Based on the saposin-A (SapA) scaffold protein, we demonstrate the suitability of a size-adaptable phospholipid membrane-mimetic system for solution NMR studies of membrane proteins (MPs) under close-to-native conditions. The Salipro nanoparticle size can be tuned over a wide pH range by adjusting the saposin-to-lipid stoichiometry, enabling maintenance of sufficiently high amounts of phospholipid in the Salipro nanoparticle to mimic a realistic membrane environment while controlling the overall size to enable solution NMR for a range of MPs. Three representative MPs, including one G-protein-coupled receptor, were successfully incorporated into SapA-dimyristoylphosphatidylcholine nanoparticles and studied by solution NMR spectroscopy.
In this study, we report the structure and function of a lectin from the sea mollusk Crenomytilus grayanus collected from the sublittoral zone of Peter the Great Bay of the Sea of Japan. The crystal structure of C. grayanus lectin (CGL) was solved to a resolution of 1.08 Å, revealing a β-trefoil fold that dimerizes into a dumbbell-shaped quaternary structure. Analysis of the crystal CGL structures bound to galactose, galactosamine, and globotriose Gb3 indicated that each CGL can bind three ligands through a carbohydrate-binding motif involving an extensive histidine- and water-mediated hydrogen bond network. CGL binding to Gb3 is further enhanced by additional side-chain-mediated hydrogen bonds in each of the three ligand-binding sites. NMR titrations revealed that the three binding sites have distinct microscopic affinities toward galactose and galactosamine. Cell viability assays showed that CGL recognizes Gb3 on the surface of breast cancer cells, leading to cell death. Our findings suggest the use of this lectin in cancer diagnosis and treatment.
Recent studies on the mechanisms by which topologically knotted proteins attain their natively knotted structures have intrigued theoretical and experimental biophysicists. Of particular interest is the finding that YibK and YbeA, two small trefoil knotted proteins, remain topologically knotted in their chemically denatured states. Using small-angle X-ray scattering (SAXS), we examine whether these chemically denatured knotted proteins are different from typical random coils. By revisiting the scaling law of radius of gyration (Rg) as a function of polypeptide chain length for chemically denatured proteins and natively folded proteins, we find that the chemically denatured knotted proteins in fact follow the same random coil-like behavior, suggesting that the formation of topological protein knots do not necessarily require global compaction while the loosely knotted polypeptide chains are capable of maintaining the correct chirality without defined secondary or tertiary structures.
Galectins are β-galactoside-binding proteins implicated in a myriad of biological functions. Despite their highly conserved carbohydrate binding motifs with essentially identical structures, their affinities for lactose, a common galectin inhibitor, vary significantly. Here, we aimed to examine the molecular basis of differential lactose affinities amongst galectins using solution-based techniques. Consistent dissociation constants of lactose binding were derived from nuclear magnetic resonance (NMR) spectroscopy, intrinsic tryptophan fluorescence, isothermal titration calorimetry and bio-layer interferometry for human galectin-1 (hGal1), galectin-7 (hGal7), and the N-terminal and C-terminal domains of galectin-8 (hGal8NTD and hGal8CTD, respectively). Furthermore, the dissociation rates of lactose binding were extracted from NMR lineshape analyses. Structural mapping of chemical shift perturbations revealed long-range perturbations upon lactose binding for hGal1 and hGal8NTD. We further demonstrated using the NMR-based hydrogen–deuterium exchange (HDX) that lactose binding increases the exchange rates of residues located on the opposite side of the ligand-binding pocket for hGal1 and hGal8NTD, indicative of allostery. Additionally, lactose binding induces significant stabilisation of hGal8CTD across the entire domain. Our results suggested that lactose binding reduced the internal dynamics of hGal8CTD on a very slow timescale (minutes and slower) at the expense of reduced binding affinity due to the unfavourable loss of conformational entropy.
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