Membrane Curvature Revisited-the Archetype of Rhodopsin Studied by Time-Resolved Electronic Spectroscopy.

G-protein-coupled receptors (GPCRs) comprise the largest and most pharmacologically targeted membrane protein family. Here, we used the visual receptor rhodopsin as an archetype for understanding membrane lipid influences on conformational changes involved in GPCR activation. Visual rhodopsin was recombined with lipids varying in their degree of acyl chain unsaturation and polar headgroup size using 1-palmitoyl-2-oleoyl-sn-glycero- and 1,2-dioleoyl-sn-glycerophospholipids with phosphocholine (PC) or phosphoethanolamine (PE) substituents. The receptor activation profile after light excitation was measured using time-resolved ultraviolet-visible spectroscopy. We discovered that more saturated POPC lipids back shifted the equilibrium to the inactive state, whereas the small-headgroup, highly unsaturated DOPE lipids favored the active state. Increasing unsaturation and decreasing headgroup size have similar effects that combine to yield control of rhodopsin activation, and necessitate factors beyond proteolipid solvation energy and bilayer surface electrostatics. Hence, we consider a balance of curvature free energy with hydrophobic matching and demonstrate how our data support a flexible surface model (FSM) for the coupling between proteins and lipids. The FSM is based on the Helfrich formulation of membrane bending energy as we previously first applied to lipid-protein interactions. Membrane elasticity and curvature strain are induced by lateral pressure imbalances between the constituent lipids and drive key physiological processes at the membrane level. Spontaneous negative monolayer curvature toward water is mediated by unsaturated, small-headgroup lipids and couples directly to GPCR activation upon light absorption by rhodopsin. For the first time to our knowledge, we demonstrate this modulation in both the equilibrium and pre-equilibrium evolving states using a time-resolved approach.

Fluorescence Resonance Energy Transfer-based Structural Analysis of the Dihydropyridine Receptor α1S Subunit Reveals Conformational Differences Induced by Binding of the ß1a Subunit.

The skeletal muscle dihydropyridine receptor α1S subunit plays a key role in skeletal muscle excitation-contraction coupling by sensing membrane voltage changes and then triggering intracellular calcium release. The cytoplasmic loops connecting four homologous α1S structural domains have diverse functions, but their structural arrangement is poorly understood. Here, we used a novel FRET-based method to characterize the relative proximity of these intracellular loops in α1S subunits expressed in intact cells. In dysgenic myotubes, energy transfer was observed from an N-terminal-fused YFP to a FRET acceptor, ReAsH (resorufin arsenical hairpin binder), targeted to each α1S intracellular loop, with the highest FRET efficiencies measured to the α1S II-III loop and C-terminal tail. However, in HEK-293T cells, FRET efficiencies from the α1S N terminus to the II-III and III-IV loops and the C-terminal tail were significantly lower, thus suggesting that these loop structures are influenced by the cellular microenvironment. The addition of the ß1a dihydropyridine receptor subunit enhanced FRET to the II-III loop, thus indicating that ß1a binding directly affects II-III loop conformation. This specific structural change required the C-terminal 36 amino acids of ß1a, which are essential to support EC coupling. Direct FRET measurements between α1S and ß1a confirmed that both wild type and truncated ß1a bind similarly to α1S These results provide new insights into the role of muscle-specific proteins on the structural arrangement of α1S intracellular loops and point to a new conformational effect of the ß1a subunit in supporting skeletal muscle excitation-contraction coupling.

Methods for labeling skeletal muscle ion channels site-specifically with fluorophores suitable for FRET-based structural analysis.

Skeletal muscle excitation-contraction coupling is triggered by the concerted action of two enormous Ca(2+) channel complexes, the dihydropyridine receptor and the type 1 ryanodine receptor. Recent advances in our understanding of the structure of these large Ca(2+) channels have been driven by fluorescence resonance energy transfer (FRET)-based analysis. A methodological challenge in conducting these FRET measurements is the ability to site-specifically label these huge ion channels with donor and acceptor fluorophores capable of undergoing energy transfer. In this chapter, we detail specific protocols for tagging large membrane proteins with these fluorescent probes using three orthogonal labeling methods: fluorescent protein fusions, biarsenical reagents directed to engineered tetracysteine tags, and Cy3/5 nitrilotriacetic acid conjugates that bind to poly-histidine tags.

Structural mapping of divergent regions in the type 1 ryanodine receptor using fluorescence resonance energy transfer.

