*4.1. Materials*

Deuterium oxide, d11-Tris acid and isopropyl-β-D-1-tiogalactopyranoside (IPTG) were purchased from Apollo Scientific (Stockport, UK). DNase, kanamycin, the Trizma base and its acid, sodium trimethylsilyl (2,2,3,3-2H4) propionate (TSP), imidazole, and the His-Select HF nickel resin were from Sigma-Aldrich (Madrid, Spain). The β-mercaptoethanol (β-ME) was provided by BioRad (Madrid, Spain). The protein marker, PAGEmark Tricolor, and Triton X-100 were from VWR (Barcelona, Spain). The protein gel-filtration-column calibration markers were from GE Healthcare (Barcelona, Spain). Dialysis tubing was from Spectrapor (Spectrum Laboratories, Shiga, Japan). Amicon centrifugal devices with a cut-off molecular weight of 3000 Da were from Millipore (Barcelona, Spain). The rest of the materials used were of analytical grade. Water was deionized and purified on a Millipore system.

#### *4.2. Protein Expression and Purification*

The C-LrtA region (residues 102-191 of LrtA) was cloned by NZytech (Lisbon, Portugal) in a pHTP1 *E. coli* expression vector (between XhoI and NcoI sites), with kanamycin resistance. The final construct contained an N-terminal His-tag to allow for purification (MGSSHHHHHHSSGPQQGLR), and had the overall sequence: MGSSHHHHHHSSGPQQGLRQHGNVKTSEIVEDKPVEENLIGDRA PELPSEVLRMKYFAMPPMAIEDALEQLQLVDHDFYMFRNKDTDEINVIYIRNHGGYGVIQPHQAS.

Expression of C-LrtA was carried out in the *E. coli* BL21 (DE3) strain (Novagen, VWR, Barcelona; Spain) strains with a final kanamycin concentration of 50 mg/mL. We used 1 L flasks to culture the cells. The expression of the protein was induced with a final concentration of 1.0 mM IPTG, when the absorbance observed for the cell culture was 0.4–0.9 at 600 nm, and the growth of the cells continued for 15–16 h at 25 ◦C 15–16 h at 25 ◦C (this temperature was chosen to decrease the possibility of aggregates in C-LrtA, as the parental LrtA had a tendency to form inclusion bodies). We harvested the cells in a JA-10 rotor (Beckman Coulter) for 15 min at 8000 rpm. We re-suspended the cellular pellet from 5 L of culture in 50 mL buffer A (500 mM NaCl, 5 mM imidazole, 20 mM Tris buffer (pH 8), 0.1% Triton X-100 and 1 mM β-ME) and adding a tablet of Sigma Protease Cocktail EDTA-free. In the first attempts to get the protocol of purification, we added 2 mg of DNase (per 1 L of culture). We incubated the mixture for 10 min, with gentle agitation in the fridge (4 ◦C). Next, we sonicated the mixture (by using a Branson sonicator, 750 W), with 10 cycles of 45 s each at 55% of maximal power output, with intervals of 15 s between the cycles, and always keeping the cells in ice. We separated the supernatant by centrifugation at 18,000 rpm for 40 min at 4 ◦C in a Beckman JSI30 centrifuge with a JA-20 rotor. C-LrtA was present in the supernatant, and we purified it by immobilized affinity chromatography (IMAC), by adding 5 mL of Ni-resin previously equilibrated in buffer A. The mixture was incubated for 20 min at 4 ◦C, and afterwards, the lysate was separated from the resin by gravity. The washing step was carried out with 20 mL of buffer B (20 mM Tris buffer (pH 8.0), 500 mM NaCl, 1 mM β-ME, and 20 mM imidazole); the protein was eluted by gravity from the column with buffer C (20 mM Tris buffer (pH 8.0), 500 mM NaCl, 1 mM β-ME, and 500 mM imidazole). The solution was dialyzed against 50 mM Tris buffer (pH 8.0). The protein was further purified in a Hi-Trap Mono Q (GE Healthcare) column by using a gradient step from 0 to 1 M NaCl (50 mM Tris buffer, pH 8.0) in 60 min and a flow of 1 mL/min, in an AKTA Basic system (GE Healthcare), while monitoring the absorbance at 280 nm. This column purification step was used based on the theoretical isoelectric point of C-LrtA (pI = 5.72).

In the initial purification attempts (carried out in the presence of DNase), we observed that the main peak coming from the Hi-Trap Mono Q column did not show an emission fluorescence spectrum expected for a protein containing only 4 tyrosine residues (i.e., with a maximum at 308 nm), but rather the spectrum of a protein containing tryptophan (a maximum at ~330 nm). Therefore, we thought of the possibility of contamination of the protein with the DNase used in the first steps of the purification, which also has a similar pI (5.2), and it has a tendency to self-associate. Then, in subsequent purifications we did not add DNase to the cell lysate. As a consequence, the protein

coming from the Hi-Trap Mono Q showed absorbance at 260 nm, probably due to the presence of oligonucleotides resulting from the sonication step; these oligonucleotides must be present in a small amount, since no evidence of sharp peaks (contaminants of low-molecular weight) were observed in the 1D 1H-NMR spectrum (Figure 1). The presence of traces of oligonucleotides is not infrequent in proteins of the same family as it has been also observed in the recombinant HPF from *S. aureus* after its purification [5]. After elution from the Hi-Trap Mono Q column, the sample was extensively dialyzed against water, and no precipitation was observed in the dialysis tubing. The concentration of the protein Pc (mg/mL) was calculated by [45]: *P*<sup>c</sup> = 1.55 A280–0.75 A260, where A280 and A260 are the absorbances of the dialyzed solution of the protein at 280 and 260 nm, respectively.

