. Gonchukov, Moscow M. Jelinek, Prague U. Keller, Zürich J. Lademann, Berlin J. T. Manassah, New York P. Meystre, Tucson R. B. Miles, Princeton P. P. Pashinin, Moscow G. Petite, Saclay L. P. Pitaevskii, Trento M. Pollnau, Enschede K. A. Prokhorov, Moscow M. Scalora, Huntsville V. M. Shalaev, West Lafayette J. E. Sipe, Toronto Ken-ichi Ueda, Tokyo I. A. Walmsley, Oxford E. Wintner, Vienna E. Yablonovitch, Los Angeles V. M. Yermachenko, Moscow I. V. Yevseyev, Moscow V. I. Yukalov, Dubna A. M. Zheltikov, Moscow
T N I R P RE
Laser Phys. Lett. 8, No. 2, 91–102 (2011) / DOI 10.1002/lapl.201010107
Abstract: To expand the field of research in biological systems development of extra-sensitive analytical methods is highly desirable. In this review, the latest advances in technologies relying on the fluorescence resonance energy transfer between fluorescent proteins (FP’s) to visualize numerous molecular processes in living cells are discussed. Variety of FP’s as well as of novel experimental techniques allows one to choose the most appropriate tools to attack concrete problems.
91
FRET
FRET
Site Donor
Acceptor
Protease
Cleavage of the linker between two fluorescent proteins by sitespecific protease resulted in essential decrease of FRET-level c 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA
Fluorescence resonance energy transfer between fluorescent proteins as powerful toolkits for in vivo studies A.L. Rusanov 1 and A.P. Savitsky 1,2,∗ 1 2
Department of Chemistry, M.V. Lomonosov Moscow State University, 1/3, Leninskie Gory, Moscow 119991, Russia A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, 33, Leninsky prospekt, Moscow 119071, Russia
Received: 6 October 2010, Revised: 14 October 2010, Accepted: 18 October 2010 Published online: 7 December 2010
Key words: fluorescent proteins; GFP; RFP; FRET; FLIM; molecular imaging; protein-protein interaction; enzymatic activity
1. Introduction Fluorescence is a tool for visualization of cell biology processes at different levels, from separate molecules to entire organisms [1]. When using traditional methods, which are based on fluorescent dyes, there is a need for invasive introduction of dyes inside a cell. This limitation was overcome by the application of green fluorescent protein (GFP) and its homologes, which possess a small compact structure. The attachment of them to proteins of interest results in weak or negligible effect on the initial properties of proteins under study [2]. In 2008, the Nobel Prize in Chemistry rewards the initial discovery of GFP and a series of important developments, which have led to its use as a tagging tool in bioscience. Application of methods based on Fluorescence Resonance Energy Transfer (FRET) between two fluorescent proteins increased substantially the possibilities of using fluorescent proteins (FP’s) as markers in living cells for ∗
monitoring of biological mechanisms and physiological functions of a cell [3]. FP-based FRET-biosensors have multiple advantages over other compounds obtained by conjugation with synthetic dyes. First, their fluorescence is spontaneous; second, they can be constructed using simple genetic manipulations; third, they can be delivered in cells by means of transfection followed by the expression; finally, these biosensors can be targeted to organelles or tissues that permits monitoring not only organelles and separate cells but also entire organisms. A potential drawback of FRET˚ [4] biosensors is that the FP’s are relatively large (∼ 40 A) ˚ as compared with synthetic dyes (∼ 5 A) and, thus, may influence the activity of proteins under study. The activity of the majority of investigated proteins remained, however, unchanged upon the attachment of FP’s. Another potential limitation is that the ability to fluoresce in these proteins appears not in the moment of their synthesis on a ribosome but only after maturation and the maturation rate is limited.
Corresponding author: e-mail:
[email protected] c 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA
92
A.L. Rusanov and A.P. Savitsky : Fluorescence resonance energy transfer between FP’s
RedOx potential, and this property allows using FP’s for monitoring these events [17, 18]. Several biosensors for studies of a wide range of molecular processes have been created on the basis of FRET between FP’s:
Figure 1 (online color at www.lphys.org) β-barrel structure of red FP mKate [4]
In some cases, the maturation (post-translational appearance of the chromophore) may last more than 12 hours that prevents investigation of processes occurring within this period of time [5]. To solve this problem, a large number of quickly-maturating FP’s have been created [6–8]. One more obstacle for the application of FP’s is their sensitivity to photobleaching, similar to the case with many organic fluorescent dyes. This drawback limits using FP’s for prolonged monitoring of cell processes during illumination with intense light sources [5].
2. Application of fluorescent proteins FP’s used to trace the processes that take part in biological systems, should correspond to several requirements. It is necessary that the expression of the proteins is non-toxic for the system. Primarily, FP’s are non-toxic for cells but they may become toxic if there is a tendency for aggregation as had been demonstrated for a large group of color proteins from corals [9]. Several FP’s tend to form aggregates [10] but there are several methods described in literature for obtaining less aggregated mutant proteins [11] and dimeric and monomeric proteins, in particular, red proteins [12–16]. Another important criterion for FP selection is the brightness of fluorescence, which is determined as a product of extinction coefficient (ε) and quantum yield (q): ε × q. GFP and similar proteins have the β-barrel structure (Fig. 1) and, therefore, the properties of the chromophore located inside the protein environment are determined by adjacent amino acid residues. Fluorescent properties of FP’s can be significantly changed under the external factors, such as change in concentration of different ions, pH,
c 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA
1) Monitoring of protein-protein interactions. Any change in the distance or mutual orientation of the fluorophore pair will influence the efficiency of energy transfer, therefore, FRET is one of the most effective methods for the study of protein-protein interactions [2, 17, 19, 20]; 2) Substrates for in vivo enzymology: a) Measuring of protease activity. In this case, fusion protein where an amino acid sequence specific for the given protease is built between the pair of FP’s, is synthesized. Proteolysis results in the change of distance between the proteins and the FRET process is disturbed; b) Measuring of kinase activity. The approaching of FP’s takes place as a result of intramolecular phosphorylation reaction significantly affecting FRET [2, 21]; c) Measuring of the activity of methylating agents. Conformational changed result in changes of distance between FP’s [22]; d) Investigation of interaction of metabolites [2]; 3) Intracellular sensors for ions of different metals. Some FP’s and constructs on their basis may significantly alter fluorescent properties upon changes of concentration of definite ions and, thus, these proteins can be used as intracellular sensors for monitoring changes in concentration of such ions as, for example, zinc or calcium ions [2, 17, 23, 24]; 4) Intracellular pH-sensors. The typical feature of all FP’s is their sensitivity to pH. Change of pH influences the fluorescence signal amplitude and this effect can be used for determination of pH inside different cellular organelles [2, 17, 24]. FP’s are also applied for improvement of photodynamic therapy of tumors (PDT). Simultaneous application of FP’s and photosensitizers [21, 25] can be used to visualize the response of tumor to PDT [24, 26, 27]. Red FP’s have an advantage over other FP’s for the methods applied for studies of intracellular processes, in particular, for monitoring of localization of proteins in a cell since the long-wave fluorescence penetrates better through tissues and background cell autofluorescence in red spectral region is significantly lower than in short-wave region. Application of red FP’s in this case permits one to reach much better contrast of the measurements [2].
