Nivel de Satisfacción según percepción de los estudiantes

Bovine β-Lg has a molecular weight of 18400 Da and a monomer-diameter of approximately 18 Å (Green et al., 1979; Verheul et al., 1999). It has 162 residues consisting of all the 20 amino acids in its primary sequence (Sawyer, 2003), and shows substantial homology with the sequence of lipocalins involved in the transport of hydrophobic ligands (Pervaiz et al., 1985). β-Lg has a free thiol group at Cys121 and two disulfide bonds, between Cys66-Cys160 and Cys106-Cys119 (Brownlow et al., 1997; Qin et al., 1998a).

β-Lg has a high proportion of β-sheets in its secondary structure comprising up to 50% of the protein conformation, the rest being 10% α-helix and 35% random coils determined from its far-UV (FUV) circular dichroism spectroscopy spectra (Qi et al., 1997). The β-sheets are primarily found on the 9 anti-parallel β-strands designated as

strands A to I, and their arrangement results in a three dimensional barrel-like structure also called the β-barrel or the β-calyx (Brownlow et al., 1997). The three- turn α-helix is located between β-strands H and E on the outside of the β-barrel in the C-terminal region of β-Lg. The random coils are mostly found on the structure- stabilizing flexible loops connecting the different β-strands in the structure (Jameson et al., 2002). The locations of the different elements of the tertiary structure are shown in Figure 2.4.

Figure 2.4 Sequence of β-Lg A (Variant B shows presence of glycine instead of aspartic acid at residue 64 and alanine instead of valine at residue 118)

adapted from the Protein Data Bank© (PDB ID 1BSO) (Qin et al.,

1998a). Dotted lines indicate disulfide bonding. Solid arrows indicate β-

strands; waveforms indicate helices and arches indicate loops.

The tertiary structure of β-Lg has been characterized in fine detail by different methods, including CD and NMR spectroscopy and X-ray crystallography. The near UV (NUV) CD spectrum of β-Lg is characterized by two sharp troughs at about 286 and 293 nm that are due to Trp19 (Manderson et al., 1999). Small troughs observed in the region between 262 and 269 nm are attributed to Phe residues (Strickland, 1974; Woody, 1978). The tertiary structure is stabilized by the two disulfide bonds between, Cys66-Cys160 linking the flexible CD loop to the outside of the β-barrel in

the C-terminal region, and Cys106-Cys119 linking the strands G and H (Brownlow et al., 1997; Qin et al., 1998a).

The free Cys121 residue is located on strand H and is hidden between strand H and the α-helix in the native β-Lg structure (Brownlow et al., 1997; Qin et al., 1998a). The reduction of the native disulfide bonds does not affect the native structure of β- Lg (Burova et al., 1998), however the effect of reduction followed by modification of cysteine residues in β-Lg depends on the modifying agent used (Iametti et al., 1996; Sakai et al., 2000). The modification of Cys121 in the native β-Lg alters its association properties (Iametti et al., 1996).

The stability of the native structure is a net result of the equilibrium between forces that stabilize the native structure e.g. hydrophobic interactions, hydrogen bonds or van der Walls interactions and those that oppose folding e.g. steric effects of side chain residues (Creighton, 1993; Damodaran, 1996). The hydrophobic non-polar residues remain buried inside the native structure (Tanford, 1991) which minimizes their thermodynamically unfavorable contact with the aqueous solution (Privalov et al., 1993). The polar residues are retained on the surface in contact with the aqueous solution stabilizing the globular structure (Damodaran, 1996).

β-Lg exhibits pH-dependent reversible structural transitions in its tertiary and quaternary structures. One of the most important changes in the tertiary structure occurs between pH 6 and 8 and is called the Tanford transition. This reversible transition in the native structure is characterized by a low sedimentation coefficient

of β-Lg (Pedersen, 1936), an increase in optical levo-rotation (Groves et al., 1951)

and change in the titration behavior of β-Lg (Tanford et al., 1959). Qin et al. (1998b) proposed that these changes resulted from the pH-dependent transitions of loop EF

(residues 85-90). At acidic pH, the EF loop is placed over the calyx, but rearranges itself by moving aside to expose the calyx at alkaline pH (Qin et al., 1998b). These authors hypothesized that this pH-dependent structural transition may have a role to play in protecting the ligands bound in the calyx from the acidic conditions of stomach (Qin et al., 1998b) since native β-Lg remains largely undigested by the proteolytic enzyme pepsin (Peram et al., 2013). The structural changes in the native structure of β-Lg at pH >8 are irreversible (Groves et al., 1951; Taulier et al., 2001). The quaternary structure of β-Lg also exhibits a pH-dependent association behavior. At low ionic strengths and neutral pH, β-Lg exists as stable non-covalently-linked dimer (Uhrinova et al., 2000) stabilized by hydrophobic interactions (Mercadante et al., 2012). Between pH 5.5 and 3.5, dimers associate to form octamers (Casal et al., 1988; McKenzie et al., 1967). Below pH 3.5 (Townend et al., 1969; Townend et al., 1960), and above 7.5 (McKenzie et al., 1967) the dimers dissociate and β-Lg primarily exists as monomers due to high electrostatic repulsion (Aymard et al., 1996; Mercadante et al., 2012; Molinari et al., 1996). These pH-dependent transitions in the quaternary structure are represented in Figure 2.5.

The factors affecting pH-dependent association of β-Lg include protein concentration (McKenzie, 1971; Verheul et al., 1999), genetic variation (Thresher et al., 1997), ionic strength of the medium (Aymard et al., 1996; Renard et al., 1998; Sakurai et al., 2001; Verheul et al., 1999) and temperature (Aymard et al., 1996; Verheul et al., 1999). Reduction of disulfide bonds, followed by their thiolation, produced β-Lg with modified properties. Thiolated β-Lg retained a native-like structure at acidic pH, but became partially unfolded at neutral pH (Sakai et al., 2000).

Figure 2.5 pH-dependent reversible association behavior of β-Lg. Adapted from Cheison et al. (2011).

At pH below its isoelectric point, the native structure of β-Lg shows high resemblance to that at neutral pH (Belloque et al., 1998; Casal et al., 1988; Matsuura et al., 1994; Uhrinova et al., 2000). The native structure of β-Lg is comparatively rigid at low pH imparting high structural stability (Boye et al., 1997; Jameson et al., 2002). Under these conditions, β-Lg retains the proportion of different elements of the secondary structures (Casal et al., 1988) and the three dimensional β-barrel fold (Ragona et al., 1997). However, minor transitions in the secondary structure of β-Lg have been noted at very low pH (< 2.0)(Taulier et al., 2001).