Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (PRh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Although arrestin-1 affinity for Rh* was seen to be low (K D > 150 μM), its affinity for P-Rh (K D ∼80 μM) was comparable to the concentration of active monomeric arrestin-1 in the outer segment, suggesting that P-Rh generated by high-gain phosphorylation is occupied by arrestin-1 under physiological conditions and will not signal upon photo-activation. Arrestin-1 was seen to bind P-Rh* and P-opsin with fairly high affinity (K D of ∼50 and 800 nM, respectively), implying that arrestin-1 dissociation is triggered only upon P-opsin regeneration with 11-cis-retinal, precluding noise generated by opsin activity. Based on their observed affinity for arrestin-1, P-opsin and inactive P-Rh very likely affect the physiological monomer-dimer-tetramer equilibrium of arrestin-1, and should therefore be taken into account when modeling photoreceptor function. The data also suggested that complex formation with either PRh* or P-opsin results in a global transition in the conformation of arrestin-1, possibly to a dynamic molten globule-like structure. We hypothesize that this transition contributes to the mechanism that triggers preferential interactions of several signaling proteins with receptor-activated arrestins.G protein-coupled receptors | nuclear magnetic resonance | TROSY | bicelles

The determination of the location and conformation of a natural ligand bound to a protein receptor is often a first step in the rational design of molecules that can modulate receptor function. NMR observables, including NOEs, often provide the basis for these determinations. However, when ligands are carbohydrates, interactions mediated by extensive hydrogen-bonding networks often reduce or eliminate NOEs between ligand and protein protons. In these cases, it is useful to look to other distance-and orientation-dependent observables that can constrain the geometry of ligand-protein complexes. Here we illustrate the use of paramagnetism-based NMR constraints, including pseudo-contact shifts (PCS) and field-induced residual dipolar couplings (RDCs). When a paramagnetic center can be attached to the protein, field-induced RDCs and PCS reflect only bound-state properties of the ligand, even when averages over small fractions of bound states and large fractions of free states are observed. The effects can also be observed over a long range, making it possible to attach a paramagnetic center to a remote part of the protein. The system studied here is a Galectin-3-lactose complex. A lanthanide-binding peptide showing minimal flexibility with respect to the protein was integrated into the C terminus of an expression construct for the Galectin-3-carbohydrate-binding domain. Dysprosium ion, which has a large magnetic susceptibility anisotropy, was complexed to the peptide, making it possible to observe both PCSs and field-induced RDCs for the protein and the ligand. The structure determined from these constraints shows agreement with a crystal structure of a Galectin-3-N-acetyllactosamine complex.

Hydroxyl radical footprinting is a valuable technique for studying protein structure, but care must be taken to ensure that the protein does not unfold during the labeling process due to oxidative damage. Footprinting methods based on sub-microsecond laser photolysis of peroxide that complete the labeling process faster than the protein can unfold have been recently described; however, the mere presence of large amounts of hydrogen peroxide can also cause uncontrolled oxidation and minor conformational changes. We have developed a novel method for sub-microsecond hydroxyl radical protein footprinting using a pulsed electron beam from a 2 MeV Van de Graaff electron accelerator to generate a high concentration of hydroxyl radicals by radiolysis of water. The amount of oxidation can be controlled by buffer composition, pulsewidth, dose, and dissolved nitrous oxide gas in the sample. Our results with ubiquitin and β-lactoglobulin A demonstrate that one submicrosecond electron beam pulse produces extensive protein surface modifications. Highly reactive residues that are buried within the protein structure are not oxidized, indicating that the protein retains its folded structure during the labeling process. Time-resolved spectroscopy indicates that the major part of protein oxidation is complete in a timescale shorter than that of large scale protein motions.

The outer membrane protein G (OmpG) is a monomeric 33 kDa 14-stranded β-barrel membrane protein functioning as a non-specific porin for the uptake of oligosaccharides in E. coli. Two different crystal structures of OmpG obtained at different values of pH suggest a pH-gated pore opening mechanism. In these structures, extracellular loop 6 extends away from the barrel wall at neutral pH, but is folded back into the pore lumen at low pH, blocking transport through the pore. Loop 6 was invisible in a previously published solution NMR structure of OmpG in DPC micelles, presumably due to conformational exchange on an intermediate NMR time-scale. Here we present an NMR paramagnetic relaxation enhancement (PRE)-based approach to visualize the conformational dynamics of loop 6 and to calculate conformational ensembles that explain the pH-gated opening and closing of the OmpG channel. The different loop conformers detected by the PRE ensemble calculations were validated by disulfide cross-linking of strategically engineered cysteines and electrophysiological single channel recordings. The results indicate a more dynamically regulated channel opening and closing than previously thought and reveal additional membrane-associated conformational ensembles at pH 6.3 and 7.0. We anticipate this approach to be generally applicable to detect and characterize functionally important conformational ensembles of membrane proteins.

