It is challenging to find membrane mimics that stabilize the native structure, dynamics, and functions of membrane proteins. In a recent advance, nanodiscs have been shown to provide a bilayer environment compatible with solution NMR. Increasing the lipid to “belt” peptide ratio expands their diameter, slows their reorientation rate, and enables the protein-containing discs to be aligned in a magnetic field for Oriented Sample solid-state NMR. Here, we compare the spectroscopic properties of membrane proteins with between one and seven trans-membrane helices in q=0.1 isotropic bi-celles, ∼10 nm diameter isotropic nanodiscs, ∼30 nm diameter magnetically aligned macrodiscs, and q=5 bicelles.

The structure of monomeric human chemokine IL-8 (residues 1–66) was determined in aqueous solution by NMR spectroscopy. The structure of the monomer is similar to that of each subunit in the dimeric full-length protein (residues 1–72), with the main differences being the location of the N-loop (residues 10–22) relative to the C-terminal α-helix and the position of the side chain of phenylalanine 65 near the truncated dimerization interface (residues 67–72). NMR was used to analyze the interactions of monomeric IL-8 (1–66) with ND-CXCR1 (residues 1–38), a soluble polypeptide corresponding to the N-terminal portion of the ligand binding site (Binding Site-I) of the chemokine receptor CXCR1 in aqueous solution, and with 1TM-CXCR1 (residues 1–72), a membrane-associated polypeptide that includes the same N-terminal portion of the binding site, the first trans-membrane helix, and the first intracellular loop of the receptor in nanodiscs. The presence of neither the first transmembrane helix of the receptor nor the lipid bilayer significantly affected the interactions of IL-8 with Binding Site-I of CXCR1.

p62/SQSTM1 is a multiprotein interaction hub forming cellular punctate structures known as p62 bodies. p62 is centrally involved in the degradation of ubiquitinated cargo through autophagy, as well as in a wide range of signaling activities as part of the cellular response to nutrient sensing, oxidative stress, infection, immunity, and inflammation. Structural work has shown that p62 forms flexible filamentous assemblies composed of an N‐terminal PB1‐domain scaffold and a C‐terminal binding platform, including folded recognition domains and structurally disordered binding motifs. In the cell, these filaments are part of cellular p62 bodies that display properties of liquid–liquid‐phase separation. Here, we review the accumulated structural and functional work of p62 and integrate them with the emerging framework of filamentous biomolecular condensates.

The use of paramagnetic constraints in protein NMR is an active area of research because of the benefits of long-range distance measurements (>10 Å). One of the main issues in successful execution is the incorporation of a paramagnetic metal ion into diamagnetic proteins. The most common metal ion tags are relatively long aliphatic chains attached to the side chain of a selected cysteine residue with a chelating group at the end where it can undergo substantial internal motions, decreasing the accuracy of the method. An attractive alternative approach is to incorporate an unnatural amino acid (UAA) that binds metal ions at a specific site on the protein using the methods of molecular biology. Here we describe the successful incorporation of the unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl) propanoic acid (HQA) into two different membrane proteins by heterologous expression in E. coli. Fluorescence and NMR experiments demonstrate complete replacement of the natural amino acid with HQA and stable metal chelation by the mutated proteins. Evidence of site-specific intra- and inter-molecular PREs by NMR in micelle solutions sets the stage for the use of HQA incorporation in solid-state NMR structure determinations of membrane proteins in phospholipid bilayers.