### *4.3. Fluorescence*

Fluorescence spectra were collected on a Cary Varian spectrofluorimeter (Agilent, Santa Clara, CA, USA), interfaced with a Peltier, at 25 ◦C. The C-LrtA concentrations used were 5 and 10 μM of protein (in protomer units) in the GdmCl denaturations carried out at pH 7.5 (50 mM Tris buffer). A 1-cm-pathlength quartz cell (Hellma; Sigma Aldrich, Madrid, Spain) was used. In the denaturation experiments with GdmCl, we prepared the samples the day before starting from a 7 M GdmCl concentrated stock that equilibrated overnight; samples were left at 25 ◦C for 1 h before performing the measurements.

The emission intensity weighted average of the inverse wavelengths (also called the spectrum mass centre, or the spectral average energy of emission), <λ>, was calculated as described [46].

(a) Steady-state spectra: The experimental set-up used in the case of the denaturation experiments with GdmCl was as previously described [46]. In brief, excitation of the protein samples was at 278 nm, and excitation and emission slits were 5 nm in all cases. The experiments were recorded between 300 and 400 nm. The signal was acquired for 1 s and the increment of wavelength was set to 1 nm.

(b) Thermal denaturations: Thermal denaturations of isolated C-LrtA were carried out with the same experimental set-up previously described [46]. These experiments were performed at constant heating rates of 60 ◦C/h, with an average time of 1 s. Thermal scans were collected at 308 nm after excitation at 278 nm from 25 to 90 ◦C and acquired every 0.2 ◦C. Protein concentration was 10 μM (in protomer units).

(c) Fluorescence quenching: Quenching by iodide was examined with concentrations ranging from 5 to 40 μM (in protomer units) at pH 7.0 (phosphate buffer, 50 mM). The experimental set-up for KI was the same described above for the intrinsic fluorescence experiments. The data were fitted to [47]: *F*0/*F* = 1 + *Ksv*[*K I*], where *K*sv is the Stern-Volmer constant for collisional quenching; *F*<sup>0</sup> 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. Fittings to the above equation were carried out by using Kaleidagraph (Synergy software).

#### *4.4. CD*

The far-UV CD spectra were recorded at 25 ◦C on a Jasco J815 spectropolarimeter (Jasco, Easton, MD, USA City, Japan) equipped with a thermostated cell holder, and interfaced with a Peltier unit. A periodical calibration was performed with (+)-10-camphorsulphonic acid. We used two concentration values for C-LrtA (10 and 20 μM, in protomer units) to check for concentration-dependence of the shape and intensity of spectra, performed at pH 7.0 (50 mM, phosphate buffer). Molar ellipticity was determined as previously indicated [46].

(a) *Steady-state spectra*: Experiments were performed using the experimental set-up described previously [46]. Spectra were corrected by subtracting the baseline in all cases. Protein concentration was 10 and 20 μM (in protomer units) for GdmCl-denaturation experiments.

(b) Thermal denaturations: Experiments were carried out with the same experimental set-up described previously [46] and a protein concentration of 10 μM (in protomer units). Briefly, thermal denaturations were performed at a constant heating rate of 60 ◦C/h from 25 to 85 ◦C, a response time of 8 s, a band width of 1 nm, acquired every 0.2 ◦C, and following the ellipticity at 222 nm.

#### *4.5. NMR Spectroscopy*

The NMR experiments were acquired at 20 ◦C on a Bruker Avance DRX-500 spectrometer equipped with a triple resonance probe and z-pulse field gradients. All spectra were processed and analysed by using TopSpin 2.1 (Bruker GmbH, Karlsruhe, Germany). We used TSP as the external chemical shift reference [26].

(a) 1D 1H-NMR experiments: The 1D-1H-NMR spectrum were acquired with a C-LrtA concentration of 120 μM (in protomer units) in 0.5 mL, 50 mM d11-Tris buffer (pH 7.2) in H2O/D2O (90%/10%, *v*/*v*), without any correction for deuterium isotope effects. The spectrum was acquired with 16 K data points. We acquired 2 K scans with a 6000 Hz spectral width (12 ppm), by using the WATERGATE sequence [48]. Before processing the data, baseline correction and zero-filling were applied.

(b) Translational diffusion measurements: The DOSY experiments of C-LrtA at pH 7.2 were performed with the pulse-field gradient (PFG) spin-echo sequence, as described previously [12,46], with sixteen gradient strengths ranging linearly from 2% to 95% of the total power of the gradient unit. The intensity of the methyl signals, *I*, was fit to: *<sup>I</sup> <sup>I</sup>*<sup>0</sup> <sup>=</sup> <sup>−</sup>exp *D*γ<sup>2</sup> *H*δ2*G*<sup>2</sup> <sup>Δ</sup> <sup>−</sup> <sup>δ</sup> <sup>3</sup> <sup>−</sup> <sup>τ</sup> 2 , where *<sup>I</sup>*<sup>0</sup> is the maximum peak intensity of the methyl resonances at the smallest gradient strength; δ is the duration (in s) of the gradient (2.7 ms); *G* is the gradient strength (in T cm−1); Δ is the time (in s) between the gradients (150 ms); γ<sup>H</sup> is the gyromagnetic constant of the proton; and, τ is the recovery delay between the bipolar gradients (100 μs). Samples were exchanged in D2O buffer (50 mM d11-Tris buffer, pH 7.2, not corrected for isotope effects) by using Amicon centrifugal devices for 4 to 6 h.
