title: Improving the resistance of a eukaryotic β-barrel protein to thermal and chemical perturbations.
abstract: β-Barrel membrane proteins have regular structures with extensive hydrogen-bond networks between their transmembrane (TM) β-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 β-barrel membrane proteins, we have identified three unfavorable β-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 β-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 β-barrel membrane proteins may be a useful tool in the field of nanopore bioengineering.
authors.affiliation: Biophysics Department, Institute of Biology, University of Stuttgart, Pfaffenwaldring 57, 70550 Stuttgart, Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Gessmann
authors.forename: Frauke
authors.initials: F
authors.lastname: Mager
authors.forename: Hammad
authors.initials: H
authors.lastname: Naveed
authors.forename: Thomas
authors.initials: T
authors.lastname: Arnold
authors.forename: Sara
authors.initials: S
authors.lastname: Weirich
authors.forename: Dirk
authors.initials: D
authors.lastname: Linke
authors.forename: Jie
authors.initials: J
authors.lastname: Liang
authors.forename: Stephan
authors.initials: S
authors.lastname: Nussberger
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D002942:Circular Dichroism; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics; D011489:Protein Denaturation / Q000187:drug effects / Q000528:radiation effects; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D044366:Transition Temperature
chemical_list: D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D050505:Mutant Proteins; C400783:TOMM40 protein, human