3. Fluorescence resonance energy transfer between FP’s Fluorescence energy transfer is a transfer of energy of excited state from a donor d to an acceptor a. The transfer
www.lphys.org
Laser Phys. Lett. 8, No. 2 (2011)
93
is a result of dipole-dipole interactions between donor and acceptor and it occurs without intermediate emission of photons. The dependence of energy transfer on distance resulted in its wide use for measurements of distances between donors and acceptors. For these measurements it is necessary that the distance in the d – a pair remained unchanged during the lifetime of the donor excited state. The rate constant of the energy transfer from d to a is determined by the equation 9000(ln 10)κ2 ϕd kT = 128π 5 n4 NA r6 τd
∞ Fd (λ)εa (λ)λ4 dλ ,
(1)
0
where φd is the fluorescence quantum yield of the donor without the acceptor; n is the refraction index of the medium; NA is the Avogadro constant; r is the distance between donor and acceptor; τd is the donor excited state lifetime in the absence of the acceptor; κ2 is the factor of mutual space orientation of transfer dipole moments of donor and acceptor; Fd (λ) is the corrected fluorescence intensity of the donor in the wavelength range λ to λ + Δλ with the total intensity normalized to unity; εa (λ) is the extinction coefficient of the acceptor at the wavelength λ. The constant terms in Eq. (1) are usually combined to determine the F¨orster radius (R0 ) – the distance between donor and acceptor, at which the efficiency of energy transfer is 50%, that is, one half of the molecules deactivates by means of the energy transfer and the other half by means of usual radiative or non-radiative mechanisms R06
9000(ln 10)κ2 ϕd = 128π 5 n4 NA
∞ Fd (λ)εa (λ)λ4 dλ .
(2)
0
The efficiency of energy transfer E is often measured; E is determined as a ratio of number of photons transferred to the acceptor to number of photons absorbed by the donor E=
R06 kT −1 = R6 + r 6 , kT + τd 0
(3)
where kT is the rate constant of the energy transfer from donor to acceptor according to Eq. (1). Eq. (1) – Eq. (3) demonstrate that the high quantum yield of the donor and larger overlapping of the emission spectrum of the donor and excitation spectrum of the acceptor are key parameters to achieve high efficiency of energy transfer. The important factor in the analysis of energy transfer is orientation factor κ2 that characterizes statistic distribution of relative fluorophore orientation. Orientation factor is 2/3 for free FRET-pairs but it is of another value for biosensors where movement of FRET-pairs is restricted [5,28]. The limits for κ2 value may be set by measuring of fluorescence anisotropy of donor and acceptor and, thus,
www.lphys.org
the uncertainty for the calculating of the distance may be minimized [5]. The second factor affecting the FRET signal is the distance R between fluorophores. The most appropriate R values lie within the interval (0.7 – 1.4)R0 that corresponds to 10–90% FRET efficiency. Usually R0 is in the interval ˚ to 70 A ˚ and, thus, conformational changes in from 30 A proteins within this range can be seen using FRET biosensors. The F¨orster radia for different protein pairs were theoretically calculated in [29] assuming that the orientation factor is equal to 2/3. It has been shown that the values of ˚ (for the radii can vary within wide interval from 31.7 A EBFP (enhanced blue fluorescent protein) – DsRed pro˚ (for EGFP (enhanced green fluorestein pair) to 56.4 A cent protein) – EYFP (enhanced yellow fluorescent protein) protein pair). The 100% efficiency of energy transfer cannot be achieved since the chromophore in the FP’s is located at ˚ from the surface. the distance approximately 12 – 15 A Therefore, the minimal possible distance between the two ˚ Introduction of additional bindfluorophores is near 25 A. ing sites on the protein surface, for example, for binding with zinc ions, increases substantially the efficiency of energy transfer [23,30,31]. The construction, in which the efficiency of energy transfer reached 98% was also reported in literature. In this case, ECFP and EYFP proteins were connected with very short linker. These results confirm that using binding sites near N- and C-termina is a good alternative to binding on back surface of β-barrels [32]. Analysis of the behavior of flexible linkers between two FP’s according to the worm-like chain or the Gaussian chain [33] model demonstrates distribution of distances between donor and acceptor; this proves the existence of multiple conformations, each with its own efficiency of energy transfer varying from 0 to 1. A similar situation occurs also when studying the interaction between molecules. The fluorescent signal is recorded both from interacting and non-interacting molecules. Usual methods of FRET registration determine only average value of transfer efficiency and weighted average fluorescence lifetimes of these pairs. In addition, the classic method of processing kinetics of fluorescence decay data using a set of discrete exponents does not allow reliable determination of more than three fluorescing components in one sample. Maximum entropy method (MEM) [34,35] permits one to carry out processing the fluorescence decay kinetics using up to 300 exponents. The possibility to use MEM method to determine fluorescence lifetimes of some FP’s and FRET pairs was shown in [36].
4. Choice of FP’s for FRET Since the discovery of GFP from Aequorea Victoria, a great number of other FP’s has been found and created [37, 38]. The diversity of FP’s is represented in Fig. 2 and
c 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA
94
A.L. Rusanov and A.P. Savitsky : Fluorescence resonance energy transfer between FP’s
Extinction, M-1cm-1, ×104
15
10
5
0 400
450
500
550
600
Maximum of excitation wavelength, nm
Figure 2 (online color at www.lphys.org) Extinction coefficients for FP’s with different maxima of excitation wavelength
1.0
Quantum yield
0.8
0.6
0.4
0.2
0 450
500
550
600
650
Wavelength, nm
Figure 3 (online color at www.lphys.org) Quantum yields for FP’s with different maxima of emission wavelength
Fig. 3. Relation between structure and spectra of fluorescent proteins is actively studied by the computer modeling [39–44] and resent progress in molecular modeling of the structural parameters and optical spectra of FP’s by methods of quantum chemistry, molecular dynamics, and combined quantum-mechanical-molecular-mechanical approaches are summarized in [45]. FP’s can be divided into six groups according to their emission maxima: blue, cyan, green, yellow, orange and red. Based on the color, four types of usual FRET pairs may be selected: a) blue and green, b) cyan and yellow/orange, c) green and orange/red, d) orange and red
c 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA
donor and acceptor, respectively. BFP-GFP pair was one of the first FRET pairs with FP’s. However, BFP has low fluorescence quantum yield, is subjected to strong bleaching, and, since its excitation maximum is near 380 nm, cell auto-fluorescence and light scattering contribute significantly to the level of noise when using this combination of proteins [3]. To overcome these problems, proteins with excitation spectra shifted to more long-wave region are used. Thus, FP’s, which are most often used for FRET can be divided into two groups. The first group comprises constructions where donor has cyan fluorescence, and acceptor fluoresces in yellow region. Orange proteins as donors and red fluorescent proteins as acceptors belong to the second group. The CFP-YFP pair is most frequently used: the emission maximum of the donor is 476 nm, the excitation maximum of the acceptor is 529 nm [3, 46, 47]. The drawback of the CFP-YFP FRET system is extended in long-wave region fluorescence spectrum that interferes with independent monitoring of YFP emission [46]. The attempts are made to obtain brighter CFP and YFP mutants with higher extinction coefficient and improved maturation and folding rates and also to obtain proteins with fluorescence lifetime that can be described with a single exponent [48–50]. Red FP’s are promising for the application as acceptors [12, 51–55]. Using RFP would allow one, first, to achieve better separation of emission peaks, second, to decrease auto-fluorescence of tissues, and, third, to increase penetration of light deeper into tissues since long-wave light is less subjected to scattering. In addition, excitation light for RFP is significantly less dangerous for cells [51, 56]. Different FRET constructs were studied in [57] with different orange proteins as donors (SYFP2, mOrange, mKO) and red proteins as acceptors (mRFP1, mStrawberry, mCherry). It was shown that these constructs could be used for monitoring protein-protein interactions inside a cell and these pairs had advantages over the CFP-YFP pairs. For each pair, the overlap integral and the F¨orster radius were calculated. The latter varied within the limits ˚ (for the SYFP2 – mRFP1 pair) to 64 A ˚ (mKO – from 56 A mCherry). One of the promising directions in the application and design of FRET biosensors is to increase the signal-tonoise ratio level. This can be achieved by the right choice of fluorescent proteins or by using the circular-mutated FP’s (cpFPs) [57–61]. Circular transition allows creating new C- and N-termini to obtain fusion proteins and, thus, to change orientation factor and distance between FRET donor and acceptor. There is a group of photo-activated FP’s (PAFPs) that are able to change their spectral properties in response to the irradiation with light with definite wavelength or intensity. Some PAFPs turn from dark to fluorescent state (photo-activation) or vise versa (photo-quenching) while other PAFPs change fluorescence color (photo-switching or photo-conversion) [62–67]. In addition to the convenience for watching protein transitions, using of PAFPs
www.lphys.org
Laser Phys. Lett. 8, No. 2 (2011)
significantly decreases the problem of photobleaching and phototoxicity [62]. Since all the monitoring regions can be photo-activated with a single excitation impulse, this procedure is applicable to watch quenching of donor in multiple cells [64].
