TY  - JOUR
A1  - Herget, Meike
A1  - Kreiβig, Nina
A1  - Kolbe, Christian
A1  - Schölz, Christian
A1  - Tampé, Robert
A1  - Abele, Rupert
T1  - Purification and reconstitution of the antigen transport complex TAP: a prerequisite for determination of peptide stoichiometry and ATP hydrolysis
T2  - Journal of biological chemistry
N2  - The transporter associated with antigen processing (TAP) is an essential machine of the adaptive immune system that translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen for loading of major histocompatibility class I molecules. To examine this ABC transport complex in mechanistic detail, we have established, after extensive screening and optimization, the solubilization, purification, and reconstitution for TAP to preserve its function in each step. This allowed us to determine the substrate-binding stoichiometry of the TAP complex by fluorescence cross-correlation spectroscopy. In addition, the TAP complex shows strict coupling between peptide binding and ATP hydrolysis, revealing no basal ATPase activity in the absence of peptides. These results represent an optimal starting point for detailed mechanistic studies of the transport cycle of TAP by single molecule experiments to analyze single steps of peptide translocation and the stoichiometry between peptide transport and ATP hydrolysis.
Y1  - 2021
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/76437
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-764376
SN  - 0021-9258
VL  - 284.2009
IS  - 49
SP  - 33740
EP  - 33749
PB  - American Society for Biochemistry and Molecular Biology Publications
CY  - Bethesda, Md
ER  -