title: Fluorinated Aromatic Amino Acids Distinguish Cation-π Interactions from Membrane Insertion.
abstract: Cation-π interactions, where protein aromatic residues supply π systems while a positive-charged portion of phospholipid head groups are the cations, have been suggested as important binding modes for peripheral membrane proteins. However, aromatic amino acids can also insert into membranes and hydrophobically interact with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-π interactions from membrane insertion of the aromatic side chains. Fluorinated aromatic amino acids destabilize the cation-π interactions by altering electrostatics of the aromatic ring, whereas their increased hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis phosphatidylinositol-specific phospholipase C definitively identifies those involved in cation-π interactions with phosphatidylcholine. This powerful method can easily be used to determine the roles of aromatic residues in other peripheral membrane proteins and in integral membrane proteins.
authors.affiliation: From the Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467.
authors.forename: Tao
authors.initials: T
authors.lastname: He
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, Massachusetts 01003.
authors.forename: Anne
authors.initials: A
authors.lastname: Gershenson
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, Massachusetts 01003.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Eyles
authors.affiliation: Department of Chemistry, Texas A&M University, College Station, Texas 77843, and.
authors.forename: Yan-Jiun
authors.initials: YJ
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Texas A&M University, College Station, Texas 77843, and.
authors.forename: Wenshe R
authors.initials: WR
authors.lastname: Liu
authors.affiliation: Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, China.
authors.forename: Jiangyun
authors.initials: J
authors.lastname: Wang
authors.affiliation: From the Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467.
authors.forename: Jianmin
authors.initials: J
authors.lastname: Gao
authors.affiliation: From the Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, mary.roberts@bc.edu.
authors.forename: Mary F
authors.initials: MF
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002412:Cations; D018360:Crystallography, X-Ray; D057927:Hydrophobic and Hydrophilic Interactions; D008563:Membrane Lipids / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry*; D054801:Phosphoinositide Phospholipase C / Q000737:chemistry*; D011485:Protein Binding; D021381:Protein Transport; D013211:Staphylococcus aureus / Q000201:enzymology; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D002412:Cations; D008563:Membrane Lipids; C025200:3,5-difluorotyrosine; D014443:Tyrosine; D010649:Phenylalanine; D054801:Phosphoinositide Phospholipase C
keywords: 3,5-difluorotyrosine; cation-π interactions; crystal structure; lipid-protein interaction; pentafluorophenylalanine; phosphatidylcholine; phospholipase C; site-directed mutagenesis