RESUMO
ß-Barrel membrane proteins have regular structures with extensive hydrogen-bond networks between their transmembrane (TM) ß-strands, which stabilize their protein fold. Nevertheless, weakly stable TM regions, which are important for the protein function and interaction with other proteins, exist. Here, we report on the apparent stability of human Tom40A, a member of the "mitochondrial porin family" and main constituent of the mitochondrial protein-conducting channel TOM (translocase of the outer membrane). Using a physical interaction model, TmSIP, for ß-barrel membrane proteins, we have identified three unfavorable ß-strands in the TM domain of the protein. Substitution of key residues inside these strands with hydrophobic amino acids results in a decreased sensitivity of the protein to chemical and/or thermal denaturation. The apparent melting temperature observed when denatured at a rate of 1 °C per minute is shifted from 73 to 84 °C. Moreover, the sensitivity of the protein to denaturant agents is significantly lowered. Further, we find a reduced tendency for the mutated protein to form dimers. We propose that the identified weakly stable ß-strands 1, 2 and 9 of human Tom40A play an important role in quaternary protein-protein interactions within the mammalian TOM machinery. Our results show that the use of empirical energy functions to model the apparent stability of ß-barrel membrane proteins may be a useful tool in the field of nanopore bioengineering.
Assuntos
Proteínas de Membrana Transportadoras/química , Proteínas Mitocondriais/química , Substituição de Aminoácidos/genética , Dicroísmo Circular , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Desnaturação Proteica/efeitos dos fármacos , Desnaturação Proteica/efeitos da radiação , Multimerização Proteica , Estrutura Secundária de Proteína , Temperatura de TransiçãoRESUMO
Tom40 proteins represent an essential class of molecules which facilitate translocation of unfolded proteins from the cytosol into the mitochondrial intermembrane space. They are part of a high-molecular mass complex that forms the protein-conducting channel in outer mitochondrial membranes. This study concerns the recombinant expression, purification and folding of amino-terminally truncated variants of the two human Tom40 isoforms for structural biology experiments. Both CD and FTIR secondary structure analysis revealed a dominant beta-sheet structure and a short alpha-helical part for both proteins together with a high thermal stability. Two secondary structure elements can be denatured independently. Reconstitution of the recombinant protein into planar lipid bilayers demonstrated ion channel activity similar to Tom40 purified from Neurospora crassa mitochondrial membranes, but conductivity fingerprints differ from the structurally closely related VDAC proteins.
Assuntos
Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Dicroísmo Circular , Eletrofisiologia , Humanos , Bicamadas Lipídicas/metabolismo , Proteínas Mitocondriais/genética , Dobramento de Proteína , Isoformas de Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
In the present work we applied a novel mass spectrometry method termed laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS) to the outer mitochondrial membrane protein translocon TOM to analyze its subunit composition and stoichiometry. With TOM core complex, purified at high pH, we demonstrate that a TOM core complex of Neurospora crassa is composed of at least two Tom40 and Tom22 molecules, respectively, and more than five small Tom subunits between 5.5 and 6.4 kDa. We show that the multiprotein complex has a total molecular mass higher than 170 depending on the number of Tom5, Tom6 and Tom7 molecules bound.