5. Methods for FRET measurement One of the most valuable properties of FRET method is that fluorescence of donor and acceptor can be measured simultaneously and the effectiveness of the transfer can be estimated by the ratio of fluorescence intensities at two wavelengths corresponding to the irradiation of donor and acceptor. The advantages of the FRET-based methods include also their spatial (sub-microns) and time (milliseconds) resolution. Among the drawbacks is the existence of energy transfer in the absence of any protein interactions. The presence of this background level narrows dynamic range of measured concentrations [17].
5.1. The spectral method Two different approaches are used for FRET studies. The first one, the spectral method, is the comparison of emission spectra of donor and acceptor. Different ways of detection are used in this case [51]: 1) induced emission of the acceptor: excitation of the donor and detection of the acceptor emission. Three components are present in real measurements: light emitted by the donor (donor that does not take part in the FRET, light emitted by the acceptor (the FRET itself), or both simultaneously (% FRET can be calculated). In the case of FRET, emission of the donor decreases and emission of the acceptor grows; 2) photobleaching of the acceptor; excitation of the donor and detection of the donor emission before and after photobleaching of the acceptor. Photoinactivation of the acceptor usually requires presence of powerful laser irradiating in the region of excitation maximum of the acceptor. This approach can be implemented in several ways. The first, FRAP (Fluorescence Recovery After Photobleaching), is based on irreversible photobleaching of the FP at a small region of a cell followed by monitoring of recovery of fluorescence at this region due to diffusion of fluorescing molecules from non-irradiated regions of the cell. This method allows one to determine such parameters as portion of mobile protein fraction and diffusion coefficient. In the Inverse FRAP variant, the whole cell is irradiated with the exception of the small region. Analyzing the decrease in fluorescence of this region with time allows estimating the rate of diffusion of the particles from the region under study [68].
www.lphys.org
95
FLIP (Fluorescence Loss in Photobleaching) is another approach that is based on the photobleaching of the fluorescent protein. In this case a small region of the cell is subjected to irradiation while the decrease in the fluorescence of the whole cell is recorded; this allows one to determine the boundaries of different cell organelles and compartments [68]. The method called FLAP (Fluorescence Localization after Photobleaching) requires two fluorescent proteins as labels. Photobleaching of one of the proteins allows monitoring the whole pool of objects that were subjected to irradiation. The obtained data may be represented in terms of efficiency of energy transfer, or percentage value from 0 to 100%, or scale value from 0 to 1, where 0 indicates the absence of interactions and 1 – maximal interactions. In real living systems, the data obtained from FRET measurements is usually within the interval from 0 to 0.3 – 0.7 [69]. The main difference between the method of induced emission of the acceptor and photobleaching of the acceptor is the means of detection of FRET signal. FRET efficiency in the second method is measured by comparison of results in the presence and in the absence of the acceptor and there is no direct determination of energy transfer. In contrast, measurement of emission of the acceptor in the first method is direct measurement of FRET, which occurs upon excitation of the donor and transfer of energy to the acceptor. However, the first method requires subsequent correction of fluorescence spectra due to the cross signal. The measured fluorescence intensities in most cases consist of both FRET constituents and components, which are not associated with FRET [69].
5.2. Measuring of fluorescence lifetimes A great number of studies of protein interactions are based on protein conjugates where the measured fluorescence intensity is different for free and bound state. Nevertheless, it is usually impossible to compare accurately the relative changes in fluorescence intensity obtained in different laboratories. This limitation can be overcome by measuring such photophysical parameters as polarization and fluorescence lifetime. These parameters can be determined using stationary and time-resolved procedures [70, 71]. Analysis of time of fluorescence decay of the fluorescing sample in excited state permits multiple analysis of protein conjugates since each component in the sample can be found by its decay rate. Lifetime of the sample will be significantly changed when the sample will take part in the FRET with the acceptor inside the protein complex [70]; in this case it is preferable to use as donors the fluorescent proteins with fluorescence decay that can be described with a single exponent [51]. In most cases FRET efficiency is determined more accurately when fluorescence lifetimes are measured [70].
c 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA
A.L. Rusanov and A.P. Savitsky : Fluorescence resonance energy transfer between FP’s
c 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA
(a) FRET
FRET
Site Acceptor
Donor
Protease
(b)
28
before cleavage after cleavage
24
Fluorescense, a.u.
An application of this method allows one to widen the field of applicable FP’s and to include the pairs that cannot be divided using microscope optical filters. The approach, which is based on measurement of fluorescent intensities, requires higher statistic averaging of results obtained from samples by using negative control, or improvement of monitoring process for calculation of signals obtained on the direct excitation of the acceptor and signals from the donor [72]. The method with time resolution is based on measurement of lifetimes of the donor in the absence (τD ) and in the presence of the acceptor (τDA ). The τDA value differs from τD and this difference allows calculating FRET efficiency [32, 57, 72–75]. Such measurements are carried out with rapidly pulsing laser (with the frequency of several megahertz) for the excitation of a sample and rapid detection to measure time from the moment of impulse to the moment of photon irradiation. The main advantage of this approach is that there is no need to carry out external calibration of measurements [70]. To achieve efficient energy transfer, a good spectral overlap is required, and the emissions of donor and acceptor should be sufficiently divided. The FRET pairs with a compromise between these two requirements are usually used, which decreases maximal achievable sensitivity of FRET. This problem can be solved by using of nonfluorescent proteins. Construction of EYFP mutants with Y145W and Y145W/H148V substitutions led to the obtaining of corresponding REACh1 and REACh2 dark proteins. Fluorescence lifetimes of these mutant proteins were significantly lower (∼ 320 ps) as compared with EYFP (∼ 2.9 ns). Such fast decay of fluorescence decreases the level of acceptor bleaching during the FRET [76]. Recently we have reported construction of a FRET pair on the basis of red proteins with practically non-fluorescent chromoprotein KFP as the acceptor [77, 78]. Cleavage of the linker under the action of protease (Fig. 4) resulted in substantial increase in intensity and fluorescence lifetime [79]. Using of dark acceptors allows one to set free the major part of the spectrum and, thus, to use additional fluorescing components; this can be considered to be additional advantage of such pairs. Fluorescence lifetimes of FP’s lie within the range of hundreds of picoseconds to nanoseconds. In a series of systems obtained by chemical methods [80, 81], the problem of background signal is solved using time discrimination principle. In such systems, Tb3+ and Eu3+ ions, capable of prolonged millisecond fluorescence, are used as donors [82]. The advantage of these pairs is the possibility to measure distances between donors as acceptor up ˚ This can be achieved due to the sharp emission to 100 A. peaks, high quantum yield, and the absence of polarization of fluorescence in the lanthanides [83]. Similar FRET pairs found application in several methods [81, 84–88]. A genetically encoded FRET pair has been obtained by us [89] where terbium-binding peptide was used as a donor, tryptophan served as a sensitizer of lanthanide fluorescence [90, 91], and RFP’s were acceptors. Two energy
20 16 12 8 4 0 540
560
580
600
620
640
660
680
700
Wavelength, nm (c)
before cleavage after cleavage
103
Intensity, a.u.
