*4.3. Fluorescence*

Fluorescence spectra were collected on a Cary Varian spectrofluorimeter (Agilent, Foster City, CA, USA), interfaced with a Peltier, at 25 ◦C. LrtA concentration in the pH- or chemical-denaturation experiments was 9.8 μM (in protomer units). For experiments with ANS, a final probe concentration of 100 μM was added. A 1-cm-pathlength quartz cell (Hellma, Mullheim, Germany) was used.

In the pH-induced unfolding curves, the pH was measured after completion of the experiments with an ultra-thin Aldrich electrode in a Radiometer pH-meter (Madrid, Spain). The acids and salts used were: pH 2.0–3.0, phosphoric acid; pH 3.0–4.0, formic acid; pH 4.0–5.5, acetic acid; pH 6.0–7.0, NaH2PO4; pH 7.5–9.0, Tris acid; pH 9.5–11.0, Na2CO3; pH 11.5–13.0, Na3PO4. Chemical and pH denaturations were repeated three times with new samples at any of the concentrations assayed. Appropriate blank corrections were made in all spectra both in pH- and chemicaldenaturation experiments.

For GdmCl-denaturation experiments the samples were prepared the day before from a 7 M GdmCl concentrated stock and left overnight to equilibrate; before experiments, samples were left to 25 ◦C for 1 h. For the refolding experiments, the sample was exchanged in 7 M GdmCl by using Amicon centrifugal devices; protein concentration was the same as in the unfolding experiments.

The emission intensity weighted average of the inverse wavelengths (also called the spectrum mass center, or the spectral average energy of emission), <1/λ>, was calculated as described [50]. Briefly, we define <1/λ> as: 1/*λ* <sup>=</sup> *<sup>n</sup>* ∑ 1 1 *λi Ii*/ *n* ∑ 1 *Ii*, where *I*<sup>i</sup> is the intensity at wavelength λi. We shall report <1/λ> in units of μm<sup>−</sup>1.

Steady-State Spectra—The experimental set-up for the intrinsic and ANS fluorescence pH-denaturation experiments has been described previously [50]. Briefly, protein samples were excited at 278 nm, for the intrinsic fluorescence, and 380 nm for the ANS experiments. In all cases, excitation and emission slits were 5 nm. The experiments were recorded between 300 and 400 nm (for the intrinsic fluorescence) and between 400 to 600 for the ANS experiments. The signal in all cases was acquired for 1 s and the increment of wavelength was set to 1 nm. For the chemical denaturations, following intrinsic fluorescence, several protein concentrations were used in the range from 1.9 to 19.6 μM (in protomer units) at 100 mM phosphate buffer (pH 7.0) and 50 mM NaCl.

Thermal Denaturations—Thermal denaturations of isolated LrtA at different pH values were carried out with the same experimental set-up described [50] and protein concentrations of 9.8 and 5 μM (in protomer units). Briefly, these experiments were performed at constant heating rates of 60 ◦C/h and an average time of 1 s. Thermal scans were collected at 308 nm after excitation at 278 nm from 25 to 95 ◦C and acquired every 0.2 ◦ C.

Fluorescence Quenching—Quenching by iodide and acrylamide was examined at different solution conditions, with an LrtA concentration of 9.8 μM (in protomer units): pH 3.5 (formic buffer, 50 mM), pH 7.0 (phosphate buffer, 50 mM), and pH 11.0 (boric buffer, 50 mM). Experiments were also carried out in the presence of 6 M GdmCl at pH 7.0 (50 mM, phosphate buffer). The experimental set-up for both quenchers was the same described above for the intrinsic fluorescence experiments. The data for KI were fitted to [12]

$$\frac{F\_0}{F} = 1 + K\_{sv}[KI] \tag{1}$$

where *K*sv is the Stern-Volmer constant for collisional quenching; *F0* is the fluorescence intensity in the absence of KI; and *F* is that at any KI concentration. The range of KI concentrations explored was 0–0.7 M. For experiments with acrylamide, the data were fitted to [12]

$$\frac{F\_0}{F} = (1 + K\_{sv}[acrylamide])e^{v[acrylamide]}\tag{2}$$

where υ is the dynamic quenching constant. Fittings to Equations (1) and (2) were carried out by using Kaleidagraph (Synergy software, Dubai, United Arab Emirates).
