Structure of two crystal forms of sheep β‐lactoglobulin with EF‐loop in closed conformation

Abstract

Ovine β-lactoglobulin has been isolated from whey fraction of sheep milk and crystallized. The high-resolution structures of two crystal forms (triclinic and trigonal) obtained at pH 7.0 have been determined revealing that ovine protein, similarly to its bovine analog, is dimeric. Access to the binding site located in the eight-stranded antiparallel β-barrel in both structures is blocked by the EF loop that has been found in closed conformation. Similarly to bovine lactoglobulin (BLG), conformation of the EF loop is stabilized by hydrogen bond between Glu89 and Ser116 indicating that Tanford transition might occur with the same mechanism. The substitution at six positions in relation to the most abundant isoform B of BLG also affects the distribution of electrostatic potentials and the total charge.

Ovine β-lactoglobulin at atomic resolution

“…Recently, Loch et al (2014) have published the structures of two crystal forms of ovine -Lg grown at high pH: a trigonal form that is identical to that reported by Rocha et al (1996) and a triclinic form (PDB entry 4nlj) that is distinct from both the bovine triclinic form (Brownlow et al, 1997) and that reported here. Interestingly, their high-pH forms both have the EF-loop in the closed position.…”

“…The space group was determined to be P1, with unit-cell parameters a = 37.40, b = 49.36, c = 49.42 Å , = 69.66, = 68.98, = 77.67 . The unit-cell parameters and the space group of the ovine -Lg crystals were distinct from those found previously for either the bovine protein (Brownlow et al, 1997) or the ovine protein (Rocha et al, 1996;Loch et al, 2014) and could not be transformed into a higher symmetry space group.…”

“…Interestingly, their high-pH forms both have the EF-loop in the closed position. As pointed out by Lewiń ski and coworkers (Loch et al, 2014), the same buried Glu89 is present in the ovine protein, so that it is likely that a similar pH-dependent transition to the transition reported by Tanford and coworkers (Tanford et al, 1959;Sakurai et al, 2009) for the bovine protein will occur in the ovine protein. To the best of our knowledge, no such solution studies have been carried out for the ovine protein as they have for the porcine protein (Ragona et al, 2003), in which the transition is shifted to significantly higher pH.…”

“…The structure has been determined from these data collected at around 100 K in the hope that clear density might emerge for the less well defined regions in other electrondensity maps of -Lg. Loch et al (2014) have recently published two high-pH structures of the sheep protein at significantly lower resolution than that presented here. A brief comparison of the triclinic form (PDB entry 4nlj) will be made here.…”

The pH, temperature, and protein structure effect on β‐lactoglobulin A and B separation in anion‐exchange chromatography

“…Along with these findings, looking at the tertiary structure of the protein for the position of the amino acid residue in the chain provides important insight as to what gives rise to the separation. The Protein Data Bank has the β ‐lactoglobulin A, and β ‐lactoglobulin B, the tertiary structure of the proteins was observed. Position 64 is on a turn in on the outside and position 118 is on a beta strand inside the core of the protein.…”