3 research outputs found

    Antigenic Peptide Recognition on the Human ABC Transporter TAP Resolved by DNP-Enhanced Solid-State NMR Spectroscopy

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    The human transporter associated with antigen processing (TAP) is a 150 kDa heterodimeric ABC transport complex that selects peptides for export into the endoplasmic reticulum and subsequent loading onto major histocompatibility complex class I molecules to trigger adaptive immune responses against virally or malignantly transformed cells. To date, no atomic-resolution information on peptideā€“TAP interactions has been obtained, hampering a mechanistic understanding of the early steps of substrate translocation catalyzed by TAP. Here, we developed a mild method to concentrate an unstable membrane protein complex and combined this effort with dynamic nuclear polarization enhanced magic angle spinning solid-state NMR to study this challenging membrane proteinā€“substrate complex. We were able to determine the atomic-resolution backbone conformation of an antigenic peptide bound to human TAP. Our NMR data also provide unparalleled insights into the nature of the interactions between the side chains of the antigen peptide and TAP. By combining NMR data and molecular modeling, the location of the peptide binding cavity has been identified, revealing a complex scenario of peptideā€“TAP recognition. Our findings reveal a structural and chemical basis of substrate selection rules, which define the crucial function of this ABC transporter in human immunity and health. This work is the first NMR study of a eukaryotic transporter protein and presents the power of solid-state NMR in this growing field

    Hostā€“Guest Complexes as Water-Soluble High-Performance DNP Polarizing Agents

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    Dynamic nuclear polarization (DNP) enhances the sensitivity of solid-state NMR (SSNMR) spectroscopy by orders of magnitude and, therefore, opens possibilities for novel applications from biology to materials science. This multitude of opportunities implicates a need for high-performance polarizing agents, which integrate specific physical and chemical features tailored for various applications. Here, we demonstrate that for the biradical bTbK in complex with captisol (CAP), a Ī²-cyclodextrin derivative, hostā€“guest assembling offers a new and easily accessible approach for the development of new polarizing agents. In contrast to bTbK, the CAP-bTbK complex is water-soluble and shows significantly improved DNP performance compared to the commonly used DNP agent TOTAPOL. Furthermore, NMR and EPR data reveal improved electron and nuclear spin relaxation properties for bTbK within the host molecule. The numerous possibilities to functionalize host molecules will permit designing novel radical complexes targeting diverse applications

    Interaction of Cisplatin with Human Superoxide Dismutase

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    <i>cis</i>-DiamminedichloroplatinumĀ­(II) (cisplatin) is able to interact with human superoxide dismutase (hSOD1) in the disulfide oxidized apo form with a dissociation constant of 37 Ā± 3 Ī¼M through binding cysteine 111 (Cys111) located at the edge of the subunit interface. It also binds to Cu<sub>2</sub>ā€“Zn<sub>2</sub> and Zn<sub>2</sub>ā€“Zn<sub>2</sub> forms of hSOD1. Cisplatin inhibits aggregation of demetalated oxidized hSOD1, and it is further able to dissolve and monomerize oxidized hSOD1 oligomers <i>in vitro</i> and <i>in cell</i>, thus indicating its potential as a leading compound for amyotrophic lateral sclerosis
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