Sunyia Hussain

Determining the Oligomeric Structure of Proteorhodopsin by Gd3+-Based Pulsed Dipolar Spectroscopy of Multiple Distances

The structural organization of the functionally relevant, hexameric oligomer of green-absorbing proteorhodopsin (G-PR) was obtained from double electron-electron resonance (DEER) spectroscopy utilizing conventional nitroxide spin labels and recently developed Gd3+ -based spin labels. G-PR with nitroxide or Gd3+ labels was prepared using cysteine mutations at residues Trp58 and Thr177. By combining reliable measurements of multiple interprotein distances in the G-PR hexamer with computer modeling, we obtained a structural model that agrees with the recent crystal structure of the homologous blue-absorbing PR (B-PR) hexamer. These DEER results provide specific distance information in a membrane-mimetic environment and across loop regions that are unresolved in the crystal structure. In addition, the X-band DEER measurements using nitroxide spin labels suffered from multispin effects that, at times, compromised the detection of next-nearest neighbor distances. Performing measurements at high magnetic fields with Gd3+ spin labels increased the sensitivity considerably and alleviated the difficulties caused by multispin interactions.

Structure and function of G protein-coupled receptor oligomers: implications for drug discovery

G protein-coupled receptor (GPCR) oligomers are promising targets for the design of new highly selective therapeutics. GPCRs have historically been attractive drug targets for their role in nearly all cellular processes, and their localization at the cell surface makes them easily accessible to most small molecule therapeutics. However, GPCRs have traditionally been considered a monomeric entity, a notion that greatly oversimplifies their function. As evidence accumulates that GPCRs tune function through oligomer formation and protein-protein interactions, we see a greater demand for structural information about these oligomers to facilitate oligomer-specific drug design. These efforts are slowed by difficulties inherent to studying membrane proteins, such as low expression yield, in vitro stability and activity. Such obstacles are amplified for the study of specific oligomers, as there are limited tools to directly isolate and characterize these receptor complexes. Thus, there is a need to develop new interdisciplinary approaches, combining biochemical and biophysical techniques, to address these challenges and elucidate structural details about the oligomer and ligand binding interfaces. In this review, we provide an overview of mechanistic models that have been proposed to underlie the function of GPCR oligomers, and perspectives on emerging techniques to characterize GPCR oligomers for structure-based drug design.

Functional Consequences of the Oligomeric Assembly of Proteorhodopsin

The plasma membrane is the crucial interface between the cell and its exterior, packed with embedded proteins experiencing simultaneous protein-protein and protein-membrane interactions. A prominent example of cell membrane complexity is the assembly of transmembrane proteins into oligomeric structures, with potential functional consequences that are not well understood. From the study of proteorhodopsin (PR), a prototypical seven-transmembrane light-driven bacterial proton pump, we find evidence that the inter-protein interaction modulated by self-association yields functional changes observable from the protein interior. We also demonstrate that the oligomer is likely a physiologically relevant form of PR, as crosslinking of recombinantly expressed PR reveals an oligomeric population within the E. coli membrane (putatively hexameric). Upon chromatographic isolation of oligomeric and monomeric PR in surfactant micelles, the oligomer exhibits distinctly different optical absorption properties from monomeric PR, as reflected in a prominent decrease in the pKa of the primary proton acceptor residue (D97) and slowing of the light-driven conformational change. These functional effects are predominantly determined by specific PR-PR contacts over nonspecific surfactant interactions. Interestingly, varying the surfactant type alters the population of oligomeric states as well as the proximity of proteins within an oligomer, as determined by sparse electron paramagnetic resonance (EPR) distance measurements. Nevertheless, the dynamic surfactant environment retains the key function-tuning property exerted by oligomeric contacts. A potentially general design principle for transmembrane protein function tuning emerges from this work, one that hinges on specific oligomeric contacts that can be modulated by protein expression or membrane composition.

Proton-Based Structural Analysis of a Heptahelical Transmembrane Protein in Lipid Bilayers

The structures and properties of membrane proteins in lipid bilayers are expected to closely resemble those in native cell-membrane environments, although they have been difficult to elucidate. By performing solid-state NMR measurements at very fast (100 kHz) magic-angle spinning rates and at high (23.5 T) magnetic field, severe sensitivity and resolution challenges are overcome, enabling the atomic-level characterization of membrane proteins in lipid environments. This is demonstrated by extensive 1H-based resonance assignments of the fully protonated heptahelical membrane protein proteorhodopsin, and the efficient identification of numerous 1H–1H dipolar interactions, which provide distance constraints, inter-residue proximities, relative orientations of secondary structural elements, and protein–cofactor interactions in the hydrophobic transmembrane regions. These results establish a general approach for high-resolution structural studies of membrane proteins in lipid environments via solid-state NMR.

