Yanke Chen

The Sugar Will Eventually be Exported Transporter (SWEET) family is a new class of transporters that plays crucial roles in the cellular sugar transport process. Their bacterial homologs, known as SemiSWEETs, are among the smallest transporters and can be used as a prototype for studying the biological properties of sugar transporters. Here, a set of dipolar-based multidimensional solid-state NMR spectra were employed to investigate the structure of Vibrio sp. SemiSWEET (Vs-SemiSWEET) reconstituted in the native-like lipid bilayers. A nearly complete (88% of the amino acid residues) backbone and side-chain ¹³C and ¹⁵ N chemical shift assignments of Vs-SemiSWEET were obtained. The overall secondary structure of Vs-SemiSWEET predicted from the obtained ¹³C and ¹⁵ N chemical shifts is similar to that from X-ray crystallography, with some differences, reflecting the influence of the membrane environments to the structure of membrane proteins.

Solid-state Nuclear Magnetic Resonance (ssNMR) is an emerging technique to investigate the structures and dynamics of membrane proteins in an artificial or native membrane environment. However, the structural studies of proteins by ssNMR are usually prolonged or impeded by signal assignments, especially the assignments of signals for collection of distance restraints, because of serious overlapping of signals in 2D ¹³C–¹³C spectra. Sparse labeling of ¹³C spins is an effective approach to simplify the ¹³C spectra and facilitate the extractions of distance restraints. Here, we propose a new reverse labeling combination of six types of amino acid residues (Ile, Leu, Phe, Trp, Tyr and Lys), and show a clean reverse labeling effect on a model membrane protein E. coli aquaporin Z (AqpZ). We further combine this reverse labeling combination and alternate ¹³C–¹²C labeling, and demonstrate an enhanced dilution effect in ¹³C–¹³C spectra. In addition, the influences of reverse labeling on the labeling of the other types of residues are quantitatively analyzed in the two strategies (1, reverse labeling and 2, reverse labeling combining alternate ¹³C–¹²C labeling). The signal intensities of some other types of residues in 2D ¹³C–¹³C spectra are observed to be 20–50% weaker because of the unwanted reverse labeling. The extensively sparse ¹³C labeling proposed in this study is expected to be useful in the collection of distance restraints using 2D ¹³C–¹³C spectra of membrane proteins.

Diacylglycerol kinase (DgkA) is a small integral membrane protein, responsible for the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid. Its structures reported in previous studies, determined in detergent micelles by solution NMR and in monoolein cubic phase by X-ray crystallography, differ significantly. These differences point to the need to validate these detergent-based structures in phospholipid bilayers. Here, we present a well-defined homo-trimeric structure of DgkA in phospholipid bilayers determined by magic angle spinning solid-state NMR (ssNMR) spectroscopy, using an approach combining intra-, inter-molecular paramagnetic relaxation enhancement (PRE)-derived distance restraints and CS-Rosetta calculations. The DgkA structure determined in lipid bilayers is different from the solution NMR structure. In addition, although ssNMR structure of DgkA shows a global folding similar to that determined by X-ray, these two structures differ in monomeric symmetry and dynamics. A comparative analysis of DgkA structures determined in three different detergent/lipid environments provides a meaningful demonstration of the influence of membrane mimetic environments on the structure and dynamics of membrane proteins.