Arrestins specifically bind activated and phosphorylated G protein-coupled receptors, and orchestrate both receptor trafficking, and channel signaling to G protein-independent pathways via direct interactions with numerous non-receptor partners. Here we report the first successful use of solution NMR to map the binding sites in arrestin-1 (visual arrestin) for two polyanionic compounds that mimic phosphorylated light-activated rhodopsin: inositol hexaphosphate (IP6) and heparin. This yielded a more complete identification of residues involved in the binding with these ligands than has previously been feasible. IP6 and heparin appear to bind to the same site on arrestin-1, centered on a positively charged region in the N-domain. We present the first direct evidence that both IP6 and heparin induced a complete release of the arrestin C-tail. These observations provide novel insight into the nature of arrestin transition from basal to active state and demonstrate the potential of NMR-based methods in the study of protein-protein interactions involving members of the arrestin family.Arrestins are soluble proteins that specifically bind to the phosphorylated forms of activated G protein-coupled receptors (GPCRs) (1), precluding further G protein activation and targeting the complex to clathrin-coated pits via direct interactions with clathrin (2) and its adaptor AP2 (3). This leads to endocytosis and receptor down-regulation. In addition to modulating signaling and trafficking of hundreds of GPCRs, arrestins also bind numerous non-receptor partners and regulate a variety of receptor-dependent and -independent signaling pathways(4). Vertebrates have four arrestin subtypes, only two of which, arrestin-2 and -3 2 , are ubiquitously expressed(5). Arrestin-1, the first member of the family to be discovered (6), is present at very high levels in both rod (7-8) and cone (9) photoreceptors where it shuts off the signaling by light-activated opsins. Arrestin-1 was originally described as a protein that binds light-activated phosphorylated rhodopsin (6). Subsequent studies have also revealed that it has additional partners. Arrestin-1 self-associates to form dimers and tetramers (10-12) and also binds microtubules (13-14), calmodulin (15), protein kinase JNK3 and ubiquitin ligase Mdm2 (16), N-ethylmaleimide sensitive factor (17), and many other proteins. Association with active phosphorhodopsin is known to induce a global † This study was supported by NIH grants GM081756 (ARRA supplement), EY011500, and GM077561 to VVG, and by PO1 GM08513 and Roadmap RO1 GM081816 to CRS. We use systematic names of arrestin proteins: arrestin-1 (historic names S-antigen, 48 kDa protein, visual or rod arrestin), arrestin-2

From roughly 1985 through the start of the new millennium, the cutting edge of solution protein nuclear magnetic resonance (NMR) spectroscopy was to a significant extent driven by the aspiration to determine structures. Here we survey recent advances in protein NMR that herald a renaissance in which a number of its most important applications reflect the broad problem-solving capability displayed by this method during its classical era during the 1970s and early 80s. “Without receivers fitted and kept in order, the air may tingle and thrill with the message, but it will not reach my spirit and consciousness.” Mary Slessor, Calabar, circa 1910

Nanopores have attracted much interest for nucleic acid sequencing, chemical sensing, and protein folding at the single molecule level. The outer membrane protein OmpG from E. coli stands out because it forms nanopores from single polypeptide chains. This property affords one to separately engineer each of the seven extracellular loops that control access to the pore. The longest of these loops, loop 6, has been recognized previously as the main gating loop that closes the pore at low pH and opens it at high pH. In the present work, we devised a method to pin each of these loops to the embedding membrane and measure the single pore conductances of these constructs. The electrophysiological and complementary NMR measurements show that the pinning of individual loops alters the structure and dynamics of neighbouring and distant loops in a correlated fashion. Pinning of loop 6 generates a constitutively open pore and patterns of concerted loop motions control access to the OmpG nanopore.

Protein nanopores have attracted much interest for nucleic acid sequencing, chemical sensing, and protein folding at the single molecule level. The outer membrane protein OmpG from E. coli stands out because it forms a nanopore from a single polypeptide chain. This property allows the separate engineering of each of the seven extracellular loops that control access to the pore. The longest of these loops, loop 6, has been recognized as the main gating loop that closes the pore at low pH values and opens it at high pH values. A method was devised to pin each of the loops to the embedding membrane and measure the single-pore conductances of the resulting constructs. The electrophysiological and complementary NMR measurements show that the pinning of individual loops alters the structure and dynamics of neighboring and distant loops in a correlated fashion. Pinning loop 6 generates a constitutively open pore and patterns of concerted loop motions control access to the OmpG nanopore.