Approximately 30% of the proteins expressed from the human genome correspond to membrane proteins. They perform a wide variety of cellular functions and are very attractive drug targets, with almost half of all pharmaceutics targeting these proteins. G protein coupled receptors (GPCRs) are the largest class of integral membrane proteins comprised of seven transmembrane-spanning helices, an extracellular N-terminus, and an intracellular C-terminus. CXCR1 is a class A, rhodopsin-like GPCR which couples to the Gi G-proteins, and has one high-affinity ligand, interleukin-8 (IL8). Many methods used to study membrane protein structure, function, and dynamics require significant modifications to be made to the protein of interest, resulting in an environment that is far from the native phospholipid bilayer. However, NMR spectroscopy is a powerful technique which allows the study of unmodified membrane proteins in near-native lipid environments and at physiological temperatures and pH.

IL8 interacts with CXCR1 at two sites - the N-terminus (Site I) and the extracellular loops (Site II). To probe these interactions, 1H fast MAS solid-state NMR was used to study IL8 bound to CXCR1 in pnhospholipid bilayers. A majority of the IL8 residues become immobilized upon receptor binding and their chemical shifts significantly perturbed, indicating that the environment and dynamics of the ligand are affected by interactions with CXCR1. Additional long-range distance restraints for the IL8-CXCR1 interaction were obtained via paramagnetic relaxation enhancement (PRE) experiments utilizing incorporation of a metal-chelating unnatural amino acid, 2-amino-3-(8-hydroxyquinolin-3-yl) propanoic acid (HQA), into various CXCR1 constructs positioned at residues near the ligand binding sites.

Sample preparation is challenging because membrane proteins are generally low expressing in heterologous systems, hydrophobic, and difficult to re-fold into their active conformations – any variation from the native structure results in inactive and/or mis-folded protein. The use of detergents can be especially detrimental to membrane proteins. We have developed styrene-maleic acid (SMA) macrodiscs which provide a detergent-free phospholipid bilayer environment for biophysical and functional studies of membrane proteins under physiological conditions. They are particularly well suited for structure determination by oriented-sample solid-state NMR and high-resolution solid-state NMR spectra of membrane proteins have been obtained in these discs.

The spread of novel coronavirus, SARS-CoV-2, a lethal and infectious respiratory syndrome, has heightened urgency in antiviral drug development. The SARS-CoV-2 envelope (E) and spike (S) proteins play a significant role in viral lifecycle and pathogenicity. The immediate threat posed by this infectious disease necessitates structural and functional characterization of these key structural membrane proteins involved in viral infection and replication. E protein’s transmembrane domain exhibits ion channel activity while its C-terminal domain is thought to participate in protein-protein interactions. S protein initiates viral infection through host-cell attachment and is proposed to participate in intermolecular interactions with E. Here, a bacterial expression and purification system is introduced to allow successful expression and purification of membrane proteins through E. coli. Use of this system accelerates production of purified full-length E and S proteins for spectroscopic NMR studies. Purification optimization allowed for high-resolution NMR spectra leading to characterization of the full-length E protein secondary structure and dynamics through assessment of CSPs. Interactions between E and known ion-channel inhibitor hexamethylene amiloride (HMA) allowed for characterization of the drug binding site. Analysis of the N-terminal transmembrane domain indicated Asn15 crucial to preserving E protein’s conformation. To evaluate potential intermolecular interactions between the C-terminal domains of E and S proteins, a truncated S protein construct was developed. Purification and expression of the S protein construct led to an initial 1H/15N HSQC spectrum, paving the way for future studies involving E and S protein-protein interactions integral to their structure and function in the pathogenicity of SARS-CoV-2.