Ryanodine receptors (RyRs) release Ca(2+) to initiate striated muscle contraction. Three highly divergent regions (DRs) in the RyR protein sequence (DR1, DR2, and DR3) may confer isoform-specific functional properties to the RyRs. We used cell-based fluorescence resonance energy transfer (FRET) measurements to localize these DRs to the cryoelectron microscopic (cryo-EM) map of the skeletal muscle RyR isoform (RyR1). FRET donors were targeted to RyR1 using five different FKBP12.6 variants labeled with Alexa Fluor 488. FRET was then measured to the FRET acceptors, Cy3NTA or Cy5NTA, targeted to decahistidine tags introduced within the DRs. DR2 and DR3 were localized to separate positions within the "clamp" region of the RyR1 cryo-EM map, which is presumed to interface with Cav1.1. DR1 was localized to the "handle" region, near the regulatory calmodulin-binding site on the RyR. These localizations provide insights into the roles of DRs in RyR allosteric regulation during excitation contraction coupling.

Site-specific labeling of the type 1 ryanodine receptor using biarsenical fluorophores targeted to engineered tetracysteine motifs.

The type 1 ryanodine receptor (RyR1) is an intracellular Ca(2+) release channel that mediates skeletal muscle excitation contraction coupling. While the overall shape of RyR1 has been elucidated using cryo electron microscopic reconstructions, fine structural details remain elusive. To better understand the structure of RyR1, we have previously used a cell-based fluorescence resonance energy transfer (FRET) method using a fused green fluorescent protein (GFP) donor and a fluorescent acceptor, Cy3NTA that binds specifically to short poly-histidine 'tags' engineered into RyR1. However, the need to permeabilize cells to allow Cy3NTA entry as well as the noncovalent binding of Cy3NTA to the His tag limits future applications of this technique for studying conformational changes of the RyR. To overcome these problems, we used a dodecapeptide sequence containing a tetracysteine (Tc) motif to target the biarsenical fluorophores, FlAsH and ReAsH to RyR1. These compounds freely cross intact cell membranes where they then bind covalently to the tetracysteine motif. First, we used this system to conduct FRET measurements in intact cells by fusing a yellow fluorescent protein (YFP) FRET donor to the N-terminus of RyR1 and then targeting the FRET acceptor, ReAsH to an adjacent Tc tag. Moderate energy transfer (∼33%) was observed whereas ReAsH incubation of a YFPRyR1 fusion protein lacking the Tc tag resulted in no detectable FRET. We also developed a FRET-based system that did not require RyR fluorescent protein fusions by labeling N-terminal Tc-tagged RyR1 with FlAsH, a FRET donor and then targeting the FRET acceptor Cy3NTA to an adjacent decahistidine (His10) tag. A high degree of energy transfer (∼66%) indicated proper binding of both compounds to these unique recognition sequences in RyR1. Thus, these two systems should provide unprecedented flexibility in future FRET-based structural determinations of RyR1.

N-terminal and central segments of the type 1 ryanodine receptor mediate its interaction with FK506-binding proteins.

We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca(2+) leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His10) "tags" placed within N-terminal (amino acid residues 76-619) or central (residues 2157-2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.

Rhodopsin activation switches in a native membrane environment.

The elucidation of structure-function relationships of membrane proteins still poses a considerable challenge due to the sometimes profound influence of the lipid bilayer on the functional properties of the protein. The visual pigment rhodopsin is a prototype of the family of G protein-coupled transmembrane receptors and a considerable part of our knowledge on its activation mechanisms has been derived from studies on detergent-solubilized proteins. This includes in particular the events associated with the conformational transitions of the receptor from the still inactive Meta I to the Meta II photoproduct states, which are involved in signaling. These events involve disruption of an internal salt bridge of the retinal protonated Schiff base, movement of helices and proton uptake from the solvent by the conserved cytoplasmic E(D)RY network around Glu134. As the equilibria associated with these events are considerably altered by the detergent environment, we set out to investigate these equilibria in the native membrane environment and to develop a coherent thermodynamic model of these activating steps using UV-visible and Fourier-transform infrared spectroscopy as complementary techniques. Particular emphasis is put on the role of protonation of Glu134 from the solvent, which is a thermodynamic prerequisite for full receptor activation in membranes, but not in detergent. In view of the conservation of this carboxylate group in family A G protein-coupled receptors, it may also play a similar role in the activation of other family members.

Two protonation switches control rhodopsin activation in membranes.

