Fluorescence spectroscopy

Fluorescence is the other side of absorbance, or the fate of a molecule after it has been excited to a higher energy state by the absorbance of light. The phenomenon is best explained with a standard Jabƚoński energy diagram. Molecules in their ground energy state are excited by the absorption of light of a specific wavelength to a higher energy state, from where they can fall back down to the ground state by several processes: initially by loss of heat by internal conversion, which is loss of energy within an energy state, across vibronic levels; and then via non-radiative or radiative transitions (Van Holde et al., 1998). The radiative transitions are either fluorescence or phosphorescence. Phosphorescence is a comparatively slow process and thus relatively rare. Fluorescence is thus basically the loss of electronic energy by emission of light, which emission occurs at a longer wavelength than the excitation radiation. This loss of energy between absorption and emission of light is termed “Stokes’ shift” and is due to processes including dissipation of vibrational energy and solvent-fluorophore interactions (Lakowicz, 1999). Generally speaking, the delocalised electrons in π orbitals of aromatic ring

structures are most easily excited to higher energy states, and these molecules are prone to fluoresce.

The tertiary structure of a protein can be characterised by fluorescence spectroscopy because of the presence of the intrinsic aromatic fluorophores, Tyr and Trp in just about every protein molecule. Fluorescence spectroscopy gives information about the packing and local environment of Tyr and Trp (Lakowicz, 1999), although protein fluorescence spectra are generally dominated by tryptophan emission. The quantum yields for Tyr and Trp - basically the ratio of the number of photons emitted to the number absorbed - are not that different (0.14 vs. 0.13 respectively) (Lakowicz, 1999), but the molar extinction coefficient for Trp is much higher, resulting in greater total yield. The indole ring of Trp is highly sensitive to solvent polarity because of the complexity of the electronic transitions to the 1La and 1Lb states and the relative orientations of the absorbing

and emission states (Lakowicz, 1999). Proteins exhibit characteristic fluorescence spectra according to the environment within which the main fluorescing species are packed. Fluorescence intensity varies according to the polarity of the environment, but the fluorescence wavelength does not change for tyrosine (Lakowicz, 1999). The contribution of tyrosine can be undetected in the folded protein, due to energy transfer to tryptophan residues, or to quenching by nearby groups, but will often become apparent in a denatured sample (Lakowicz, 1999). Fluorescence emission intensity and wavelength for tryptophan change according to the polarity of the environment within which the tryptophan is found, and the degree of quenching experienced by the fluorophore as a result of that environment (Lakowicz, 1999). The greater the exposure of Trp to the polar aqueous environment, the longer its wavelength of maximum emission will be, since the polar solvent molecules lower the energy of the excited state (Royer, 1995). Tryptophan can be selectively excited at 295 nm since Tyr absorbs well below this wavelength. CLIC1 has eight Tyr and one Trp residue. The tyrosine residues are mostly in the C-terminal domain, with only two in the N-terminal domain, and Trp35 is in the putative transmembrane region in the N-terminal domain, acting as a local reporter for that region.

2.2.4.2.1. Intrinsic fluorescence

All fluorescence measurements on CLIC1 were performed on a Jasco FP6300 spectrofluorometer with Spectra Manager v. 1.54.03 software, using a quartz cuvette. Scan speed was 200 nm/min over a wavelength range of 280-450 nm using a quartz cuvette of path length 1 cm. Excitation was at 280 nm (Ex.280) or 295 nm (Ex.295), with excitation and emission slit widths at 5 nm. Sensitivity was set to manual with the photomultiplier (PMT) voltage set to 350 V, and response set to fast. The data pitch was 0.5 nm. Native and unfolded spectra of 2

µM CLIC1 in CLIC1 storage buffer (50 mM sodium phosphate, 0.02% NaN3, 1

mM DTT), pH 7.0 and pH 5.5, and in 3.8 M and 8.0 M urea made up with CLIC1 storage buffer, were measured. All data were buffer-corrected.