The chemokine Interleukin-8 is a major mediator in inflammation. It interacts with the G Protein-Coupled Receptor CXCR1, which is expressed on neutrophils and other cells. Activation of CXCR1 leads to the neutrophils traveling to the site of infection and degranulation. The molecular mechanism between IL-8 – CXCR1 binding and receptor activation is not well understood. IL-8 contacts the two main interaction sites on CXCR1; one located on the flexible N-terminal domain (Binding Site-I) and the other on extracellular loop 2 and 3 (Binding Site-II). Solution and solid-state NMR spectroscopy have been used to better understand the IL-8 – CXCR1 complex. The solution NMR structure of monomeric IL-8 was solved and its interaction with constructs only composed of Binding Site-I studied. The presence of a membrane or the first transmembrane helix was found not to affect the interaction of IL-8. Proton-detected fast MAS solid-state NMR spectroscopy was used to map the interaction between IL-8 and Binding Site-II and as well as the wildtype CXCR1 receptor. Around 11 residues in IL-8 were immobilized upon contact with Binding Site-II, however almost all residues of IL-8 were immobilized upon contact with both Binding Site-I and Binding Site-II. Interestingly, IL-8 does not undergo a major conformational change upon binding to CXCR1. MAS solid-state NMR spectroscopy was used to study the interaction site and the change in dynamics in 1TM-CXCR1(1-72) upon IL-8 binding. Part of the flexible N-terminal domain was immobilized upon interaction with IL-8, but no tilt angle changes in the transmembrane helix were found. Oriented sample solid-state NMR spectroscopy was used to study the orientation of IL-8 bound to CXCR1. The receptor was reconstituted in uniaxially aligned bicelles and orientation dependent parameters were measured in IL-8. Additionally, a new membrane mimetic, called macrodisc, was developed based on the nanodisc. By changing the ratio of belt protein to lipid, larger discs were created that could be aligned in a magnetic field and used for oriented sample solid-state NMR spectroscopy.

Solid-state nuclear magnetic resonance (NMR) spectroscopy is a powerful method for the study of membrane proteins under native environments. G protein-coupled receptors (GPCRs) are highly challenging integral membrane proteins that are often hard to study using the available biophysical techniques. Limited information about their structures and dynamics has been elucidated with x-ray crystallography as well as various biochemical techniques, such as Föster resonance energy transfer (FRET), and mutation studies. Obtaining site resolved NMR spectra for GPCRs can help to better understand their structures and behaviors. Here CXC-chemokine receptor 2 (CXCR2) is studied with various NMR tools, and the functional relevance of the obtained protein is verified with ligand binding assays as well as NMR. A comparison of CXCR2 interaction with two of its ligands: interleukin 8 and CXCL5 is also presented. At the same time, methods such as protein perdeuteration, and 1H detection were developed for the study of complex membrane proteins using the oriented-sample solid-state NMR technique. NMR developments are also made on the MerFTC chimeric protein, to help experiments for the more complex CXCR2. Combining various NMR techniques, optimized NMR spectra are presented, providing insights into the structure and function of the CXCR2 protein.

Hepatitis C virus (HCV) proteins are targets for potential pharmaceutical drugs since there are currently no vaccines against the virus, and drug treatments are expensive and prone to viral resistance. The channel-forming behavior and role of the viroporin p7 from HCV in virus assembly and release make it a promising choice for structure-based drug design. The structures of viroporins have only been successfully solved using nuclear magnetic spectroscopy (NMR). In the case of p7 and other membrane proteins, the lipid environment plays a crucial role in the folding of the protein. Micellar environments have been known to distort membrane protein structures whereas bilayers provide a more native-like membrane environment. The structure of p7 has been solved in two micelle environments, but the distance between the transmembrane helices differ greatly between the two structures. One way to obtain long distance structural restraints in NMR is by obtaining paramagnetic relaxation enhancement (PRE) measurements through the incorporation of a metal-chelating unnatural amino acid (UAA). Here, an UAA with an 8-hydroxyquinoline moiety was successfully incorporated into the transmembrane portion of p7, and intramolecular paramagnetic relaxation enhancements were observed by solution NMR. The next step is to solve the structure of p7 in a lipid bilayer by solid-state NMR. In this research, efforts were made to move from isotropic detergent environments to larger native-like bilayer ones, with the goal of obtaining PREs with the UAA-incorporated p7 by solid-state NMR.