**3.1.2 Registration system**

190 Macro to Nano Spectroscopy

A homemade laser fluorimeter (Fig. 3) has been built for the experiments. The fluorimeter consist of a laser source, optical elements for light conversion, a fiber-optic cable, a cuvette

Fig. 3. Nanosecond laser fluorescence spectrometer scheme (for details see text). The letters

The pulsed single mode Nd:YAG laser (the fundamental wavelength is 1064 nm) with the set of nonlinear crystals for generating the 2nd (532 nm), the 3d (355 nm) and the 4th (266 nm) harmonics of the fundamental radiation was used. At a pulse repetition frequency of 10 Hz, the average pulse energy was: 5 mJ (532 nm), 2 mJ (355 nm) and 0.7 mJ (266 nm). The 2 mm-diameter Nd:YAG crystal with the diaphragm (the diameter is 1.5 mm) on the outlet face was used to obtain the light generation in a single mode regime (single transverse mode). Q-switching was made by the electro-optical shutter, working based on the Pockels effect. Between the Nd:YAG crystal and the shutter, the polarizer, transmitting light only with horizontal polarization, was placed. Thus, at the generator output, we had a single mode beam with horizontal polarization. After the generator, the light beam passed through the linear telescope, which also worked as a rotating prism. The main purpose of this component was to decrease the beam divergence in the horizontal plane, which is necessary for effective conversion of the fundamental frequency to its harmonics. Then, the laser beam was sent to the 90 degree polarization rotator, which changed the horizontal polarization to vertical one (required for reducing energy losses on rotating mirrors). Passed through the rotator and reflected from the rotating mirror beam was directed through the round-trip amplifier, which was composed of the Nd:YAG crystal (5 mm in diameter) and the rear

H, V and C denote the horizontal, vertical and circular light polarization.

**3. Experimental** 

**3.1 Nanosecond laser fluorescence spectrometer** 

and an optical multichannel analyser.

**3.1.1 Laser excitation system** 

The laser radiation was focused on the cuvette with the sample solution by a long-focallength lens (the focal length is 20 cm). For collecting the fluorescence photons, the lightguide cable (the length is 5 m), consisting of seven quartz fibers (the diameter of each fiber is 600 micron), which were laid out in a row (like a slit) at both ends, was used. The cable inlet was fixed at the cuvette side, and the outlet was clasped to the polychromator entrance slit; the sample solution was excited by transmitted laser radiation. As a detector of radiation, the optical multichannel analyser (OMA) was used. The optical chamber (DeltaTech, Scientific Park of MSU) of the analyser consists of the electro-optical converter based on a gated microchannel plate (MCP), CCD matrix, and optical device for transferring an image from the MCP to the CCD matrix (a pixel size is 11×11µm2). The chamber was fixed to the polychromator (MUM without the output slit, reciprocal linear dispersion is 0.15 nm per channel) optical output. The multichannel analyser was connected to the PC. As a result, optical image in the polychromator output slit plane could be obtained as a 2D picture on the PC monitor. The software installed on the PC allowed the OMA to operate both in the continuous and gated modes. When working in gated mode, a part of light was sent to the silica photodiode PD-265 (the building-up time of the leading edge is less than 2 ns). The photodiode was connected to the nanogenerator triggering inlet (the trigger level is 0.6 V). The gating of the MCP was implemented by high-voltage pulses from the nanogenerator (the amplitude is 800 V). The detector gate delay time could be adjusted through the nanogenerator over the range of 50 ns (the dead time of the detector) to 1200 ns with the step *tstep*=2.5 ns. Exactly because of the dead time of the registration system, when it operates in the gated mode (i.e. in the case of kinetic curves measurements), the light-guide cable as the dead time compensator (the optical delay line) was used. The detector gate width *tg* could be varied from 10 to 1200 ns; in our experiments it was set to 10 ns.

#### **3.1.3 Laser radiation parameters**

The laser pulse duration at the wavelengths of 532 and 266 nm were fitted well by Gaussian function with the full width at half maximum (*tp*) of 12 and 10 ns, respectively. When measuring (i) the kinetic curves, the laser beam diameter was 3 mm and (ii) the saturation curves, the beam was focused to a spot with a diameter from 600 to 800 µm, depending on the protein under investigation. The values of the photon flux density *F* were determined according to the equation *F-1=E-1ћStp*, where *ћ*is a photon energy, *E* is average pulse

Laser Fluorescence Spectroscopy:

**4.2 Red fluorescent proteins** 

energy transfer (FRET (Valeur, 2002)) mechanism possible.

