RESUMO
The 14-kDa sterol carrier protein 2 (SCP2) domain is present in Eukaria, Bacteria and Archaea, and has been implicated in the transport and metabolism of lipids. We report the cloning, expression, purification and physicochemical characterization of a SCP2 from the yeast Yarrowia lipolytica (YLSCP2). Analytical size-exclusion chromatography, circular dichroism and fluorescence spectra, indicate that recombinant YLSCP2 is a well-folded monomer. Thermal unfolding experiments show that SCP2 maximal stability is at pH 7.0-9.0. YLSCP2 binds cis-parinaric acid and palmitoyl-CoA with KD values of 81+/-40 nM and 73+/-33 nM, respectively, sustaining for the first time the binding of fatty acids and their CoA esters to a nonanimal SCP2. The role of yeast SCP2 and other lipid binding proteins in transport, storage and peroxisomal oxidation of fatty acids is discussed.
Assuntos
Acil-CoA Desidrogenase/química , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Ácidos Graxos/química , Modelos Químicos , Modelos Moleculares , Yarrowia/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/análise , Simulação por Computador , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Acyl-CoA binding protein (ACBP) and fatty acid binding protein (FABP) are intracellular transporters of activated and free fatty acids, respectively. Unlike other tissues with active lipid metabolism, armadillo Harderian gland contains much more ACBP than FABP. To characterize armadillo ACBP structure and binding properties, we produced it in Escherichia coli and carried out detailed fluorescence and circular dichroism spectroscopy studies. The K(D) for palmitoyl-CoA, measured directly by fluorescence and rotatory power, was 34+/-12 and 75+/-39 nM, respectively. The structure of armadillo ACBP appears to be very similar to that of bovine and rat liver ACBPs.