title: Conformational stabilization of the membrane embedded targeting domain of the lysosomal peptide transporter TAPL for solution NMR.
abstract: The ATP binding cassette transporter TAPL translocates cytosolic peptides into the lumen of lysosomes driven by the hydrolysis of ATP. Functionally, this transporter can be divided into coreTAPL, comprising the transport function, and an additional N-terminal transmembrane domain called TMD0, which is essential for lysosomal targeting and mediates the interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. To elucidate the structure of this unique domain, we developed protocols for the production of high quantities of cell-free expressed TMD0 by screening different N-terminal expression tags. Independently of the amino acid sequence, high expression was detected for AU-rich sequences in the first seven codons, decreasing the free energy of RNA secondary structure formation at translation initiation. Furthermore, avoiding NGG codons in the region of translation initiation demonstrated a positive effect on expression. For NMR studies, conditions were optimized for high solubilization efficiency, long-term stability, and high quality spectra. A most critical step was the careful exchange of the detergent used for solubilization by the detergent dihexanoylphosphatidylcholine. Several constructs of different size were tested in order to stabilize the fold of TMD0 as well as to reduce the conformation exchange. NMR spectra with sufficient resolution and homogeneity were finally obtained with a TMD0 derivative only modified by a C-terminal His10-tag and containing a codon optimized AT-rich sequence.
authors.affiliation: Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue-Str. 9, 60438, Frankfurt am Main, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Tumulka
authors.forename: Christian
authors.initials: C
authors.lastname: Roos
authors.forename: Frank
authors.initials: F
authors.lastname: Löhr
authors.forename: Christoph
authors.initials: C
authors.lastname: Bock
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.forename: Rupert
authors.initials: R
authors.lastname: Abele
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D002474:Cell-Free System; D003062:Codon / Q000235:genetics; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000737:chemistry; D012995:Solubility; D012996:Solutions
chemical_list: C412573:ABCB9 protein, human; D018528:ATP-Binding Cassette Transporters; D003062:Codon; D008565:Membrane Proteins; D012333:RNA, Messenger; D012996:Solutions