Activation of the G protein-coupled receptor (GPCR) rhodopsin is initiated by light-induced isomerization of the retinal ligand, which triggers 2 protonation switches in the conformational transition to the active receptor state Meta II. The first switch involves disruption of an interhelical salt bridge by internal proton transfer from the retinal protonated Schiff base (PSB) to its counterion, Glu-113, in the transmembrane domain. The second switch consists of uptake of a proton from the solvent by Glu-134 of the conserved E(D)RY motif at the cytoplasmic terminus of helix 3, leading to pH-dependent receptor activation. By using a combination of UV-visible and FTIR spectroscopy, we study the activation mechanism of rhodopsin in different membrane environments and show that these 2 protonation switches become partially uncoupled at physiological temperature. This partial uncoupling leads to approximately 50% population of an entropy-stabilized Meta II state in which the interhelical PSB salt bridge is broken and activating helix movements have taken place but in which Glu-134 remains unprotonated. This partial activation is converted to full activation only by coupling to the pH-dependent protonation of Glu-134 from the solvent, which stabilizes the active receptor conformation by lowering its enthalpy. In a membrane environment, protonation of Glu-134 is therefore a thermodynamic rather than a structural prerequisite for activating helix movements. In light of the conservation of the E(D)RY motif in rhodopsin-like GPCRs, protonation of this carboxylate also may serve a similar function in signal transduction of other members of this receptor family.

Functional role of the "ionic lock"--an interhelical hydrogen-bond network in family A heptahelical receptors.

Activation of family A G-protein-coupled receptors involves a rearrangement of a conserved interhelical cytoplasmic hydrogen bond network between the E(D)RY motif on transmembrane helix 3 (H3) and residues on H6, which is commonly termed the cytoplasmic "ionic lock." Glu134(3.49) of the E(D)RY motif also forms an intrahelical salt bridge with neighboring Arg135(3.50) in the dark-state crystal structure of rhodopsin. We examined the roles of Glu134(3.49) and Arg135(3.50) on H3 and Glu247(6.30) and Glu249(6.32) on H6 on the activation of rhodopsin using Fourier transform infrared spectroscopy of wild-type and mutant pigments reconstituted into lipid membranes. Activation of rhodopsin is pH-dependent with proton uptake during the transition from the inactive Meta I to the active Meta II state. Glu134(3.49) of the ERY motif is identified as the proton-accepting group, using the Fourier transform infrared protonation signature and the absence of a pH dependence of activation in the E134Q mutant. Neutralization of Arg135(3.50) similarly leads to pH-independent receptor activation, but with structural alterations in the Meta II state. Neutralization of Glu247(6.30) and Glu249(6.32) on H6, which are involved in interhelical interactions with H3 and H7, respectively, led to a shift toward Meta II in the E247Q and E249Q mutants while retaining the pH sensitivity of the equilibrium. Disruption of the interhelical interaction of Glu247(6.30) and Glu249(6.32) on H6 with H3 and H7 plays its role during receptor activation, but neutralization of the intrahelical salt bridge between Glu134(3.49) and Arg135(3.50) is considerably more critical for shifting the photoproduct equilibrium to the active conformation. These conclusions are discussed in the context of recent structural data of the beta(2)-adrenergic receptor.

Structural impact of the E113Q counterion mutation on the activation and deactivation pathways of the G protein-coupled receptor rhodopsin.

Disruption of an interhelical salt bridge between the retinal protonated Schiff base linked to H7 and Glu113 on H3 is one of the decisive steps during activation of rhodopsin. Using previously established stabilization strategies, we engineered a stabilized E113Q counterion mutant that converted rhodopsin to a UV-absorbing photoreceptor with deprotonated Schiff base and allowed reconstitution into native-like lipid membranes. Fourier-transform infrared difference spectroscopy reveals a deprotonated Schiff base in the photoproducts of the mutant up to the active state Meta II, the absence of the classical pH-dependent Meta I/Meta II conformational equilibrium in favor of Meta II, and an anticipation of active state features under conditions that stabilize inactive photoproduct states in wildtype rhodopsin. Glu181 on extracellular loop 2, is found to be unable to maintain a counterion function to the Schiff base on the activation pathway of rhodopsin in the absence of the primary counterion, Glu113. The Schiff base becomes protonated in the transition to Meta III. This protonation is, however, not associated with a deactivation of the receptor, in contrast to wildtype rhodopsin. Glu181 is suggested to be the counterion in the Meta III state of the mutant and appears to be capable of stabilizing a protonated Schiff base in Meta III, but not of constraining the receptor in an inactive conformation.

