David L. Farrens

Requirement of Rigid-Body Motion of Transmembrane Helices for Light Activation of Rhodopsin

Conformational changes are thought to underlie the activation of heterotrimeric GTP-binding protein (G protein)—coupled receptors. Such changes in rhodopsin were explored by construction of double cysteine mutants, each containing one cysteine at the cytoplasmic end of helix C and one cysteine at various positions in the cytoplasmic end of helix F. Magnetic dipolar interactions between spin labels attached to these residues revealed their proximity, and changes in their interaction upon rhodopsin light activation suggested a rigid body movement of helices relative to one another. Disulfide cross-linking of the helices prevented activation of transducin, which suggests the importance of this movement for activation of rhodopsin.

Agonist-induced conformational changes in the G-protein-coupling domain of the β2 adrenergic receptor

The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the β2 adrenergic receptor (β2AR), adjacent to the G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-β2AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction between the fluorescein at Cys-265 and fluorescence-quenching reagents localized to different molecular environments of the receptor. These observations are consistent with a rotation and/or tilting of TM6 on agonist activation. Our studies, when compared with studies of activation in rhodopsin, indicate a general mechanism for GPCR activation; however, a notable difference is the relatively slow kinetics of the conformational changes in the β2AR, which may reflect the different energetics of activation by diffusible ligands.

Conformational Changes in Rhodopsin MOVEMENT OF HELIX F DETECTED BY SITE-SPECIFIC CHEMICAL LABELING AND FLUORESCENCE SPECTROSCOPY

A recent proposal for the formation of functionally active rhodopsin has placed critical importance on a movement of one of its transmembrane helices (Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. L., and Khorana, H. G. (1996) Science 274, 768–770). We investigated this hypothesis using a series of eight rhodopsin mutants containing single reactive cysteine residues in the region (helix F) where movement was previously detected. The cysteine mutants were studied in two ways, by measuring their reactivity to a cysteine-specific reagent (PyMPO-maleimide), and by labeling the cysteines with a fluorescent label (monobromobimane) followed by fluorescence spectroscopic analysis. The chemical reactivity data showed sequence-specific variations in reactivity for the mutants in the dark state, resulting in a pattern suggestive of an α helix. Interestingly, only upon photoactivation to the MII form did residues found on the inner “face” of this helix react with the PyMPO-maleimide. The ability of the dark state mutants to react with the fluorescent label monobromobimane followed a similar pattern. Furthermore, fluorescence measurements indicate that a bimane label on the inner face of the helix (at V250C) detects changes in the polarity of its environment and accessibility to a fluorescence quenching agent upon MII formation. Viewed together, the data provide further direct evidence that rhodopsin activation involves a conformational change at helix F.

Biosensor reveals multiple sources for mitochondrial NAD

A fluorescent sensor for NAD+ in living cells Roles of cellular nicotinamide adenine dinucleotide (NAD+) in metabolism, aging, and disease have garnered much interest, but methods have been lacking to measure the amounts of NAD+ in living cells. Cambronne et al. developed a genetically encoded biosensor that can be used to monitor concentrations of free NAD+ in various compartments of a cell (see the Perspective by Guarente). Such concentrations of NAD+ appear to be important in regulating the activity of NAD+-consuming enzymes such as sirtuins and ADP-ribosyltransferases. The authors used the sensor to demonstrate that NAD+ concentrations in mitochondria of cultured human cells can be controlled by multiple mechanisms. Science, this issue p. 1474; see also p. 1396 A fluorescent sensor allows measurement of NAD+ concentrations in cultured mammalian cells. Nicotinamide adenine dinucleotide (NAD+) is an essential substrate for sirtuins and poly(adenosine diphosphate–ribose) polymerases (PARPs), which are NAD+-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD+ concentrations within these subcellular compartments are thought to regulate the activity of NAD+-consuming enzymes; however, the challenge in measuring compartmentalized NAD+ in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD+ concentrations in subcellular compartments. We found that the concentrations of free NAD+ in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD+ biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD+ concentrations.

Monomeric Rhodopsin Is the Minimal Functional Unit Required for Arrestin Binding

We have tested whether arrestin binding requires the G-protein-coupled receptor be a dimer or a multimer. To do this, we encapsulated single-rhodopsin molecules into nanoscale phospholipid particles (so-called nanodiscs) and measured their ability to bind arrestin. Our data clearly show that both visual arrestin and beta-arrestin 1 can bind to monomeric rhodopsin and stabilize the active metarhodopsin II form. Interestingly, we find that the monomeric rhodopsin in nanodiscs has a higher affinity for wild-type arrestin binding than does oligomeric rhodopsin in liposomes or nanodiscs, as assessed by stabilization of metarhodopsin II. Together, these results establish that rhodopsin self-association is not required to enable arrestin binding.

Dynamics of Arrestin-Rhodopsin Interactions ARRESTIN AND RETINAL RELEASE ARE DIRECTLY LINKED EVENTS

In this study, we address the mechanism of visual arrestin release from light-activated rhodopsin using fluorescently labeled arrestin mutants. We find that two mutants, I72C and S251C, when labeled with the small, solvent-sensitive fluorophore monobromobimane, exhibit spectral changes only upon binding light-activated, phosphorylated rhodopsin. Our analysis indicates that these changes are probably due to a burying of the probes at these sites in the rhodopsin-arrestin or phospholipid-arrestin interface. Using a fluorescence approach based on this observation, we demonstrate that arrestin and retinal release are linked and are described by similar activation energies. However, at physiological temperatures, we find that arrestin slows the rate of retinal release ∼2-fold and abolishes the pH dependence of retinal release. Using fluorescence, EPR, and biochemical approaches, we also find intriguing evidence that arrestin binds to a post-Meta II photodecay product, possibly Meta III. We speculate that arrestin regulates levels of free retinal in the rod cell to help limit the formation of damaging oxidative retinal adducts. Such adducts may contribute to diseases like atrophic age-related macular degeneration (AMD). Thus, arrestin may serve to both attenuate rhodopsin signaling and protect the cell from excessive retinal levels under bright light conditions.

