Ligand binding and self‐association cooperativity of β‐lactoglobulin

Abstract

Unlike most small globular proteins, lipocalins lack a compact hydrophobic core. Instead, they present a large central cavity that functions as the primary binding site for hydrophobic molecules. Not surprisingly, these proteins typically exhibit complex structural dynamics in solution, which is intricately modified by intermolecular recognition events. Although many lipocalins are monomeric, an increasing number of them have been proven to form oligomers. The coupling effects between self-association and ligand binding in these proteins are largely unknown. To address this issue, we have calorimetrically characterized the recognition of dodecyl sulfate by bovine β-lactoglobulin, which forms weak homodimers at neutral pH. A thermodynamic analysis based on coupled-equilibria revealed that dimerization exerts disparate effects on the ligand-binding capacity of β-lactoglobulin. Protein dimerization decreases ligand affinity (or, reciprocally, ligand binding promotes dimer dissociation). The two subunits in the dimer exhibit a positive, entropically driven cooperativity. To investigate the structural determinants of the interaction, the crystal structure of β-lactoglobulin bound to dodecyl sulfate was solved at 1.64 Å resolution.

A Thermodynamic Model of Auto‐regulated Protein Assembly by a Supramolecular Scaffold

“…The use of Bayesian MCMC and simultaneous fitting of the model to both isotherms facilitated robust determination of the multiple affinities and enthalpies of the model. There is increasing interest in thermodynamic binding models that treat multiple bodies ,,,,,,,,. By generalizing the model developed here we have provided an approach that may be useful to describe other auto‐regulated systems.…”

“…There is increasing interest in thermodynamic binding models that treat multiple bodies. [4,20,23,25,28,29,34,36,[38][39][40][41] By generalizing the model developed here we have provided an approach that may be useful to describe other auto-regulated systems. The anionic sclx 8 prompts the assembly of two cationic proteins, cytochrome c [11] and PAF, [22] suggesting its utility as a generic mediator of auto-regulated assembly for cationic proteins.…”

Ligand entry into the calyx of β‐lactoglobulin

“…The entire scaffold of βlg tends to be very flexible. The loops flanking the cavity entrance are particularly mobile in the apo form (C‐D, E‐F, and G‐H) . In contrast, greater conformational fluctuation has been observed for loop A‐B in the free monomeric from than in the dimeric form .…”

Ovine β-lactoglobulin at atomic resolution

“…Of all the -Lg structures in the PDB (http://www.rcsb.org), the current structure has (just) the highest resolution [1.12 Å , with the next being the 1.17 Å PDB entry 4tlj and then the 1.64 Å PDB entry 4gny (Gutierrez-Magdaleno et al, 2013)] and a good R factor of 0.139 (R = 0.132 for PDB entry 4tlj, with the next best R factor being 0.181 for PDB entry 1beb) and R free = 0.169 (R free = 0.159 for PDB entry 4tlj, with the next best R free being 0.218 for PDB entry 4nlj). Despite this high resolution, it is clear that there is still some mobility in the structure at 100 K. The current structure appears to have the highest number of alternative conformations of all -Lg structures in the PDB, reflecting the real mobility of the structure.…”