Functionally Active Membrane Proteins Incorporated in Mesostructured Silica Films

A versatile synthetic protocol is reported that allows high concentrations of functionally active membrane proteins to be incorporated in mesostructured silica materials. Judicious selections of solvent, surfactant, silica precursor species, and synthesis conditions enable membrane proteins to be stabilized in solution and during subsequent coassembly into silica-surfactant composites with nano- and mesoscale order. This was demonstrated by using a combination of nonionic ( n-dodecyl-β-d-maltoside or Pluronic P123), lipid-like (1,2-diheptanoyl- s n-glycero-3-phosphocholine), and perfluoro-octanoate surfactants under mild acidic conditions to coassemble the light-responsive transmembrane protein proteorhodopsin at concentrations up to 15 wt % into the hydrophobic regions of worm-like mesostructured silica materials in films. Small-angle X-ray scattering, electron paramagnetic resonance spectroscopy, and transient UV-visible spectroscopy analyses established that proteorhodopsin molecules in mesostructured silica films exhibited native-like function, as well as enhanced thermal stability compared to surfactant or lipid environments. The light absorbance properties and light-activated conformational changes of proteorhodopsin guests in mesostructured silica films are consistent with those associated with the native H+-pumping mechanism of these biomolecules. The synthetic protocol is expected to be general, as demonstrated also for the incorporation of functionally active cytochrome c, a peripheral membrane protein enzyme involved in electron transport, into mesostructured silica-cationic surfactant films.

Protein-Protein and Protein-Lipid Interactions Tune Proteorhodopsin Function by Altering Water Dynamics

Currently, the role of water and the membrane assembly in tuning the function of seven-helical transmembrane (7TM) proteins is not well-understood. Here, we focus on the light activation and functional properties of a prototypical example, the Proteorhodopsin (PR) proton pump from marine bacteria, observing how the protein and surrounding hydration water rearrange upon activation. This is made possible by the application of the powerful residue-specific magnetic resonance methods of electron paramagnetic resonance (EPR), which measures protein segment mobility, and Overhauser dynamic nuclear polarization (ODNP), as recently developed for probing local water diffusivity within 10 A of a nitroxide spin-label. We investigate further how these dynamics are affected by the surrounding environment, encompassing both protein-protein and protein-lipid interactions.With these techniques together with optical absorption spectroscopy, we find that water dynamics (both ps scale translational motion and ns scale “bound” water) at the membrane protein surface is dramatically affected by the lipid bilayer or surfactant micelle environment. Furthermore, hydration is correlated to functional changes such that water could modulate the timescale of conformational motion. Specifically, the slowdown of translational water motion at the membrane surface, coupled to a lack of bound waters, may facilitate proton uptake by PR.The association of PR with other PR molecules within the membrane, or oligomerization, has similar functional consequences in addition to effects on the protonation properties of key residues for ion transport (pKa of D97). The implication of our study is that PR-PR association alters the hydrogen bond network within the channel, possibly mediated by an altered interaction with the surfactant and surface hydration water upon oligomerization. This result, combined with the homology of PR with sensory receptors, elicits the intriguing possibility that PR has a functional flexibility mediated by oligomerization.

Dynamics and Oligomerization Tune Seven-Transmembrane Protein Function: Studies Based on Proteorhodopsin

Seven-transmembrane (7TM) proteins have diverse and important functions, ranging from signaling receptors to ion pumps. They share a reversible switching property, epitomized by the solar-powered microbial proton pump Proteorhodopsin (PR), which uses light energy to facilitate transport by a conformational “switch”. Here, we use PR as a model to capture the elusive details of activation and oligomerization necessary for the function of physiologically important membrane proteins.We have preliminary spectroscopic evidence that suggests altered photocycle kinetics and shifted pKa values for hexameric and monomeric forms of detergent-solubilized PR. These findings prove that PR function is tuned by self-association, and we apply magnetic resonance together with functional assays of proton transport to further understand dynamics changes that occur upon oligomerization. Our unique magnetic resonance techniques of electron paramagnetic resonance (EPR) and dynamic nuclear polarization (DNP) provide insight into the protein segment mobility and local hydration water dynamics of an amino acid residue spin-labeled with nitroxide-based radicals.Using these methods, we have found that PRs third cytoplasmic (E-F) loop is a short α-helical segment that experiences conformational change upon photoactivation. This structure is a common motif to the non-homologous G-protein coupled receptor bovine rhodopsin (Rh), where it is a docking point for a signal G-protein. Towards understanding how function hinges on dynamics, we developed a PR-Rh chimera by replacing the E-F loop of PR with the corresponding loop of Rh. The chimera successfully expresses and maintains optical properties. We evaluate its capability to activate the G-protein transducin, and apply EPR and DNP to obtain unique information about the biophysics of receptor/G-protein interactions. By controlling the oligomeric form of the PR-Rh chimera, we measure any changes in G-protein activation caused by varying the amount of receptor-receptor interactions.