96
103
103
0
5
10
15
20
25
Time, ns
Figure 4 (online color at www.lphys.org) (a) – cleavage of the linker between two fluorescent proteins by site-specific protease resulted in essential decrease of FRET-level. This leads to growth of fluorescence intensity at donor emission wavelength (b) and changes in fluorescence lifetime (c)
transfer processes occur in this construction. The first of them is the energy transfer from the excited tryptophan to the Tb3+ ion according to the induction-resonance energy transfer mechanism. The excitation energy of Tb3+ ion can be, in turn, passed to the red fluorescent protein chromophore according to the same mechanism and this leads to the increase of total duration of the light emission
www.lphys.org
Laser Phys. Lett. 8, No. 2 (2011)
at the emission wavelength of the RFP to micro- and milliseconds [92]. This approach was implemented in the experiments with living cells and the possibility to record energy transfer between terbium and GFP has been demonstrated [93].
97
Orientation factor
(a) Intramolecular FRET CFP
Distance factor
“Open” conformation “Closed” conformation
6. Genetically engineered constructions for FRET YFP
All constructions for FRET can be divided into two types: 1) Sequences encoding donor and acceptor are located in different plasmids. FRET is possible only upon simultaneous co-expression of these two genes. This approach is mainly used to study protein-protein interactions; 2) Both proteins are encoded in one plasmid and are expressed in a cell as a chimeric protein containing both donor and acceptor. This method is most often used to monitor change in concentration of some ions, in particular Ca2+ and Zn2+ ions, and pH and also for monitoring enzymatic activity and conformational changes in protein molecules. Protein interactions can be divided into intramolecular one when the donor and the acceptor are in the same molecule and the conformations of the molecule can change, for example, can co-exist in the “open” and “closed” forms; and intermolecular one when the donor is attached to one molecule (Protein 1 in Fig. 5) and the acceptor – to another (Protein 2). Energy transfer occurs upon protein binding and, on the contrary, protein dissociation results in the decrease of FRET [3].
6.1. Intermolecular FRET Structural interactions between proteins play an important role in signal transmission and catalytic activation. To study these interactions, FRET biosensors are created by the attachment of donor to one of the interacting proteins of interest and acceptor to another protein. This biosensor type is, thus, based on the formation of complexes between the interacting molecules due to hydrophobic, hydrophilic, and other interactions. Upon binding of these proteins, the donor and the acceptor are brought together to the distance where intermolecular energy transfer is possible that permits monitoring localization and interaction time. Biosensors of this class are essential for the studies of mechanisms of association of membrane receptors and cytoplasmic proteins [94–100]. In addition to investigation of protein association, intermolecular FRET is also used to study effect of small molecules on mechanism of transmission of cell signals. The mechanism of action of the biosensor is based on the fact that binding of two different domains depends on the
www.lphys.org
(b) Intermolecular FRET
Protein 1
Protein 2
Protein-protein complex
Figure 5 (online color at www.lphys.org) Intramolecular and intermolecular FRET. (a) Intramolecular FRET may take place when both donor and acceptor are attached to the same molecule, for which changes in conformation are possible, for example, transition between “open” and “closed” conformations. The FRET level depends strongly on the relative orientation of the chromophores and the distance between them. (b) Intermolecular FRET can occur between the molecule containing attached donor and the molecule with the acceptor
presence of small molecules. Binding results in the approaching of the FRET pair and, respectively, in the increase in FRET signal. This mechanism was taken into account at the construction of cAMP sensors [101,102]. One of the limitations of the intermolecular FRET biosensors is that one pair of FP’s allows studying only one pair of associating proteins. Using of multiple FRET may solve this problem [5]. It is noteworthy that nonradiative energy transfer may occur between dipoles of two identical samples. This takes place in the case when the emission and absorption spectra are overlapping. The so-called homo-FRET can be used to study protein interactions with a single fluorescence label and this method is especially useful for quantitative assay of formation of homodimers or high oligomers of the same protein. Nevertheless, homo-FRET does not result in changes of fluorescence lifetime of the donor since the excitation energy is reversibly transferred between the identical samples [70, 103]. This process, therefore, can be traced only by using fluorescence anisotropy measurements [104]. Photo-physical properties of the emission di-
c 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA
98
A.L. Rusanov and A.P. Savitsky : Fluorescence resonance energy transfer between FP’s
rectly from the excited donor sample (for example, GFP) in the case of homo-FRET are, mainly, the same as of the appearing emission of GFP-acceptor. Nevertheless, since the orientation of dipole moments of the two interacting proteins may differ, the fluorescence polarization value is a function of a number of occurring homo-FRET energy transfers. It has been shown [104] that picosecond time-resolved polarization fluorescence microscopy is an effective method for monitoring subcellular monomerdimer transitions and oligomerization of proteins inside the cell. Though homo-FRET effect is rarely used for highthroughput screening (HTS), application of this method is rather promising [70]. Constructs for the intermolecular FRET can be also used for determination of DNA concentrations [105], detection of level of RNA transcription [106], and for monitoring of transition of individual mRNA using molecular beacons technology [107].
6.2. Intramolecular FRET Cleavage of linker between the pair of FP’s results in significant change of the FRET level. Sensors for determining the activity of proteases such as caspase, calpain, Factor Xa protease, thrombin, etc. belong to the constructions of this type [5,108–110]. Decrease in the efficiency of energy transfer can be also followed by measuring fluorescence lifetimes [72]. FRET-based pH sensors are also described in literature. For example, the fusion protein consisting of two GFP variants has been obtained, one GFP variant being less pH-sensitive (GFPuv) than the other (EYFP). Fluorescence intensity and efficiency of energy transfer in this construction, called YFpH, appeared to be pH-dependent (pKa = 6.8). This indicator was applicable for determination of cytoplasmic pH within 6.0 – 8.5 range [111]. Some proteins may form complexes with metabolites or metal ions that results in significant conformational changes. By monitoring of these conformational changes using FRET, it is possible to observe molecular processes and change of concentrations of different substances [112–123]. In the mentioned examples, the molecules under study were directly bound to biosensors. Other intramolecular FRET-sensor were constructed in such a way that a sensor domain, instead of binding, was modified under the action of enzymes, and observation of processes associated with the activity of modifying enzyme became possible. Using biosensors of this type, it is possible to study properties and functions of kinases and phosphatases responsible for the corresponding phosphorylation and dephosphorylation processes, and determination of their localization and activity within the living cell [124–128]. Monitoring the activation and action of GTP-ases in the cells [129–132] and methylation of histone proteins [133] are also described.
c 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA
The mechanism of action of different biosensors for Ca2+ ions is based on monitoring of reversible conformational changes in proteins in Ca2+ -dependent reaction [3, 60, 134–138]. Such biosensors have been also used for studies of cell processes in transgenic insects [139], mice, [140,141], and nematodes [142] under the influence of external parameters. The intramolecular FRET between two FP’s is also applied to view different signaling molecules, for example, such as Smad proteins, which take part in transition of signal from membrane to nucleus [143], and for the detection of amino acids, which serve not only as building blocks of proteins but also as precursors of different primary and secondary metabolites [144]. Conformational changes in proteins may take place not only on binding to proteins or other compounds but also under the action of other factors. For example, voltsensitive domain of potassium channel protein changes conformation upon changing of membrane potential. The membrane potential biosensor has been constructed, in which reversible conformational changes cause changes of orientation factor and changes in FRET. Fourth transmembrane helix (S4) of potential-sensitive potassium channel contains large amount of positively charged residues. Change in transmembrane potential results in the turn of S4 causing redistribution of positive charges along electric field direction. This, in turn, results in change of efficiency of energy transfer between the pair of fluorescent proteins attached to the S4 helix; the energy transfer efficiency depends on relative orientation of dipole moments of these proteins and, therefore, is sensitive to rotation [145]. Presently, the intramolecular biosensors are mainly applied for determination of enzymatic activity and concentration of small molecules. From the technical point of view, it is easier to define FRET signal for intramolecular biosensors since in this case the signal is less sensitive to relative biosensor concentration as compared with intermolecular biosensors. This type of biosensors is, therefore, more preferable for quantitative estimation of molecular and cell biology processes. One of the directions for the development of this method is improvement of dynamic range of intracellular biosensors in order to increase signal/noise ratio [5].
