PROCEDIMIENTO PARA LA SOLICITUD Y VALIDACIÓN DE PERMISOS Y AUSENCIAS (RESOLUCIÓN 171 DE 2009 Y RESOLUCIÓN 1740 DE 2009)

While our attempts to express the entire NS protease (aa 1000-1300 of P150 were fruitless, we successfully expressed as a GST-fusion protein and purified the minimal metal binding domain (RUBCa, aa 1143-1252, see Fig. 5.2A), which contains the Ca2+ binding loop, both catalytic residues, and the ligands proposed to be involved in Zn2+ binding. Purified RUBCa was used to characterize the predicted EF-hand Ca2+-binding motif in its native protein environment. When purified RUBCa was reconstituted in the presence of Ca2+ and subjected to ICP-MS to measure its metal content, it was found to bind Ca2+ with a molar ratio (Ca2+/protein) of 0.7 ± 0.2 to 1 (n=2).

RUBCa contains three Trp residues and according to the model of the structure of the RUB NS protease (Figure. 5.2C), one of these aromatic residues, W1153, is in close proximity to the predicted Ca2+-binding loop (3.5 Å). This feature enabled us to probe the metal-binding properties of RUBCa using Tb3+- FRET. As shown in Figure 5A, a significant enhancement of Tb3+ fluorescence intensity was observed when Tb3+ was added to RUBCa, indicating Tb3+ binding to the predicted loop. A Kd for Tb3+ of 3±1 μM was obtained (inset, Fig. 5.7A).

In comparison with the 355 nm for free Trp, the emission maximum of the purified RUBCa blue-shifted to 339 nm, suggesting that at least some Trp in RUBCa was partly shielded from the solvent though not fully buried inside the hydrophobic core (Fig. 5.7B). The shoulder at 355 nm suggests that some Trp residues were exposed to the solvent. No Ca2+-induced emission peak position

Figure 5.7. Metal ions titration of the minimal metal-binding domain RUBCa monitored by aromatic residue sensitized Tb3+ fluorescence (A) and intrinsic Trp fluorescence (B). (A), Normalized Tb3+ fluorescence spectra of RUBCa with increasing concentration of Tb3+ (from bottom to top: 0, 1.0, 4.0, 9.9, 14.8, 19.6, 14.4 μM, respectively). The inset showed the Tb3+ fluorescence enhancement at 545 nm due to energy transfer as a function of the concentration of Tb3+. (B), Intrinsic Trp fluorescence emission spectra of RUBCa (2.5 μM) with increasing concentration of Ca2+

(From top to bottom: 0, 49.8, 291.3, 566.0, 740.7 and 909.1 μM, respectively). Inset: the intrinsic Trp fluorescence intensity plotted as a function of the concentration of Ca2+. An average dissociation constant of 316 μM was obtained by assuming a 1:1 binding model. The excitation wavelength was set at 282 nm. All the buffers used in metal titration consist of 20 mM PIPES, 10 mM KCl, pH 6.8.

change was observed. However, the addition of Ca2+ led to a decrease in the emission intensity, indicating the local changes of the chemical environment around the aromatic residues (Fig. 5.7B). By monitoring this intensity change, a dissociation constant of 316 ± 4 µM for Ca2+ was obtained (Fig. 5.7B, inset), which was in agreement with the Ca2+-binding affinity obtained using the grafted protein CD2.RUBCa (Table 5.1).

Far UV CD was performed to reveal any possible changes of secondary structure of RUBCa induced by Ca2+. As seen in Fig. 5.8A, the spectra of both EGTA-treated and Ca2+-loaded RUBCa had two troughs at 222 and 206 nm, indicating the existence of α-helical secondary structure. Using the program DICHROWEB (66), the best fit of the CD spectrum of RUBCa indicated that 17.8% was α-helix and 19.5% was β-sheet, whereas the remainder was random coil. In excess Ca2+, the CD signal of RUBCa at 208 nm and 222 nm was 5% more negative than that of RUBCa in 1 mM EGTA and the DICHROWEB- predicted α-helix and β-sheet contents were 23.8% and 14.9%, respectively. Thus, the observed gain in negative ellipticity could be attributed to the formation of a higher degree of α-helical content induced by Ca2+

binding. The anionic amphiphile ANS was further used as a hydrophobic probe to examine the conformational properties of RUBCa. As shown in Fig. 5.8B, upon the addition of RUBCa, the emission peak of ANS fluorescence blue-shifted from 510 nm to 500 nm and the maximal emission intensity increased by 30%, suggesting that part of the hydrophobic regions of the purified RUBCa were exposed to the solvent and thus accessible to ANS. The addition of excess Ca2+ did not cause significant

Figure 5.8. Ca2+-induced conformational changes and thermal unfolding of the putative Ca2+-binding domain RUBCa. (A), Far UV CD spectra of RUBCa with 1 mM EGTA (open circle) or 1 mM Ca2+ (closed circle) in 10 mM Tris-HCl, 10 mM KCl. Inset: Normalized CD signal at 222 nm plotted as a function of increasing temperature (5-90 ºC) in the presence of 1 mM EGTA (open circle) or 1 mM Ca2+ (closed circle). (B), Fluorescence emission spectra of 40 μM ANS (open square) and ANS:RUBCa complex with 1 mM EGTA (open circle) or 1 mM Ca2+ (closed circle). The excitation wavelength was set at 390 nm. The buffer consists of 10 mM Tris-HCl, 10 mM KCl (pH 7.4).

conformational changes in these hydrophobic regions considering the overlapping emission fluorescence spectra of RUBCa in the presence of 1 mM EGTA or 1 mM Ca2+. Taken together, these data indicated that the binding of Ca2+ induced a local conformational change, whereas the secondary structure and hydrophobic surface were not significantly altered.

In order to gain more insight into the possible role of Ca2+ binding, thermal unfolding was carried out by monitoring the CD signal change at 222 nm as a function of temperature under Ca2+-depleted or Ca2+-loaded conditions. With increasing temperature from 5 to 90 °C, RUBCa gradually underwent thermal denaturation, leading to the decrease of CD signals. Compared with 1 mM EGTA, the melting temperature (Tm) of RUBCa with Ca2+ increased from 37.7 ±

0.8 to 41.8 ± 0.4 °C (Fig. 5.8B). The observed increase of melting temperature suggested that the binding of Ca2+ stabilized the overall structure of RUBCa under physiological conditions (100 mM KCl).

5.2.7. Mutating the Potential Ca2+-Binding Ligands in Infectious cDNA