(Cambel et al., 2002), which made it an object of the research.

state spectra and fluorescence life-times on a picoseconds laser fluorimeter.

Application in Determining the Individual Photophysical Parameters of Proteins 193

fact makes the intramolecular energy transfer between them using the Forster resonance

Fluorescent proteins are a class of proteins that have a distinguishing property of forming their chromophore without involvement of any additional cofactors and ferments (autocatalytic reaction), except for molecular oxygen. In recent years, FPs have gained enormous popularity as genetically encoded fluorescence markers that enable to visualize a broad range of biological processes in cells and tissues. The most popular for practical applications are FPs whose fluorescence is shifted to the red (red FPs) and whose molecules are monomers (Piatkevich et al, 2010). The mRFP1 protein possesses these properties

In this work the red FP mRFP1 and its mutants at 66 amino acid residue (glutamine 66) have been used. The seven mutants with substitution of the glutamine 66 for the serine (protein mRFP1/Q66S2), cysteine (mRFP1/Q66C), asparagine (mRFP1/Q66N), histidine (mRFP1/Q66H), alanine (mRFP1/Q66A), leucine (mRFP1/Q66L) and phenylalanine (mRFP1/Q66F) have been created. The method for fabrication and purification of proteins is described in (Vrzheshch et al., 2008). All the experiments were performed in a 0.06 M phosphate buffer at a temperature of 25±1 °C. The Bradford method (McCluskey, 2003) was used to determine initial concentration of the proteins. The proteins concentration was: (1) 10-9 M when measured fluorescence saturation curves; (2) 5.5×10-10 3×10-10 M (depending on protein) when measured fluorescence quantum yield; (3) 10-6 M when measured steady-

The formation of a fluorescent molecule of red FPs is a complicated process (usually called maturation) consisting of several stages (Verkhusha et al., 2004; Strack et al., 2010). At some stages, intermediate protein forms are produced, which remain in the resultant specimen (solution) of protein. In other words, the solution of red FPs is an ensemble of protein molecules consisting of several chemically non-equivalent subensembles (a mixture of different spectral forms of FPs). At neutral pH, the solution of mRFP1-like proteins contains three different spectral forms, namely, the mature form (will denote below as R form), the immature form with protonated chromophore (GH form) and the immature form with deprotonated chromophore (G form). The detailed information about formation of the chromophore of red FPs and analysis of composition of the samples can be found elsewhere (Verkhusha et al., 2004; Strack et al., 2010). According to a widely used terminology (Verkhusha et al., 2004), I'll also call the mature form as a red form and the immature forms as a green form in this work. The spectral forms can be detected in an absorption spectrum as the bands with maxima in the blue (360-420 nm, GH form), green (450-540 nm, G form) and red (550-600 nm, R form) spectral ranges. It is known (Pakhomov et al., 2004) that the balance between GH and G forms of FP can be disturbed with the changing of the solution acidity or as a result of light (from the UV, blue, or green spectral ranges) influence on the

2 In this work, the name of each mRFP1 mutant is designated like mRFP1/Q66#, where Q66 is the abbreviation of the glutamine (single letter code is Q) at position 66, which is substituted. In place of the symbol #, the single letter code of amino acid the glutamine is substituted for is written down. The list

of the amino acids abbreviation can be found elsewhere (Zamyatin, 1972).

energy, *S* is cross section area of a laser beam, *tp* is a pulse duration. The photon flux density *Fmax* at a maximum of the saturation curve was measured before each experiment. Then, the photon flux density was gradually decreased. The *F* value at each point was obtained by the value of the Raman scattering signal from water molecules *NRS*: *F=FmaxNRS/NmaxRS*, where *NmaxRS* is the Raman scattering signal at *F=Fmax*. In nonlinear laser fluorimetry experiments, the fluorescence saturation curves were measured in the following ranges of *F*: (i) 5102371025 s-1cm-2 when the excitation wavelength was 532 nm and (ii) 2102451025 s-1cm-2 when excited at the wavelength of 266 nm.
