Technological advances in site-directed spin labeling of proteins

Hubbell, Wayne L.; López, Carlos J.; Altenbach, Christian; Yang, Zhongyu

doi:10.1016/j.sbi.2013.06.008

Cited by 283 publications

(316 citation statements)

References 54 publications

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“…In the background of the rhodopsin C140S/C316S mutant, in which surface-exposed reactive cysteine residues were replaced by nonreactive serine residues, the R1 nitroxide side chain was introduced pairwise using SDSL methods (33) to monitor the intramolecular distances between a reference site at residue 74 in TM2 to sites 225, 252, and 308 in TM5, TM6, and TM7, respectively (Fig. 1B), using DEER spectroscopy.…”

Section: Resultsmentioning

confidence: 99%

Conformational equilibria of light-activated rhodopsin in nanodiscs

Eps

Lydia

Morizumi

et al. 2017

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

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Conformational equilibria of G-protein-coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron-electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH + form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20°C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.G -protein-coupled receptors (GPCRs) are central components of protein-protein interaction networks and can serve as hubs that link the extracellular environment of cells to intracellular signaling events (1). As such, they interact with several intracellular proteins (i.e., arrestins, G proteins, kinases). To obtain such diversity in molecular interaction, GPCRs are thought to be in equilibrium between multiple conformations at the cytoplasmic surface (2-4), enabling conformation-dependent interactions with different partners.The rod photoreceptor rhodopsin has served as a model for members in the class A family of GPCRs. In native membranes (5-7) and reconstituted liposomes (8, 9), photoactivated rhodopsin within milliseconds reaches a pH-dependent quasi-equilibrium between states designated MI and MII, which are distinguished by their optical absorbance maxima and signature absorbances in the infrared (10-12). The optically identified MII state has been found to consist of isochromic substates, namely MIIa, MIIb, and MIIbH + (13-15) (Fig. 1A). MIIbH + , populated at pH 6.0, is thought to be the functionally active substate that recognizes the cognate G-protein transducin (8,15,16).Early data relating global protein structural changes to activation in a GPCR were provided by continuous wave (CW) sitedirected spin labeling (SDSL)-Electron Paramagnetic Resonance (EPR) studies in rhodopsin, where it was shown that a generally outward motion of TM6 and a smaller inward motion of TM7 at the cytoplasmic surface were hallmarks of activation in dodecyl maltoside (DDM) micelles (17, 18). High-resolution distance mapping with SDSL and double electron...

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Section: Resultsmentioning

confidence: 99%

Conformational equilibria of light-activated rhodopsin in nanodiscs

Eps

Lydia

Morizumi

et al. 2017

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

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“…In the background of Hexa I Gαi1, in which all native surfaceexposed and reactive cysteine residues were mutated (26), SDSL methods (27) were used to introduce pairs of nitroxide side chains (R1) at selected sites in nonmyristoylated GDP-bound Gαi1; sites were selected pairwise from the set shown in Fig. 1.…”

Section: Resultsmentioning

confidence: 99%

The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

Eps

Thomas

Hubbell

et al. 2015

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

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Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (Gα), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β 2 adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP-and GTP-bound states of Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF.G protein | guanine nucleotide exchange factor | double electron electron resonance spectroscopy | tertiary structure | protein dynamics

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“…Coupled with site-directed spin-labeling (SDSL), EPR is oftentimes used to characterize protein and nucleic acid structures and dynamics, conformational changes, molecule folding, macromolecule complexes, and oligomeric structures [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]17]. The majority of biomolecules do not contain unpaired electrons from which one can obtain an EPR signal; therefore, spin-labeling approaches have been developed [15,[18][19][20][21][22][23][24][25] where site-specific persistent radicals or paramagnetic metal-probes are incorporated at specific locations within a biomolecule. Properties of the EPR spectra that originate from these probes, positioned at welldefined vantage points, provide structural and dynamic constraints.…”

Section: Introductionmentioning

confidence: 99%

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

Song

Liu

Kaur

et al. 2016

Journal of Magnetic Resonance

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High-field, high-frequency electron paramagnetic resonance (EPR) spectroscopy at W-(~95 GHz) and D-band (~140 GHz) is important for investigating the conformational dynamics of flexible biological macromolecules because this frequency range has increased spectral sensitivity to nitroxide motion over the 100 ps to 2 ns regime. However, low concentration sensitivity remains a roadblock for studying aqueous samples at high magnetic fields. Here, we examine the sensitivity of a non-resonant thin-layer cylindrical sample holder, coupled to a quasi-optical induction-mode W-band EPR spectrometer (HiPER), for continuous wave (CW) EPR analyses of: (i) the aqueous nitroxide standard, TEMPO; (ii) the unstructured to -helical transition of a model IDP protein; and (iii) the base-stacking transition in a kink-turn motif of a large 232 nt RNA. For sample volumes of ~50 L, concentration sensitivities of 2-20 µM were achieved, representing a ~10-fold enhancement compared to a cylindrical TE 011 resonator on a commercial Bruker W-band spectrometer. These results therefore highlight the sensitivity of the thin-layer sample holders employed in HiPER for spin-labeling studies of biological macromolecules at high fields, where applications can extend to other systems that are facilitated by the modest sample volumes and ease of sample loading and geometry.2

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Technological advances in site-directed spin labeling of proteins

Cited by 283 publications

References 54 publications

Conformational equilibria of light-activated rhodopsin in nanodiscs

Conformational equilibria of light-activated rhodopsin in nanodiscs

The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

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