Consensus and Variant cAMP-regulated Enhancers Have Distinct CREB-binding Properties

Recent determination of the cAMP response element-binding protein (CREB) basic leucine zipper (bZIP) consensus CRE crystal structure revealed key dimerization and DNA binding features that are conserved among members of the CREB/CREM/ATF-1 family of transcription factors. Dimerization appeared to be mediated by a Tyr307–Glu312interhelical hydrogen bond and a Glu319–Arg314electrostatic interaction. An unexpected hexahydrated Mg2+ion was centered above the CRE in the dimer cavity. In the present study, we related these features to CREB dimerization and DNA binding. A Y307F substitution reduced dimer stability and DNA binding affinity, whereas a Y307R mutation produced a stabilizing effect. Mutation of Glu319 to Ala or Lys attenuated dimerization and DNA binding. Mg2+ ions enhanced the binding affinity of wild-type CREB to the palindromic CRE by ∼20-fold but did not do so for divergent CREs. Similarly, mutation of Lys304, which mediates the CREB interaction with the hydrated Mg2+, blocked CREB binding to the palindromic but not the variant CRE sequences. The distinct binding characteristics of the K304A mutants to the consensus and variant CRE sequences indicate that CREB binding to these elements is differentially regulated by Mg2+ ions. We suggest that CREB binds the consensus and variant CRE sequences through fundamentally distinct mechanisms.

Distance Mapping in Proteins Using Fluorescence Spectroscopy: The Tryptophan-Induced Quenching (TrIQ) Method

Studying the interplay between protein structure and function remains a daunting task. Especially lacking are methods for measuring structural changes in real time. Here we report our most recent improvements to a method that can be used to address such challenges. This method, which we now call tryptophan-induced quenching (TrIQ), provides a straightforward, sensitive, and inexpensive way to address questions of conformational dynamics and short-range protein interactions. Importantly, TrIQ only occurs over relatively short distances (∼5-15 Å), making it complementary to traditional fluorescence resonance energy transfer (FRET) methods that occur over distances too large for precise studies of protein structure. As implied in the name, TrIQ measures the efficient quenching induced in some fluorophores by tryptophan (Trp). We present here our analysis of the TrIQ effect for five different fluorophores that span a range of sizes and spectral properties. Each probe was attached to four different cysteine residues on T4 lysozyme, and the extent of TrIQ caused by a nearby Trp was measured. Our results show that, at least for smaller probes, the extent of TrIQ is distance dependent. Moreover, we also demonstrate how TrIQ data can be analyzed to determine the fraction of fluorophores involved in a static, nonfluorescent complex with Trp. Based on this analysis, our study shows that each fluorophore has a different TrIQ profile, or sphere of quenching, which correlates with its size, rotational flexibility, and the length of attachment linker. This TrIQ-based sphere of quenching is unique to every Trp-probe pair and reflects the distance within which one can expect to see the TrIQ effect. Thus,TrIQ provides a straightforward, readily accessible approach for mapping distances within proteins and monitoring conformational changes using fluorescence spectroscopy.

Role of the Retinal Hydrogen Bond Network in Rhodopsin Schiff Base Stability and Hydrolysis

Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin. We report here our investigation into this process focusing on the role of amino acids involved in a hydrogen bond network around the retinal Schiff base. We find conservative mutations in this network (T94I, E113Q, S186A, E181Q, Y192F, and Y268F) increase the activation energy (Ea) and abolish the concave Arrhenius plot normally seen for Schiff base hydrolysis in dark state rhodopsin. Interestingly, two mutants (T94I and E113Q) show dramatically faster rates of Schiff base hydrolysis in dark state rhodopsin, yet slower hydrolysis rates in the active MII form. We find deuterium affects the hydrolysis process in wild-type rhodopsin, exhibiting a specific isotope effect of ∼2.5, and proton inventory studies indicate that multiple proton transfer events occur during the process of Schiff base hydrolysis for both dark state and MII forms. Taken together, our study demonstrates the importance of the retinal hydrogen bond network both in maintaining Schiff base integrity in dark state rhodopsin, as well as in catalyzing the hydrolysis and release of retinal from the MII form. Finally, we note that the dramatic alteration of Schiff base stability caused by mutation T94I may play a causative role in congenital night blindness as has been suggested by the Oprian and Garriga laboratories.

Dynamics of Arrestin-Rhodopsin Interactions LOOP MOVEMENT IS INVOLVED IN ARRESTIN ACTIVATION AND RECEPTOR BINDING

In this study we investigate conformational changes in Loop V-VI of visual arrestin during binding to light-activated, phosphorylated rhodopsin (Rho*-P) using a combination of site-specific cysteine mutagenesis and intramolecular fluorescence quenching. Introduction of cysteines at positions in the N-domain at residues predicted to be in close proximity to Ile-72 in Loop V-VI of arrestin (i.e. Glu-148 and Lys-298) appear to form an intramolecular disulfide bond with I72C, significantly diminishing the binding of arrestin to Rho*-P. Using a fluorescence approach, we show that the steady-state emission from a monobromobimane fluorophore in Loop V-VI is quenched by tryptophan residues placed at 148 or 298. This quenching is relieved upon binding of arrestin to Rho*-P. These results suggest that arrestin Loop V-VI moves during binding to Rho*-P and that conformational flexibility of this loop is essential for arrestin to adopt a high affinity binding state.