6.3. Structure of linkers FP’s in many FRET sensors are attached to interacting protein domains by flexible peptide linkers [127]. FRET sensors of other type contain flexible peptide linker with a site specific to different proteases [146, 147]. Understanding conformational behavior of flexible linkers that divide FP’s facilitates to predict changes in the energy transfer and, thus, assists in obtaining of new effective sensors. Information about distances between proteins is also important for rational design of fusion proteins. In many cases amino acid sequences composed of flexible and hydrophylic residues (glycine and serine random sequences)
www.lphys.org
Laser Phys. Lett. 8, No. 2 (2011)
are used since they are supposed to form statistical ball and do not interact with protein domains [33]. Application of linkers enriched with proline residues reinforces linker conformation and this can be also used to increase the efficiency of energy transfer [148]. Long polyproline linkers (more than 17 proline resudies) became flexible enough and this results in the increase of a set of possible distances between donor and acceptor [149, 150]. Efficiency of energy transfer also depends significantly on linker length since a linker that is too short, prevents proper protein folding and using of a fairly long linker results in a decrease in FRET due to the increase in the distance between FP’s [151, 152]. In addition, increase in linker length influences not only the efficiency of FRET but also leads to a decrease in the expression level of chimeric constructions in the cell [147]. Changes in the efficiency of FRET between two FP’s ECFP and EYFP have been studied to determine the dependence of distribution of distances between protein domains in the presence of linkers with different lengths containing from one to nine GGSGGS repeats. The behavior of these flexible linkers can be described as a behavior of random coil according to the worm-like chain or Gaussian chain model [33]. The analysis of results obtained indicated that the presence of long flexible linkers may also lead to high level of energy transfer efficiency. Distances between proteins are not strictly defined values but vary within definite range [33]. FRET-based sensors can be used to study unstructured proteins since the conformational mobility of their polypeptide chain permits detection of FRET signal between donor and acceptor attached to these proteins [153]. Thus, length, structure [120], and structuring of a linker [153] are the key issues for the improvement of sensor properties in linker design.
7. Conclusion Biosensors based on resonance energy transfer between FP’s allow monitoring of wide range of molecular events such as protein-protein interactions, conformational changes in molecules, catalytic functions of enzymes, and changes of concentrations of biomolecules in living cells. Due to their unique advantages involving genetically encoded fluorescence, simplicity of genetic manipulations and delivery inside a cell, easy introduction of label into organelles and tissues under study, these biosensors became an important tool in modern cell and molecular biology. However, all known so far FRET pairs possess certain drawbacks that narrow dynamic range of measurements. One of the directions aimed to improve FRET biosensors is preparation of new FRET pairs and modify the properties of the existing pairs by changing orientation and distance factors.
www.lphys.org
99
FRET-based sensors can be used to monitor complex molecular processes such as signal transduction or metabolic paths inside transgenic animals. Possibility of real-time in vivo imaging allows deep analysis of biological mechanisms and physiological functions of a cell. Acknowledgements This study was supported by the Federal Agency of Science and Innovations of Russian Federation state contract no 02.740.11.0291 in the frame of the Program “Scientific, Academic, and Teaching Staff of Innovative Russia, duration 2009-2013” and state contract no 02.512.12.2036 in the frame of the Program “The Investigations and Advancing of Priority Directions of Scientific-Technological Complex Development of Russia, 2007-2012”, the Program of the Russian Academy of Sciences “Molecular and Cellular Biology”.
References [1] B.N.G. Giepmans, S.R. Adams, M.H. Ellisman, and R.Y. Tsien, Science 312, 217 (2006). [2] W. Du, Y. Wang, Q.M. Luo, and B.-F. Liu, Anal. Bioanal. Chem. 386, 444 (2006). [3] K. Truong and M. Ikura, Curr. Opin. Struct. Biol. 11, 573 (2001). [4] S. Pletnev, D. Shcherbo, D.M. Chudakov, N. Pletneva, E.M. Merzlyak, A. Wlodawer, Z. Dauter, and V. Pletnev, J. Biol. Chem. 283, 28980 (2008). [5] I.T. Li, E. Pham, and K. Truong, Biotechnol. Lett. 28, 1971 (2006). [6] T. Nagai, K. Ibata, E.S. Park, M. Kubota, K. Mikoshiba, and A. Miyawaki, Nat. Biotechnol. 20, 87 (2002). [7] B.J. Bevis and B.S. Glick, Nat. Biotechnol. 20, 83 (2002). [8] J.-D. P´edelacq, S. Cabantous, T. Tran, T.C. Terwilliger, and G.S. Waldo, Nat. Biotechnol. 24, 79 (2006). [9] N.C. Shaner, P.A. Steinbach, and R.Y. Tsien, Nat. Methods 2, 905 (2005). [10] N.N. Zubova, V.A. Korolenko, A.A. Astafyev, A.N. Petrukhin, L.M. Vinokurov, O.M. Sarkisov, and A.P. Savitsky, Biochem. 44, 3982 (2005). [11] Yu.G. Yanushevich, D.B. Staroverov, A.P. Savitsky, A.F. Fradkov, N.G. Gurskaya, M.E. Bulina, K.A. Lukyanov, and S.A. Lukyanov, FEBS Lett. 511, 11 (2002). [12] R.E. Campbell, O. Tour, A.E. Palmer, P.A. Steinbach, G.S. Baird, D.A. Zacharias, and R.Y. Tsien, Proc. Natl. Acad. Sci. USA 99, 7877 (2002). [13] J. Wiedenmann, B. Vallone, F. Renzi, K. Nienhaus, S. Ivanchenko, C. R¨ocker, and G.U. Nienhaus, J. Biomed. Opt. 10, 014003 (2005). [14] J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. R¨ocker, A. Salih, K.-D. Spindler, and G.U. Nienhaus, Proc. Natl. Acad. Sci. USA 101, 15905 (2004). [15] T. Kogure, S. Karasawa, T. Araki, K. Saito, M. Kinjo, and A. Miyawaki, Nat. Biotechnol. 24, 577 (2006). [16] E. Galperin, V.V. Verkhusha, and A. Sorkin, Nat. Methods 1, 209 (2004). [17] N.N. Zubova and A.P. Savitsky, Usp. Biol. Khim. 45, 391 (2005), in Russian.
