**4.2 Red fluorescent proteins**

192 Macro to Nano Spectroscopy

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

*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

The fluorescence lifetime measurements were performed with a streak camera (Agat SF 3M, VNIIOFI, Russia). A Nd:YAG laser with excitation wavelengths of 532 and 266 nm (the second and fourth harmonic of fundamental radiation) was used as a light source. The laser radiation parameters of the fluorimeter were as follows (for 532 nm): pulse energy 160 µJ, duration 20 ps (fwhm), beam diameter 5 mm. The error in determining the fluorescence lifetimes in time intervals of several nanoseconds did not exceed 5 %. In addition to the laser equipment, the Cary 100 spectrophotometer (Varian, Inc., USA) and the Cary Eclipse spectrofluorimeter (Varian, Inc., USA; slits width was 5 nm) were used for optical density

In this work, the solutions of human serum albumin (HSA) (>96%, Sigma) and of bovine serum albumin (BSA) (>98%, MP Biomedicals) in a phosphate buffer (0.01 M, pH 7.4) have been used. The proteins concentrations were 10–5 (absorption spectra measurement) and 10–9 M (fluorescence measurement at the nanosecond laser fluorimeter). All of the experiments were performed at a temperature of 25±1 0C. The structure and biological functions of HSA and BSA can be found in (Peters, 1996). Tryptophan, tyrosine, and phenylalanine (with relative contents of 1:18:31 in HSA and 2:20:27 in BSA) are the absorption groups in these proteins (as in many other natural proteins). The tyrosine fluorescence in HSA and BSA (as in many other natural proteins) is quenched due to the effect of adjacent peptide bonds, polar groups (such as CO, NH2), and other factors, and phenylalanine has a low fluorescence quantum yield (0.03) (Permyakov, 1992). Therefore, the fluorescence signal in these proteins is determined mainly by tryptophan groups. In that case the fluorescence, registered in nonlinear and kinetic laser fluorimetry measurements, correspond to

As described in (Peters, 1996), HSA and BSA have similar structure and amino acid sequences that differ insignificantly by location of some certain amino acids. However, HSA contains one tryptophan residue in the protein matrix (Trp-214), and BSA contains two residues (Trp-212 and Trp-134). Trp-212 in BSA and Trp-214 in HSA have a similar microenvironment and, hence, their spectral properties are similar (Eftink et al., 1977). Tryptophans of BSA are not spectrally identical due to the stronger integration of Trp-212 into the protein's structure and the more hydrophobic environment of Trp-212 in comparison with Trp-134. The distance between tryptophans in BSA is about 3.5 nm. This

*NRS/NmaxRS*, where

value of the Raman scattering signal from water molecules *NRS*: *F=Fmax*

**3.2 Picosecond time-resolved and steady-state spectral measurements** 

s-1cm-2 when excited at the wavelength of 266 nm.

measurements and fluorescence registration, respectively.

tryptophan residues (this fact will be used in Section 6.1).

**4. The object 4.1 Albumins** 

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

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

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

<sup>2</sup> 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).

Laser Fluorescence Spectroscopy:

Application in Determining the Individual Photophysical Parameters of Proteins 195

One can see from the absorption spectra of the mRFP1 (Fig. 5(a)) that in the wavelength range from 370 to 650 nm, there exist three absorption bands, which are explained by the presence of three spectral forms in the solution (Verkhusha et al., 2004), i.e. R, G and GH forms (the corresponding absorption maxima at 584, 503 and 380 nm). One can also see that the absorption bands of G and R forms overlap. Excitation of fluorescence in the absorption band of each form indicates that G and GH forms do not fluoresce, the R form fluoresces with maxima at 607 nm. The excitation of the protein solution by irradiation at a wavelength of 270 nm (tryptophan absorption band) leads to the appearance in the signal spectrum not only of an UV band (maximum at 330 nm), which corresponds to the tryptophan fluorescence in the protein matrix, but also of a band in the visible region of wavelengths (maximum at 607 nm) corresponding to fluorescence of the chromophore mRFP1 R form (Fig. 5(b)). The chromophore of GH and G forms is non-fluorescent (Campbell et al., 2002).

Fig. 5. (a) Absorption (solid) and fluorescence excitation (dotted, registration at 607 nm) spectra of mRFP1. The spectra are normalized to the signal at 584 nm. (b) Emission of mRFP1 (excitation by 270 nm). (c) Absorption spectra of the mutants with polar and (d) nonpolar substitutions at position 66. The spectra are normalized to optical density at 270 nm.

Absorption spectra of the variants of mRFP1 with mutation at position 66 are shown in the Fig. 5(c, d).The absorption spectra structure of the mutants is similar to one for mRFP1. One can see that for all proteins in the wavelength range from 370 to 650 nm, the spectrum contains three bands with the maxima at 360-420 nm, 450-540 nm and 550-600 nm depending on the protein. These absorption peaks are corresponding to the GH, G and R forms. There only signal in the red spectral range were observed in the fluorescence emission and excitation spectra (i.e. the G form was found to be non-fluorescent) for all proteins except mRFP1/Q66F. For mRFP1/Q66F, the additional band in fluorescence excitation (maxima at 502 nm) and emission (maxima at 512 nm) spectra was detected,

**5.2 Monomeric red fluorescent protein and its mutants at residue 66** 

protein. Irradiation of the protein molecules by the light from the green spectral range provokes a conversion of G form molecules to the molecules of GH form; as a result, the protein solution will represent a mixture of only two forms, namely, GH and R. In current work, for conversion the radiation of an Ar laser (LG-106-M1) at 488 nm (300mW) was used. An increment of the protein solution pH to 9 leads to a practically total transfer of the GH form molecules to the G form molecules (in that case, the protein solution is a mixture of the G and R forms). These procedures will be used as a way to decrease the amount of simultaneously existing forms in the mRFP1 solution when the method of the nonlinear fluorimetry will be implemented (see Section 6).
