RESUMO
Amyloid light-chain (AL) amyloidosis is a protein-misfolding disease characterized by accumulation of immunoglobulin light chains (LCs) into amyloid fibrils. Dimerization of a full length or variable domain (VL ) of LC serves to stabilize the native state and prevent the formation of amyloid fibrils. We here analyzed the thermodynamic properties of dimerization and unfolding reactions by nonamyloidogenic VL from REI LC or its monomeric Y96K mutant using sedimentation velocity and circular dichroism. The data indicate that the equilibrium shifts to native dimerization for wild-type REI VL by elevating temperature due to the negative enthalpy change for dimer dissociation (-81.2 kJ·mol-1 ). The Y96K mutation did not affect the stability of the monomeric native state but increased amyloidogenicity. These results suggest that the heat-induced native homodimerization is the major factor preventing amyloid formation by wild-type REI VL . Heat-induced native oligomerization may be an efficient strategy to avoid the formation of misfolded aggregates particularly for thermostable proteins that are used at elevated temperatures under conditions where other proteins tend to misfold. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 5XP1 and 5XQY.
Assuntos
Amiloide/química , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Sequência de Aminoácidos , Amiloide/genética , Amiloide/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Temperatura Alta , Humanos , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Cinética , Modelos Moleculares , Mutação , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Desdobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica , Difração de Raios XRESUMO
Protein disulfide isomerase (PDI) is a multifunctional protein that catalyzes the formation of a disulfide bond in nascent and misfolded proteins and is also known to bind to the thyroid hormone triiodothyronine (T3). When T3 is bound to PDI its catalytic activity is inhibited, but the biological function of this binding is not well understood. In previous studies, it was found that T3 binds to the bb' fragment of PDI. Therefore, to clarify the structure of the complex consisting of PDI bound to T3, a crystallographic analysis of the three-dimensional structure of the T3-rat PDI bb' complex was performed. Native bb' crystals and T3-bb' complex crystals were both obtained using the hanging-drop vapour-diffusion technique with 1.6 M trisodium citrate pH 6.2 as a precipitant. The space group of the native bb' crystals was found to be C222, with unit-cell parameters a = 94.8, b = 114.9, c = 182.9 Å, while the space group of the T3-bb' complex crystals was P2(1)2(1)2(1), with unit-cell parameters a = 99.9, b = 184.5, c = 232.2 Å. Diffraction data for the native and complex crystals were collected to resolutions of 3.06 and 3.00 Å, respectively.