c 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA
100
A.L. Rusanov and A.P. Savitsky : Fluorescence resonance energy transfer between FP’s
[18] A.M. Bogdanov, A.S. Mishin, I.V. Yampolsky, V.V. Belousov, D.M. Chudakov, F.V. Subach, V.V. Verkhusha, S. Lukyanov, and K.A. Lukyanov, Nat. Chem. Biol. 5, 459 (2009). [19] R.Y. Tsien, Annu. Rev. Biochem. 67, 509 (1998). [20] M. Zimmer, Chem. Rev. 102, 759 (2002). [21] M.C. Milanetto, H. Imasato, and J.R. Perussi, Laser Phys. Lett. 6, 611 (2009). [22] C.-W. Lin, C.Y. Jao, and A.Y. Ting, J. Am. Chem. Soc. 126, 5982 (2004). [23] T.H. Evers, M.A.M. Appelhof, P.T.H.M. de GraafHeuvelmans, E.W. Meijer, and M. Merkx, J. Mol. Biol. 374, 411 (2007). [24] A.P. Castano, Q. Liu, and M.R. Hamblin, Br. J. Cancer 94, 391 (2006). [25] Y. Liu, P. Chen, F. Zhang, L. Lin, G.-Q. Tang, and G.-G. Mu, Laser Phys. Lett. 6, 465 (2009). [26] S. Mitra, S.E. Cassar, D.J. Niles, J.A. Puskas, J.G. Frelinger, and T.H. Foster, Mol. Cancer Ther. 5, 3268 (2006). [27] B. Chen, C. Crane, C. He, D. Gondek, P. Agharkar, M.D. Savellano, P.J. Hoopes, and B.W. Pogue, Int. J. Cancer 123, 695 (2008). [28] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed. (Kluwer Academic/Plenum Publishers, New York, 1999), p. 367. [29] G.H. Patterson, D.W. Piston, and B.G. Barisas, Anal. Biochem. 284, 438 (2000). [30] K.K. Jensen, L. Martini, and T.W. Schwartz, Biochem. 40, 938 (2001). [31] J.L. Vinkenborg, T.H. Evers, S.W.A. Reulen, E.W. Meijer, and M. Merkx, ChemBioChem 8, 1119 (2007). [32] S. Shimozono, H. Hosoi, H. Mizuno, T. Fukano, T. Tahara, and A. Miyawaki, Biochem. 45, 6267 (2006). [33] T.H. Evers, E.M.W.M. van Dongen, A.C. Faesen, E.W. Meijer, and M. Merkx, Biochem. 45, 13183 (2006). [34] A.K. Livesey and J.C. Brochon, Biophys. J. 52, 693 (1987). [35] J.-C. Brochon, Methods Enzymol. 240, 262 (1994). [36] A.J.W.G. Visser, S.P. Laptenok, N.V. Visser, A. van Hoek, D.J.S. Birch, J.-C. Brochon, and J.W. Borst, Eur. Biophys. J. 39, 241 (2010). [37] M.V. Matz, A.F. Fradkov, Yu.A. Labas, A.P. Savitsky, A.G. Zaraisky, M.L. Markelov, and S.A. Lukyanov, Nat. Biotechnol. 17, 969 (1999). [38] Y.A. Labas, N.G. Gurskaya, Y.G. Yanushevich, A.F. Fradkov, K.A. Lukyanov, S.A. Lukyanov, and M.V. Matz, Proc. Natl. Acad. Sci. USA 99, 4256 (2002). [39] A. Sinicropi, T. Andruniow, N. Ferr´e, R. Basosi, and M. Olivucci, J. Am. Chem. Soc. 127, 11534 (2005). [40] B. Grigorenko, A. Savitsky, I. Topol, S. Burt, and A. Nemukhin, J. Phys. Chem. B 110, 18635 (2006). [41] B. Grigorenko, A. Savitsky, I. Topol, S. Burt, and A. Nemukhin, Chem. Phys. Lett. 424, 184 (2006). [42] K.B. Bravaya, A.V. Bochenkova, A.A. Granovsky, A.P. Savitsky, and A.V. Nemukhin, J. Phys. Chem. A 112, 8804 (2008). [43] A.V. Nemukhin, I.A. Topol, B.L. Grigorenko, A.P. Savitsky, and J.R. Collins, J. Mol. Struct. THEOCHEM 863, 39 (2008).
c 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA
[44] S. Olsen and S.C. Smith, J. Am. Chem. Soc. 129, 2054 (2007). [45] A.V. Nemukhin, B.L. Grigorenkoa, and A.P. Savitsky, Acta Naturae 2, 33 (2009). [46] B.A. Pollok and R. Heim, Trends Cell Biol. 9, 57 (1999). [47] S. Shimozono and A. Miyawaki, in: K.F. Sullivan (ed.), Fluorescent Proteins, Methods in Cell Biology series, vol. 85, 2nd ed. (Academic Press, 2008), p. 382. [48] M.A. Rizzo, G.H. Springer, B. Granada, and D.W. Piston, Nat. Biotechnol. 22, 445 (2004). [49] G.-J. Kremers, J. Goedhart, E.B. van Munster, and T.W.J. Gadella, Jr., Biochem. 45, 6570 (2006). [50] M. Lelimousin, M. Noirclerc-Savoye, C. Lazareno-Saez, B. Paetzold, S. Le Vot, R. Chazal, P. Macheboeuf, M.J. Field, D. Bourgeois, and A. Royant, Biochem. 48, 10038 (2009). [51] A. M¨uller-Taubenberger and K.I. Anderson, Appl. Microbiol. Biotechnol. 77, 1 (2007). [52] H. Mizuno, A. Sawano, P. Eli, H. Hama, and A. Miyawaki, Biochem. 40, 2502 (2001). [53] N.G. Gurskaya, A.F. Fradkov, A. Terskikh, M.V. Matz, Yu.A. Labas, V.I. Martynov, Yu.G. Yanushevich, K.A. Lukyanov, and S.A. Lukyanov, FEBS Lett. 507, 16 (2001). [54] R. Steinmeyer, A. Noskov, C. Krasel, I. Weber, C. Dees, and G.S. Harms, J. Fluoresc. 15, 707 (2005). [55] R.L. Strack, B. Hein, D. Bhattacharyya, S.W. Hell, R.J. Keenan, and B.S. Glick, Biochem. 48, 8279 (2009). [56] A. Doerr, Nat. Methods 6, 482 (2009). [57] J. Goedhart, J.E.M. Vermeer, M.J.W. Adjobo-Hermans, L. van Weeren, and T.W.J. Gadella, Jr., PLoS One 2, e1011 (2007). [58] O. Zapata-Hommer and O. Griesbeck, BMC Biotechnol. 3, 5 (2003). [59] T. Nagai, A. Sawano, E.S. Park, and A. Miyawaki, Proc. Natl. Acad. Sci. USA 98, 3197 (2001). [60] M. Mank, D.F. Reiff, N. Heim, M.W. Friedrich, A. Borst, and O. Griesbeck, Biophys. J. 90, 1790 (2006). [61] Y.-M. Huang and C. Bystroff, Biochem. 48, 929 (2009). [62] S.J. Remington, Curr. Opin. Struct. Biol. 16, 714 (2006). [63] K.A. Lukyanov, D.M. Chudakov, S. Lukyanov, and V.V. Verkhusha, Nat. Rev. Mol. Cell Biol. 6, 885 (2005). [64] I.A. Demarco, A. Periasamy, C.F. Booker, and R.N. Day, Nat. Methods 3, 519 (2006). [65] R. Bizzarri, M. Serresi, F. Cardarelli, S. Abbruzzetti, B. Campanini, C. Viappiani, and F. Beltram, J. Am. Chem. Soc. 132, 85 (2010). [66] T. Brakemann, G. Weber, M. Andresen, G. Groenhof, A.C. Stiel, S. Trowitzsch, C. Eggeling, H. Grubm¨uller, S.W. Hell, M.C. Wahl, and S. Jakobs, J. Biol. Chem. 285, 14603 (2010). [67] F.V. Subach, G.H. Patterson, M. Renz, J. LippincottSchwartz, and V.V. Verkhusha, J. Am. Chem. Soc. 132, 6481 (2010). [68] J. Lippincott-Schwartz and G.H. Patterson, Science 300, 87 (2003). [69] I.B. Autenrieth, Int. J. Med. Microbiol. 293, 1 (2004). [70] Y.L. Yan and G. Marriott, Curr. Opin. Chem. Biol. 7, 635 (2003). [71] D. Chorvat, Jr. and A. Chorvatova, Laser Phys. Lett. 6, 175 (2009).
