Electron Paramagnetic Resonance Studies of Functionally Active, Nitroxide Spin-Labeled Peptide Analogues of the C-Terminus of a G-Protein α Subunit

Eps, Ned Van; Anderson, Lori L.; Kisselev, Oleg G.; Baranski, Thomas J.; Hubbell, Wayne L.; Marshall, Garland R.

doi:10.1021/bi100846c

Cited by 27 publications

(39 citation statements)

References 69 publications

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“…One striking molecular feature in the x-ray structure of the ␤ 2 AR-G s complex is that G␣AH is completely displaced from G␣Ras and moves in a direction toward the N terminus of G␣ near the IC membrane surface. Such displacement was predicted from the x-ray structure of G␣ (4) and experimentally confirmed by site-directed spin labeling and deuterium exchange mass spectroscopy studies (15). GPCR-mediated GDP-GTP exchange, where GDP release is the rate-determining step (16), is the key event in the G protein activation cycle.…”

mentioning

confidence: 68%

Molecular Basis of Cannabinoid CB1 Receptor Coupling to the G Protein Heterotrimer Gαiβγ

Shim

Ahn

Kendall

2013

Journal of Biological Chemistry

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Background: The molecular basis of CB1 coupling to its cognate G protein is unknown. Results: Using an approach combining mutagenesis and molecular dynamics simulations, we identified CB1 residues critical for G protein signaling. Conclusion: Tight interactions between CB1 and the C-terminal helix ␣ 5 of G␣ i are crucial for G protein signaling. Significance: This is the first reported molecular description of CB1 receptor coupling at the receptor-G i interface.

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mentioning

confidence: 68%

Molecular Basis of Cannabinoid CB1 Receptor Coupling to the G Protein Heterotrimer Gαiβγ

Shim

Ahn

Kendall

2013

Journal of Biological Chemistry

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“…3. Previous studies demonstrate that the C terminus undergoes a disorder-to-order transition upon binding to activated receptors, inducing structural changes that are important for efficient GDP release (27)(28)(29). G αi with a flexible 5-glycine linker inserted at the base of the α5 helix (at residue 343, Fig.…”

Section: Resultsmentioning

confidence: 99%

Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit

Eps

Preininger

Alexander

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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150

149

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In G-protein signaling, an activated receptor catalyzes GDP/GTP exchange on the G α subunit of a heterotrimeric G protein. In an initial step, receptor interaction with G α acts to allosterically trigger GDP release from a binding site located between the nucleotide binding domain and a helical domain, but the molecular mechanism is unknown. In this study, site-directed spin labeling and double electron-electron resonance spectroscopy are employed to reveal a large-scale separation of the domains that provides a direct pathway for nucleotide escape. Cross-linking studies show that the domain separation is required for receptor enhancement of nucleotide exchange rates. The interdomain opening is coupled to receptor binding via the C-terminal helix of G α , the extension of which is a high-affinity receptor binding element.signal transduction | structural polymorphism T he α-subunit (G α ) of heterotrimeric G proteins (G αβγ ) mediates signal transduction in a variety of cell signaling pathways (1). Multiple conformational states of G α are involved in the signal transduction pathway shown in Fig. 1A. In the inactive state, the G α subunit contains a bound GDP [G α ðGDPÞ] and has a high affinity for G βγ . When activated by an appropriate signal, a membrane-bound G-protein coupled receptor (GPCR) binds the heterotrimer in a quaternary complex, leading to the dissociation of GDP and formation of an "empty complex" [G α ð0Þ βγ ], which subsequently binds GTP. The affinity of G α ðGTPÞ for G βγ is dramatically reduced relative to G α ðGDPÞ, resulting in functional dissociation of active G α ðGTPÞ from the membrane-bound complex. The active G α ðGTPÞ subsequently binds downstream effector proteins to trigger a variety of regulatory events, depending on the particular system. Thus, the GPCR acts to catalyze GDP/GTP exchange via an empty complex. Crystallographic (2-7), biochemical (8), and biophysical (9-11) studies have elucidated details of the conformational states of G α that correspond to the discrete steps indicated in Fig. 1A, but the mechanism by which receptor interaction leads to release of the bound GDP from G α and the structure of the empty complex remain a major target of research in the field.The G α subunit has two structural domains, namely a nucleotide binding domain and a helical domain that partially occludes the bound nucleotide (Fig. 1B). From the initial G α crystal structure in 1993, Noel et al. (2) recognized that nucleotide release would probably require an opening between the two domains in the empty complex, but in the intervening 18 years there has been little compelling experimental support for this idea. Nevertheless, some constraints on the general topology of the complex are known. For example, numerous studies indicate that the C terminus of G α is bound tightly to the receptor in the empty complex (9). In addition, the N-terminal helix of G α is associated with G βγ and with the membrane via N-terminal myristoylation (12,13). Together, these constraints fix the position of the nucleot...

