Structural determinants of nitroxide motion in spin‐labeled proteins: Tertiary contact and solvent‐inaccessible sites in helix G of T4 lysozyme

Guo, Zhefeng; Cascio, Duilio; Hideg, Kálmán; Kálai, Tamás; Hubbell, Wayne L.

doi:10.1110/ps.062739107

Cited by 103 publications

(209 citation statements)

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“…The EPR spectra for the selected sites show little or no change upon photoactivation, as previously reported (12,(37)(38)(39)(40). Because the spectra are sensitive to changes in backbone dynamics (41,42) and local interactions within the protein (29)(30)(31)43), the lack of spectral change upon photoactivation strongly suggests that the conformation of R1, the immediate local environment, and the secondary structure to which it is attached remain the same under rhodopsin activation. Thus, changes in interspin distances between pairs of these nitroxides report relative motion of the structures to which they are attached.…”

Section: Experimental Design and Resultssupporting

confidence: 71%

“…1), and interspin distances were measured in the inactive (R) and light-activated state (R*), using DEER spectroscopy. A global analysis of the data localized individual spins in the structure, and the results for R are in excellent agreement with the rhodopsin crystal structure from Li et al [Protein Data Bank (PDB) entry 1GZM] (8) and models of R1 based on the known structure of the side chain (29)(30)(31)(32). For R*, the data reveal an outward motion of R1 in TM6 by 5 Å and smaller movements of R1 in TM1, TM7, and in the C-terminal domain following helix H8.…”

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confidence: 73%

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High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

Altenbach

Kusnetzow

Ernst

et al. 2008

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

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431

454

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Site-directed spin labeling has qualitatively shown that a key event during activation of rhodopsin is a rigid-body movement of transmembrane helix 6 (TM6) at the cytoplasmic surface of the molecule. To place this result on a quantitative footing, and to identify movements of other helices upon photoactivation, double electron-electron resonance (DEER) spectroscopy was used to determine distances and distance changes between pairs of nitroxide side chains introduced in helices at the cytoplasmic surface of rhodopsin. Sixteen pairs were selected from a set of nine individual sites, each located on the solvent exposed surface of the protein where structural perturbation due to the presence of the label is minimized. Importantly, the EPR spectra of the labeled proteins change little or not at all upon photoactivation, suggesting that rigid-body motions of helices rather than rearrangement of the nitroxide side chains are responsible for observed distance changes. For inactive rhodopsin, it was possible to find a globally minimized arrangement of nitroxide locations that simultaneously satisfied the crystal structure of rhodopsin (Protein Data Bank entry 1GZM), the experimentally measured distance data, and the known rotamers of the nitroxide side chain. A similar analysis of the data for activated rhodopsin yielded a new geometry consistent with a 5-Å outward movement of TM6 and smaller movements involving TM1, TM7, and the C-terminal sequence following helix H8. The positions of nitroxides in other helices at the cytoplasmic surface remained largely unchanged.DEER ͉ G protein-coupled receptor ͉ photoreceptor ͉ spin labeling G protein-coupled receptors (GPCRs) constitute a large family of membrane proteins that mediate cellular responses to a variety of both physical and chemical signals.Interaction of a GPCR with a cognate signal produces a conformational change leading to an activated receptor. The activated state subsequently binds to and activates a corresponding heterotrimeric G protein, which in turn triggers events that ultimately modulate an amplified cellular response (for recent reviews, see refs. 1 and 2). The nature of the conformational switch or switches that underlie receptor activation is a subject of much interest (3, 4). Crystal structures of an inactive state of the GPCRs rhodopsin (5-9) and ␤ 2 -adrenergic receptor (10, 11) have been reported, and they provide a crucial foundation for designing and interpreting the results of spectroscopic experiments aimed at revealing conformational changes associated with activation.Site-directed spin-labeling (SDSL) studies of rhodopsin provided the first direct structural evidence for an outward tilt of the cytoplasmic end of transmembrane helix 6 (TM6) as a major event in the activation of rhodopsin, both in solutions of dodecyl maltoside (DM) and in phospholipid bilayers (12-15). Data from site-directed fluorescent labeling and chemical reactivity (16), UV spectroscopy (17), zinc cross-linking of histidines (18), and disulfide cross-linking (13) offered s...

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Section: Experimental Design and Resultssupporting

confidence: 71%

supporting

confidence: 73%

High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

Altenbach

Kusnetzow

Ernst

et al. 2008

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

Self Cite

431

454

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“…1A shows a model of the L121A/L133A mutant based on the crystal structure, along with the location of R1 sensor sites and the spectra compared with those of the WT′. The spectra for the WT′ protein have been published previously and interpreted in terms of the protein structure and dynamics (16)(17)(18)(19)(20); the basic principles of spectral analysis are outlined in SI Materials and Methods. The spectra of 131R1, 132R1, 140R1, and 151R1 in the WT′ reflect a simple anisotropic motion characteristic of R1 at solvent-exposed sites in relatively rigid helical segments, whereas the spectra of 48R1, 109R1, 123R1, 128R1, and 135R1 reflect higher mobility of the nitroxide consistent with their location near or at the helix termini (16).…”