Interaction of a G protein-coupled receptor with a G protein-derived peptide induces structural changes in both peptide and receptor: a Fourier-transform infrared study using isotopically labeled peptides.

G protein-coupled receptor signaling involves productive interaction between agonist-activated receptor and G protein. We have used Fourier-transform infrared difference spectroscopy to examine the interaction between the active Meta II state of the visual pigment rhodopsin with a peptide analogue corresponding to the C terminus of the alpha-subunit of the G protein transducin. Formation of the receptor-peptide complex evokes a spectral signature consisting of conformationally sensitive amide I and amide II difference bands. In order to distinguish between amide backbone contributions of the peptide and of the receptor moiety to the vibrational spectra, we employed complete (13)C,(15)N-labeling of the peptide. This isotopic labeling downshifts selectively the bands of the peptide, which can thus be extracted. Our results show that formation of the complex between the activated Meta II receptor state and the peptide is accompanied by structural changes of the peptide, and of the receptor, indicating that the conformation of the Meta II.peptide complex is different from that of Meta II. This result implies that the activated receptor state has conformational flexibility. Binding of the peptide to the activated receptor state stabilizes a substate that deviates from that stabilized only by the agonist.

The all-trans-15-syn-retinal chromophore of metarhodopsin III is a partial agonist and not an inverse agonist.

Meta III is formed during the decay of rhodopsin's active receptor state at neutral to alkaline pH by thermal isomerization of the retinal Schiff base C15=N bond, converting the ligand from all-trans 15-anti to all-trans 15-syn. The thereby induced change of ligand geometry switches the receptor to an inactive conformation, such that the decay pathway to Meta III contributes to the deactivation of the signaling state at higher pH values. We have examined the conformation of Meta III over a wider pH range and found that Meta III exists in a pH-dependent conformational equilibrium between this inactive conformation at neutral to alkaline pH and an active conformation similar to that of Meta II, which, however, is assumed at very acidic pH only. The apparent pKa of this transition is around 5.1 and thus several units lower than that of the Meta I/Meta II photoproduct equilibrium with its all-trans 15-anti ligand, but still about 1 unit higher than that of the opsin conformational equilibrium in the absence of ligand. The all-trans-15-syn-retinal chromophore is therefore not an inverse agonist like 11-cis- or 9-cis-retinal, which lock the receptor in an inactive conformation, but a classical partial agonist, which is capable of activating the receptor, yet with an efficiency considerably lower than the full agonist all-trans 15-anti. As the Meta III chromophore differs structurally from this full agonist only in the isomeric state of the C15=N bond, this ligand represents an excellent model system to study principal mechanisms of partial agonism which are helpful to understand the partial agonist behavior of other ligands.

Characterization of phylogenetically distant members of the adenylate cyclase family from mycobacteria: Rv1647 from Mycobacterium tuberculosis and its orthologue ML1399 from M. leprae.

Analysis of the genome sequence of Mycobacterium tuberculosis H37Rv has identified 16 genes that are similar to the mammalian adenylate and guanylate cyclases. Rv1647 was predicted to be an active adenylate cyclase but its position in a phylogenetically distant branch from the other enzymes characterized so far from M. tuberculosis makes it an interestingly divergent nucleotide cyclase to study. In agreement with its divergence at the sequence level from other nucleotide cyclases, the cloning, expression and purification of Rv1647 revealed differences in its biochemical properties from the previously characterized Rv1625c adenylate cyclase. Adenylate cyclase activity of Rv1647 was activated by detergents but was resistant to high concentrations of salt. Mutations of substrate-specifying residues to those present in guanylate cyclases failed to convert the enzyme into a guanylate cyclase, and did not alter its oligomeric status. Orthologues of Rv1647 could be found in M. leprae, M. avium and M. smegmatis. The orthologue from M. leprae (ML1399) was cloned, and the protein was expressed, purified and shown biochemically to be an adenylate cyclase, thus representing the first adenylate cyclase to be described from M. leprae. Importantly, Western-blot analysis of subcellular fractions from M. tuberculosis and M. leprae revealed that the Rv1647 and ML1399 gene products respectively were expressed in these bacteria. Additionally, M. tuberculosis was also found to express the Rv1625c adenylate cyclase, suggesting that multiple adenylate cyclase proteins may be expressed simultaneously in this organism. These results suggest that class III cyclase-like gene products probably have an important role to play in the physiology and perhaps the pathology of these medically important bacteria.