2.2.4.2.2. Extrinsic fluorescence

8-Anilino-1-naphthalene sulphonate (ANS) (Figure 2.2), an amphipathic dye with a tendency to bind hydrophobic surfaces, has long been used as an extrinsic fluorescent probe of protein conformations because of its greatly enhanced quantum yield upon binding to exposed hydrophobic patches (Rosen and Weber, 1969; Brand and Gohlke, 1972). In particular, ANS is widely used for probing intermediate states in the unfolding transitions of proteins since clusters of hydrophobic residues not normally exposed in the native conformation of the protein become available for ANS binding (Semisotnov et al., 1991). ANS has complex photophysical properties. The polarity of the environment affects both the quantum yield and energy of emission (and thus wavelength of emission) of the molecule (Gasymov and Glasgow, 2007). In aqueous solution the dye’s fluorescence is quenched, but in a hydrophobic environment the intensity of fluorescence is greatly enhanced, and the maximum emission wavelength (λmax) is

blue-shifted. This is due to different excited states of the molecule. The first excited state – the non-polar (NP) state - is localised on the naphthalene ring, and high energy emission from this state occurs in non-polar solvents (Kosower and Kanety, 1983; Kosower, 1986). If the solvent is more polar, an intramolecular electron-transfer reaction from the NP state forms the charge-transfer state (CT), a

low energy state. In aqueous solution, intermolecular electron-transfer results in electron solvation and subsequent radiationless decay from the CT state, explaining the low quantum yield in aqueous solution (Kosower and Kanety, 1983; Kosower, 1986).

Figure 2.2. The structure of ANS.

ANS is charged, but is highly hydrophobic due to its triple ring structure. Electronic transitions of the delocalised π electrons within these rings are the basis of its fluorescent properties, but the polarity of the solvent will affect electron transfer reactions which determine the energy and intensity of emission.

ANS stocks were made up to between 2 mM and 6 mM using the 50 mM sodium phosphate storage buffer, 0.02 % NaN3 with 1 mM fresh DTT into which CLIC1

had been dialysed that week. Separate stocks were prepared using pH 5.5 or pH 7.0 buffers. The stocks were warmed slightly during stirring and then filtered through 0.45 µm filters because ANS does not dissolve easily in aqueous solution, and black precipitate remains even after extensive stirring. The pH was checked and adjusted using NaOH and the ANS concentration was confirmed spectrophotometrically using a series of dilutions of stock ANS and an extinction coefficient of ε350 = 5000 M-1 cm-1 (Weber and Young, 1964). Dilution factors

solutions to the initial concentration of the stock solution, where the latter was taken as 1 (Reed et al., 2003).

Fluorescence spectra of ANS in the presence and absence of CLIC1 in 0.0 M, 3.8 M and 8.0 M urea were recorded at pH 5.5 and pH 7.0 on a Jasco FP6300 spectrofluorometer with Spectra Manager v. 1.54.03 software, using a 1 cm quartz cuvette. The scan speed was 200 nm/min over a wavelength range of 400-650 nm. Excitation was at 390 nm, with excitation and emission slit widths at 5 nm. Sensitivity was set to manual with PMT voltage at 350 V, and response set to fast. The data pitch was 0.5 nm.

Equilibrium unfolding in the presence of ANS was performed by allowing 2 µM samples of CLIC1 to unfold in 0-8 M urea for 1-2 hours as described in Section 2.2.5.4. The samples were then brought to 200 µM ANS and incubated at room temperature (20 °C) for a further hour before performing measurements. Equivalent samples containing no CLIC1 were prepared and treated in the same manner. These are necessary to correct for the increasing free ANS emission signal in increasing concentrations of urea. Samples were excited at 390 nm and the emission signal monitored at 460 nm (F460) using the time drive function on a Perkin Elmer LS50-B spectrofluorometer (pH5.5) or spectrum measurement on a Jasco FP6300 spectrofluorometer (pH 7.0). A quartz cuvette with a 1 cm path length was used, and both excitation and emission monochromator slit widths, respectively, were set to 5 nm on both machines. On the Perkin Elmer LS50-B, recordings were made at a data pitch of 0.5 sec for 60 seconds, and the free ANS- corrected F460 plotted as the average emission over this time period. On the Jasco FP6300, scan speed was 200 nm/min, data pitch 0.5 nm, response fast and sensitivity manual with PMT voltage at 350 V. F460 data were extracted, corrected for free ANS and plotted as a function of urea concentration using Sigmaplot v. 11.0.

The binding affinity of ANS for CLIC1 was determined by titrating aliquots of 2 mM stock ANS into 2 µM CLIC1 in 50 mM sodium phosphate buffer, 0.02 %

NaN3 and 1 mM DTT at pH 5.5 and 7.0 in the presence and absence of 3.8 M

urea. The same titrations were performed in the absence of CLIC1 to correct for free ANS. Data were collected on a Perkin Elmer LS50-B spectrofluorometer using the time drive function with excitation at 390 nm and emission at 460 nm. Recordings were made for 60 secs at a data pitch of 0.5 sec, with excitation and emission slit widths both at 5 nm. The averaged time drive data was corrected for free ANS and plotted using Sigmaplot v. 11.0.