Estudo de difração de raios-x a alta resolução da beta-lactoglobulina bovina
AUTOR(ES)
Kesley Moraes Godinho de Oliveira
DATA DE PUBLICAÇÃO
1999
RESUMO
Beta-lactoglobulin (BLG), a very abundant protein found in several species of mammals belongs to the lipocalin family. Under physiological conditions, BLG is a dimmer of 162 aminoacids per monomer, with a molecular mass of approximately 18kDa in each subunit. Although BLG has important physicochemical properties, its biological function remains unclear. BLG undergoes a number of conformational changes near pH 7,5 as indicated by specific optical rotation and titration curve. This transition, termed Tanford transition, defined the first goal of this work: to solve crystallographic structures of BLG near acidic transition. Bovine milk has many genetic variants of BLG, but the most prevalents are variants A and B. Variant A differs in the amino acid sequence at positions 64 (Asp® Gly) and 118 (Val® Ala) from variant B. These aminoacid changes cause differences in physicochemical properties of these variants and influence milk processing and milk derivatives. The second goal of this work was to solve the crystallographic structures of variants A and B with the purpose of exploring possible variants of structural origin, which could justify the physicochemical differences between both of them. Crystals of beta-lactoblobulin were grown under acidic and basic pH. Under acidic pH, the crystal was obtained from a mixture of isoforms A and B, and belongs to space group P1. Under basic pH, the crystals belong to space group C2221 and were grown from the mixture A/B, variant A and variant B. Crystals were exposed to X-rays and diffraction data were obtained. The crystal structures of beta-lactoglobulin were solved by the molecular replacement method and the initial model obtained was refined. The mixture of isoforms A/B (P1), under acidic pH, was determined at 1,6 Å resolution and refined to a crystallographic R factor of 19,8% (Rfree=25,0%) with 171 solvent molecules. The corresponding structure in basic pH was determined at 2,0 Å resolution and refined to a crystallographic R factor of 19,4% (Rfree=27,2%) with 135 solvent molecules. The crystal structures of variants A and B were determined at 2,0 Å e 1,95 Å resolution and refined to an crystallographic R factor of 19,2% (Rfree=28,7% and 123 solvent molecules) and 18,9% (Rfree=25,2% and 107 solvent molecules) respectively. The comparison between structures solved under different pHs allowed the visualization of the effect caused by Tanford transition: two loops of the structure formed by residues 84 to 90 and 123 to 131 open up and expose the carbonyl groups of residues Glu 89 and Asp 129. These two residues are considered responsible for the anomalous behavior observed to BLG when it is in pH 7,5. The superposition of the structure in the variants A and B revealed that residues 108 to 117 adopt a different conformation in the variants. Another aspect that was observed is the creation of an internal cleft close to residue 118, due to the change of one Valine by an Alanine. This cleft may be related to the stability of these variants
ASSUNTO(S)
raios x - difração proteinas cristalografia