Section: Experimental Strategy and Resultsmentioning

confidence: 99%

“…1A, Inset) was introduced at solvent-exposed sites to serve as a sensor of local structure. At such sites, R1 typically has weak or no interactions with the protein, as reflected by a singlecomponent electron paramagnetic resonance (EPR) spectrum, and introduces little structural perturbation (16)(17)(18)(19). The R1 sensor Significance Analysis of protein packing reveals the prevalence of cavities, some of which are evolutionarily conserved.…”

mentioning

confidence: 99%

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants

López

Yang

Altenbach

et al. 2013

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

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The studies presented here explore the relationship between protein packing and molecular flexibility using ligand-binding cavity mutants of T4 lysozyme. Although previously reported crystal structures of the mutants investigated show single conformations that are similar to the WT protein, site-directed spin labeling in solution reveals additional conformational substates in equilibrium exchange with a WT-like population. Remarkably, binding of ligands, including the general anesthetic halothane shifts the population to the WT-like state, consistent with a conformational selection model of ligand binding, but structural adaptation to the ligand is also apparent in one mutant. Distance mapping with double electron-electron resonance spectroscopy and the absence of ligand binding suggest that the new substates induced by the cavity-creating mutations represent alternate packing modes in which the protein fills or partially fills the cavity with side chains, including the spin label in one case; external ligands compete with the side chains for the cavity space, stabilizing the WT conformation. The results have implications for mechanisms of anesthesia, the response of proteins to hydrostatic pressure, and protein engineering.EPR | site-directed spin labeling | DEER | benzene | saturation recovery G lobular proteins have overall packing densities similar to those of crystalline solids (1) but nevertheless contain cavities and pockets that can range from a few to hundreds of cubic angstroms (2, 3). These native packing defects are generally destabilizing (4, 5), and improving the packing of the protein interior may be a general way to increase protein stability. Indeed, small-to-large mutations that fill native cavities can increase stability (6-8), but with a concomitant loss of function (7,8). Thus, cavities in the protein interior can play a critical role in function. One role of cavities may be to allow alternative packing arrangements of the core. The resulting ensemble of conformational substates could give rise to promiscuity in protein-protein interactions (9) and allosteric behavior (7,8,10). In addition, large-to-small mutations that generate cavities may have played an important role in the evolution of protein function (11).Considering the potentially important role of cavities in sculpting the energy landscape of proteins, the present study was undertaken to investigate, in solution, the structural and dynamical response of a protein to engineered cavities introduced by large-tosmall mutations. T4 lysozyme (T4L) was selected for study because of the large database of crystal structures of ligand-binding cavity mutations and the corresponding thermodynamic characterization from Matthews and coworkers (4,(12)(13)(14)(15). Remarkably, comparison of the WT and mutant structures showed very little difference or limited local relaxation near the cavity, although the mutations were strongly destabilizing (4, 15).In the present study, we examined the cavity-creating mutants L121A/L133A, L133G, and W138A in sol...

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“…Structure of the nitroxide side chain R1. The label is linked to the protein backbone through five rotatable bonds; however, interactions between the distal sulfur and the Ca proton 20 restrict rotameric conversions to X4 and X5. 21 differences that are seen between spectroscopic and crystallographic methods and suggest that regions where the two crystal structures of BtuB differ represent sites of conformational exchange.…”

Section: Introductionmentioning

confidence: 99%

Osmolytes modulate conformational exchange in solvent‐exposed regions of membrane proteins

Jiménez¹,

Cao²,

Kim³

et al. 2010

Protein Science

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Site-directed spin labeling (SDSL) was used to investigate local structure and conformational exchange in two bacterial outer-membrane TonB-dependent transporters, BtuB and FecA. Protecting osmolytes, such as polyethylene glycols (PEGs) are known to modulate a substrate-dependent conformational equilibrium in the energy coupling motif (Ton box) of BtuB. Here, we demonstrate that a segment that is N-terminal to the Ton box in BtuB, is in conformational exchange between ordered and disordered states with or without substrate. Protecting osmolytes shift this equilibrium to favor the more ordered, folded state. However, a segment of BtuB that is C-terminal to the Ton box that is not solvent exposed is insensitive to PEGs. Protecting osmolytes also modulate a conformational equilibrium in the Ton box of FecA, with larger molecular weight PEGs producing the largest shifts in the conformational free energy. These data indicate that solvent-exposed regions of these transporters undergo conformational exchange and that regions of these transporters that are involved in protein-protein interactions sample multiple conformational substates. The sensitivity to solute provides an explanation for differences seen between two high-resolution structures of BtuB, which each likely represent one conformation from a subset of states that are normally sampled by the protein. This work also illustrates how SDSL and osmolytes may be used to characterize and quantitate conformational equilibria in membrane proteins.

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Structural determinants of nitroxide motion in spin‐labeled proteins: Tertiary contact and solvent‐inaccessible sites in helix G of T4 lysozyme

Cited by 103 publications

References 64 publications

High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants

Osmolytes modulate conformational exchange in solvent‐exposed regions of membrane proteins

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