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“…For these purposes, a number of laboratories are exploring the use of an amino acid with a 6-membered nitroxide ring called TOAC (2,2,6,6-tetramethylpiperidine-1-oxy-4-amino-4-carboxylic acid), which can be incorporated into peptides during synthesis and is directly fused to the peptide backbone (48)(49)(50). Unlike RX, however, TOAC cannot be easily incorporated into selected sites of large proteins.…”

Section: Discussionmentioning

confidence: 99%

Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy

Fleissner

Bridges

Brooks

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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250

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A disulfide-linked nitroxide side chain (R1) is the most widely used spin label for determining protein topology, mapping structural changes, and characterizing nanosecond backbone motions by site-directed spin labeling. Although the internal motion of R1 and the number of preferred rotamers are limited, translating interspin distance measurements and spatial orientation information into structural constraints is challenging. Here, we introduce a highly constrained nitroxide side chain designated RX as an alternative to R1 for these applications. RX is formed by a facile cross-linking reaction of a bifunctional methanethiosulfonate reagent with pairs of cysteine residues at i and i þ 3 or i and i þ 4 in an α-helix, at i and i þ 2 in a β-strand, or with cysteine residues in adjacent strands in a β-sheet. Analysis of EPR spectra, a crystal structure of RX in T4 lysozyme, and pulsed electron-electron double resonance (ELDOR) spectroscopy on an immobilized protein containing RX all reveal a highly constrained internal motion of the side chain. Consistent with the constrained geometry, interspin distance distributions between pairs of RX side chains are narrower than those from analogous R1 pairs. As an important consequence of the constrained internal motion of RX, spectral diffusion detected with ELDOR reveals microsecond internal motions of the protein. Collectively, the data suggest that the RX side chain will be useful for distance mapping by EPR spectroscopy, determining spatial orientation of helical segments in oriented specimens, and measuring structural fluctuations on the microsecond time scale.pulsed EPR | protein dynamics S ite-directed spin labeling (SDSL) is a general method for characterizing protein topography, local and global structure, and dynamics by electron paramagnetic resonance (EPR) spectroscopy (1-5). SDSL can be applied to both soluble and membrane proteins of arbitrary molecular weight under physiological conditions. In traditional SDSL, a unique cysteine residue is introduced into a recombinant protein via site-directed mutagenesis, and subsequently reacted with a sulfhydryl-specific nitroxide reagent to generate a paramagnetic side chain. In addition, an SDSL strategy based on a genetically encoded unnatural amino acid was recently reported, a method that allows labeling in the presence of native thiols (6).The most widely used spin label for SDSL studies is a disulfidelinked side chain designated R1, in part because its inherent flexibility allows for introduction at virtually any site within a protein-even buried ones-typically with no more energetic cost than a natural amino acid substitution at that site (7,8). Indeed, R1 has proven useful for determining protein topology (2, 9), characterizing nanosecond backbone motions (4,10,11), mapping structural changes (3,(12)(13)(14), and predicting protein structure de novo (15). Although the amplitude of nanosecond internal motion and the number of preferred rotamers of R1 are limited (16), these features nevertheless make it problematic t...

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Electron Paramagnetic Resonance Studies of Functionally Active, Nitroxide Spin-Labeled Peptide Analogues of the C-Terminus of a G-Protein α Subunit

Cited by 27 publications

References 69 publications

Molecular Basis of Cannabinoid CB1 Receptor Coupling to the G Protein Heterotrimer Gαiβγ

Molecular Basis of Cannabinoid CB1 Receptor Coupling to the G Protein Heterotrimer Gαiβγ

Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit

Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy

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