[NMR paper] Conformational stabilization of the membrane embedded targeting domain of the lysosomal peptide transporter TAPL for solution NMR.

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Conformational stabilization of the membrane embedded targeting domain of the lysosomal peptide transporter TAPL for solution NMR.

Conformational stabilization of the membrane embedded targeting domain of the lysosomal peptide transporter TAPL for solution NMR.

J Biomol NMR. 2013 Sep 7;

Authors: Tumulka F, Roos C, Löhr F, Bock C, Bernhard F, Dötsch V, Abele R

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.


PMID: 24013930 [PubMed - as supplied by publisher]



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