The rhodopsin-arrestin-1 interaction in bicelles.

G-protein-coupled receptors (GPCRs) are essential mediators of information transfer in eukaryotic cells. Interactions between GPCRs and their binding partners modulate the signaling process. For example, the interaction between GPCR and cognate G protein initiates the signal, while the interaction with cognate arrestin terminates G-protein-mediated signaling. In visual signal transduction, arrestin-1 selectively binds to the phosphorylated light-activated GPCR rhodopsin to terminate rhodopsin signaling. Under physiological conditions, the rhodopsin-arrestin-1 interaction occurs in highly specialized disk membrane in which rhodopsin resides. This membrane is replaced with mimetics when working with purified proteins. While detergents are commonly used as membrane mimetics, most detergents denature arrestin-1, preventing biochemical studies of this interaction. In contrast, bicelles provide a suitable alternative medium. An advantage of bicelles is that they contain lipids, which have been shown to be necessary for normal rhodopsin-arrestin-1 interaction. Here we describe how to reconstitute rhodopsin into bicelles, and how bicelle properties affect the rhodopsin-arrestin-1 interaction.

Control of the conductance of engineered protein nanopores through concerted loop motions.

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.

NMR-based conformational ensembles explain pH-gated opening and closing of OmpG channel.

The outer membrane protein G (OmpG) is a monomeric 33 kDa 14-stranded ß-barrel membrane protein functioning as a nonspecific porin for the uptake of oligosaccharides in Escherichia 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 n-dodecylphosphocholine 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.

The quiet renaissance of protein nuclear magnetic resonance.

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 1980s.

Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin.

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 (P-Rh*). 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 P-Rh* 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.

Solution NMR approaches for establishing specificity of weak heterodimerization of membrane proteins.

Solution NMR provides a powerful approach for detecting complex formation involving weak to moderate intermolecular affinity. However, solution NMR has only rarely been used to detect complex formation between two membrane proteins in model membranes. The impact of specific binding on the NMR spectrum of a membrane protein can be difficult to distinguish from spectral changes that are induced by nonspecific binding and/or by changes that arise from forced cohabitation of the two proteins in a single model membrane assembly. This is particularly the case when solubility limits make it impossible to complete a titration to the point of near saturation of complex formation. In this work experiments are presented that provide the basis for establishing whether specific complex formation occurs between two membrane proteins under conditions where binding is not of high avidity. Application of these methods led to the conclusion that the membrane protein CD147 (also known as EMMPRIN or basigin) forms a specific heterodimeric complex in the membrane with the 99-residue transmembrane C-terminal fragment of the amyloid precursor protein (C99 or APP-ßCTF), the latter being the immediate precursor of the amyloid-ß polypeptides that are closely linked to the etiology of Alzheimer's disease.

Elucidation of inositol hexaphosphate and heparin interaction sites and conformational changes in arrestin-1 by solution nuclear magnetic resonance.

Arrestins specifically bind activated and phosphorylated G protein-coupled receptors and orchestrate both receptor trafficking and channel signaling through G protein-independent pathways via direct interactions with numerous nonreceptor partners. Here we report the first successful use of solution NMR in mapping 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 an identification of residues involved in the binding with these ligands that was more complete than what 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 the transition of arrestin from the 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.

Pulsed electron beam water radiolysis for submicrosecond hydroxyl radical protein footprinting.

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 submicrosecond 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 submicrosecond 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 beta-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 time scale shorter than that of large scale protein motions.

Structure determination of a Galectin-3-carbohydrate complex using paramagnetism-based NMR constraints.

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.

Bound-state residual dipolar couplings for rapidly exchanging ligands of His-tagged proteins.

The study of bound-state conformations of ligands interacting with proteins is important to the understanding of protein function and the design of drugs that alter function. Traditionally, transferred nuclear Overhauser effects (trNOEs), measured from NMR spectra of ligands in rapid exchange between bound and free states, have been used in these studies, owing to the inherent heavy weighting of bound-state data in the averaged ligand signals. In principle, residual dipolar couplings (RDCs) provide a useful complement to NOE data in that they provide orientational constraints as opposed to distance constraints, but use in ligand-binding applications has been limited due to the absence of heavy weighting of bound-state data. A widely applicable approach to increasing the weighting of bound-state data in averaged RDCs measured on ligands is presented. The approach rests on association of a His-tagged protein with a nickel-chelate-carrying lipid inserted into the lipid bilayer-like alignment media used in the acquisition of RDCs. The approach is validated through the observation of bound-state RDCs for the disaccharide, lactose, bound to the carbohydrate recognition domain of the mammalian lectin, galectin-3.

Enhancement of bound-state residual dipolar couplings: conformational analysis of lactose bound to Galectin-3.

Residual dipolar couplings (RDCs) have proven to be a valuable NMR tool that can provide long-range constraints for molecular structure determination. The constraints are orientational in nature and are, thus, highly complementary to conventional distance constraints from NOE data. This complementarity would seem to extend to the study of the geometry of ligands bound to proteins. However, unlike transferred NOEs, where collection, even with a large excess of free ligand, results in measurements dominated by bound contributions, RDCs of exchanging ligands can be dominated by free-state contributions. Here we present a strategy for enhancement of RDCs from bound states that is based on specifically enhancing the alignment of the protein to which a ligand will bind. The protein is modified by addition of a hydrophobic alkyl tail that anchors it to the bicelles that are a part of the ordering medium needed for RDC measurement. As an illustration, we have added a propyl chain to the C terminus of the carbohydrate recognition domain of the protein, Galectin-3, and report enhanced RDCs that prove consistent with known bound-ligand geometries for this protein.