www.lphys.org
Laser Phys. Lett. 8, No. 2 (2011)
[72] A.G. Harpur, F.S. Wouters, and P.I.H. Bastiaens, Nat. Biotechnol. 19, 167 (2001). [73] M. Pedersen, M. Carmosino, and B. Forbush, J. Biol. Chem. 283, 2663 (2007). [74] E. Zelazny, J.W. Borst, M. Muylaert, H. Batoko, M.A. Hemminga, and F. Chaumont, Proc. Natl. Acad. Sci. USA 104, 12359 (2007). [75] S. Scolari, S. Engel, N. Krebs, A. Pia Plazzo, R.F.M. De Almeida, M. Prieto, M. Veit, and A. Herrmann, J. Biol. Chem. 284, 15708 (2009). [76] S. Ganesan, S.M. Ameer-beg, T.T.C. Ng, B. Vojnovic, and F.S. Wouters, Proc. Natl. Acad. Sci. USA 103, 4089 (2006). [77] K.A. Lukyanov, A.F. Fradkov, N.G. Gurskaya, M.V. Matz, Yu.A. Labas, A.P. Savitsky, M.L. Markelov, A.G. Zaraisky, X.N. Zhao, Y. Fang, W.Y. Tan, and S.A. Lukyanov, J. Biol. Chem. 275, 25879 (2000). [78] T.A. Sch¨uttrigkeit, T. von Feilitzsch, C.K. Kompa, K.A. Lukyanov, A.P. Savitsky, A.A. Voityuk, and M.E. Michel-Beyerle, Chem. Phys. 323, 149 (2006). [79] A.L. Rusanov, T.V. Ivashina, L.M. Vinokurov, I.I. Fiks, A.G. Orlova, I.V. Turchin, I.G. Meerovich, V.V. Zherdeva, and A.P. Savitsky, J. Biophoton. (2010), DOI: 10.1002/jbio.201000065. [80] R.T. Cummings, H.M. McGovern, S. Zheng, Y.W. Park, and J.D. Hermes, Anal. Biochem. 269, 79 (1999). [81] E. Heyduk and T. Heyduk, Anal. Biochem. 248, 216 (1997). [82] P.R. Selvin, Annu. Rev. Biophys. 31, 275 (2002). [83] P.R. Selvin, Nat. Struct. Biol. 7, 730 (2000). [84] K. Lundin, K. Blomberg, T. Nordstr¨om, and C. Lindqvist, Anal. Biochem. 299, 92 (2001). [85] K. Stenroos, P. Hurskainen, S. Eriksson, I. Hemmil¨a, K. Blomberg, and C. Lindqvist, Cytokine 10, 495 (1998). [86] K. Blomberg, P. Hurskainen, and I. Hemmil¨a, Clin. Chem. 45, 855 (1999). [87] S.A. Kane, C.A. Fleener, Y.S. Zhang, L.J. Davis, A.L. Musselman, and P.S. Huang, Anal. Biochem. 278, 29 (2000). [88] E. Lopez-Crapez, H. Bazin, E. Andre, J. Noletti, J. Grenier, and G. Mathis, Nucl. Acid Res. 29, e70 (2001). [89] L.R. Arslanbaeva, V.V. Zherdeva, T.V. Ivashina, L.M. Vinokurov, A.L. Rusanov, and A.P. Savitsky, Appl. Biochem. Microbiol. 46, 154 (2010). [90] L.J. Martin, B.R. Sculimbrene, M. Nitz, and B. Imperiali, QSAR Comb. Sci. 24, 1149 (2005). [91] I.D. Clark, I. Hill, M. Sikorska-Walker, J.P. MacManus, and A.G. Szabo, FEBS Lett. 333, 96 (1993). [92] B.P. Maliwal, Z. Gryczynski, and J.R. Lakowicz, Anal. Chem. 73, 4277 (2001). [93] H.E. Rajapakse, N. Gahlaut, S. Mohandessi, D. Yu, J.R. Turner, and L.W. Miller, Proc. Natl. Acad. Sci. USA 107, 13582 (2010). [94] J.M. Kramer, L. Yi, F. Shen, A. Maitra, X.M. Jiao, T. Jin, and S.L. Gaffen, J. Immunol. 176, 711 (2006). [95] I. Azpiazu and N. Gautam, J. Biol. Chem. 279, 27709 (2004). [96] T.S. Weiss, C.E. Chamberlain, T. Takeda, P. Lin, K.M. Hahn, and M.G. Farquhar, Proc. Natl. Acad. Sci. USA 98, 14961 (2001).
www.lphys.org
101
[97] B. Camuzeaux, C. Spriet, L. H´eliot, J. Coll, and M. Duterque-Coquillaud, Biochem. Biophys. Res. Commun. 332, 1107 (2005). [98] R. Latif, P. Graves, and T.F. Davies, J. Biol. Chem. 276, 45217 (2001). [99] I. Majoul, M. Straub, S.W. Hell, R. Duden, H.-D. Soeling, Dev. Cell 1, 139 (2001). [100] T. Wurch, A. Matsumoto, and P.J. Pauwels, FEBS Lett. 507, 109 (2001). [101] V. Lissandron, A. Terrin, M. Collini, L. D’alfonso, G. Chirico, S. Pantano, and M. Zaccolo, J. Mol. Biol. 354, 546 (2005). [102] M. Zaccolo, T. Cesetti, G. Di Benedetto, M. Mongillo, V. Lissandron, A. Terrin, and I. Zamparo, Biochem. Soc. Trans. 33, 1323 (2005). [103] E.K.L. Yeow and A.H.A. Clayton, Biophys. J. 92, 3098 (2007). [104] I. Gautier, M. Tramier, C. Durieux, J. Coppey, R.B. Pansu, J.-C. Nicolas, K. Kemnitz, and M. CoppeyMoisan, Biophys. J. 80, 3000 (2001). [105] K. Yoshitake, S. Waki, and H. Ueda, Biosens. Bioelectron. 23, 1266 (2008). [106] T. Endoh, M. Mie, and E. Kobatake, J. Biotechnol. 133, 413 (2008). [107] A.J. Rodriguez, J. Condeelis, R.H. Singer, and J.B. Dictenberg, Semin. Cell Dev. Biol. 18, 202 (2007). [108] M. Rehm, H. D¨ußmann, R.U. J¨anicke, J.M. Tavar´e, D. K¨ogel, and J.H.M. Prehn, J. Biol. Chem. 277, 24506 (2002). [109] K.Q. Luo, V.C. Yu, Y.M. Pu, and D.C. Chang, Biochem. Biophys. Res. Commun. 304, 217 (2003). [110] J.J.-H. Chiang and K. Truong, Biotechnol. Lett. 27, 1219 (2005). [111] T. Awaji, A. Hirasawa, H. Shirakawa, G. Tsujimoto, and S. Miyazaki, Biochem. Biophys. Res. Commun. 289, 457 (2001). [112] T.P. Remus, A.V. Zima, J. Bossuyt, D.J. Bare, J.L. Martin, L.A. Blatter, D.M. Bers, and G.A. Mignery, J. Biol. Chem. 281, 608 (2006). [113] A. Tanimura, A. Nezu, T. Morita, R.J. Turner, and Y. Tojyo, J. Biol. Chem. 279, 38095 (2004). [114] S. De, I.G. Macara, and D.A. Lannigan, J. Steroid Biochem. Mol. Biol. 96, 235 (2005). [115] T. Endoh, H. Funabashi, M. Mie, and E. Kobatake, Anal. Chem. 77, 4308 (2005). [116] M. Fehr, S. Lalonde, I. Lager, M.W. Wolff, and W.B. Frommer, J. Biol. Chem. 278, 19127 (2003). [117] M. Fehr, S. Lalonde, D.W. Ehrhardt, and W.B. Frommer, J. Fluoresc. 14, 603 (2004). [118] M. Fehr, W.B. Frommer, and S. Lalonde, Proc. Natl. Acad. Sci. USA 99, 9846 (2002). [119] S. Okumoto, L.L. Looger, K.D. Micheva, R.J. Reimer, S.J. Smith, and W.B. Frommer, Proc. Natl. Acad. Sci. USA 102, 8740 (2005). [120] H. Takanaga, B. Chaudhuri, and W.B. Frommer, Biochim. Biophys. Acta – Biomembranes 1778, 1091 (2007). [121] P.J. Dittmer, J.G. Miranda, J.A. Gorski, and A.E. Palmer, J. Biol. Chem. 284, 16289 (2009). [122] A. Tanimura, T. Morita, A. Nezu, A. Shitara, N. Hashimoto, and Y. Tojyo, J. Biol. Chem. 284, 8910 (2009).
c 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA
102
A.L. Rusanov and A.P. Savitsky : Fluorescence resonance energy transfer between FP’s
[123] H. Imamura, K.P.H. Nhat, H. Togawa, K. Saito, R. Iino, Y. Kato-Yamada, T. Nagai, and H. Noji, Proc. Natl. Acad. Sci. USA 106, 15651 (2009). [124] A. Yamada, K. Hirose, A. Hashimoto, and M. Iino, Biochem. J. 385, 589 (2005). [125] D.C. Braun, S.H. Garfield, and P.M. Blumberg, J. Biol. Chem. 280, 8164 (2005). [126] M. Sato, T. Ozawa, K. Inukai, T. Asano, and Y. Umezawa, Nat. Biotechnol. 20, 287 (2002). [127] A.Y. Ting, K.H. Kain, R.L. Klemke, and R.Y. Tsien, Proc. Natl. Acad. Sci. USA 98, 15003 (2001). [128] Y.X. Wang, E.L. Botvinick, Y.H. Zhao, M.W. Berns, S. Usami, R.Y. Tsien, and S. Chien, Nature 434, 1040 (2005). [129] B. Hall, M.A. McLean, K. Davis, J.E. Casanova, S.G. Sligar, and M.A. Schwartz, Anal. Biochem. 374, 243 (2007). [130] R.E. Itoh, K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda, Mol. Cell. Biol. 22, 6582 (2002). [131] A.D. Collinson, S.W.A. Bligh, D.L. Graham, H.R. Mott, P.A. Chalk, N. Korniotis, and P.N. Lowe, ASSAY Drug Dev. Technol. 2, 659 (2004). [132] A. Seth, T. Otomo, H.L. Yin, and M.K. Rosen, Biochem. 42, 3997 (2003). [133] C.-W. Lin, C.Y. Jao, and A.Y. Ting, J. Am. Chem. Soc. 126, 5982 (2004). [134] K. Osibow, R. Malli, G.M. Kostner, and W.F. Graier, J. Biol. Chem. 281, 5017 (2006). [135] A. Miyawaki, J. Llopis, R. Heim, J.M. McCaffery, J.A. Adams, M. Ikura, and R.Y. Tsien, Nature 388, 882 (1997). [136] A. Miyawaki, O. Griesbeck, R. Heim, and R.Y. Tsien, Proc. Natl. Acad. Sci. USA 96, 2135 (1999). [137] R. Rudolf, M. Mongillo, P.J. Magalh˜aes, and T. Pozzan, J. Cell Biol. 166, 527 (2004). [138] R.L. Cornea, F. Nitu, S. Gruber, K. Kohler, M. Satzer, D.D. Thomas, and B.R. Fruen, Proc. Natl. Acad. Sci.
USA 106, 6128 (2009). [139] S. Diegelmann, A. Fiala, C. Leibold, T. Spall, and E. Buchner, Genesis 34, 95 (2002). [140] M. Hara, V. Bindokas, J.P. Lopez, K. Kaihara, L.R. Landa, Jr., M. Harbeck, and M.W. Roe, Am. J. Physiol. Cell Physiol. 287, C932 (2004). [141] N. Tsujino, A. Yamanaka, K. Ichiki, Y. Muraki, T.S. Kilduff, K. Yagami, S. Takahashi, K. Goto, and T. Sakurai, J. Neurosci. 25, 7459 (2005). [142] R. Kerr, V. Lev-Ram, G. Baird, P. Vincent, R.Y. Tsien, and W.R. Schafer, Neuron 26, 583 (2000). [143] K.V. Gromova, M. Friedrich, A. Noskov, and G.S. Harms, Biochim. Biophys. Acta – Mol. Cell Res. 1773, 1759 (2007). [144] M. Bogner and U. Ludewig, J. Fluoresc. 17, 350 (2007). [145] R. Sakai, V. Repunte-Canonigo, C.D. Raj, and T. Kn¨opfel, Eur. J. Neurosci. 13, 2314 (2001). [146] R.D. Mitra, C.M. Silva, and D.C. Youvan, Gene 173, 13 (1996). [147] B. Zhang, Biochem. Biophys. Res. Commun. 323, 674 (2004). [148] A.Yu. Gorokhovatsky, V.V. Marchenkov, N.V. Rudenko, T.V. Ivashina, V.N. Ksenzenko, N. Burkhardt, G.V. Semisotnov, L.M. Vinokurov, and Yu.B. Alakhov, Biochem. Biophys. Res. Commun. 320, 703 (2004). [149] B. Schuler, E.A. Lipman, P.J. Steinbach, M. Kumke, and W.A. Eaton, Proc. Natl. Acad. Sci. USA 102, 2754 (2005). [150] R.B. Best, K.A. Merchant, I.V. Gopich, B. Schuler, A. Bax, and W.A. Eaton, Proc. Natl. Acad. Sci. USA 104, 18964 (2007). [151] D.L. Graham, P.N. Lowe, and P.A. Chalk, Anal. Biochem. 296, 208 (2001). [152] F.K.-M. Chan, R.M. Siegel, D. Zacharias, R. Swofford, K.L. Holmes, R.Y. Tsien, and M.J. Lenardo, Cytometry 44, 361 (2001). [153] T. Ohashi, S.D. Galiacy, G. Briscoe, and H.P. Erickson, Protein Sci. 16, 1429 (2007).
Alexander L. Rusanov received his degree in chemistry in 2007 from M.V. Lomonosov Moscow State University. He is currently Ph.D. student of the same University. His research interests include fluorescence resonance energy transfer, fluorescence lifetime imaging microscopy, and fluorescent proteins.
Alexander P. Savitsky, professor of biochemistry, received his Ph.D. in 1979 and Doctor of Science in 1990 both in Biochemistry from M.V. Lomonosov Moscow State University. In 1991 he became the head of the laboratory of physical biochemistry at the Institute of Biochemistry of the Russian Academy of Sciences. His research interests include fluorescence spectroscopy, fluorescent proteins, micro- and macroscopic in vivo imaging, molecular nanobiosensors, lanthanide, and phosphorescent labeling technology.
c 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA
www.lphys.org
