Estimation of Inter-Residue Distances in Spin Labeled Proteins at Physiological Temperatures:  Experimental Strategies and Practical Limitations

Altenbach, Christian; Oh, Kyoung Joon; Trabanino, René J.; Hideg, Kálmán; Hubbell, Wayne L.

doi:10.1021/bi011544w

Cited by 238 publications

(308 citation statements)

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“…The main advantage of the SDSL approach is that spin interactions allow for distance estimates between nitroxide labels (23,24). Thus, if a single label is present on each protein molecule, EPR spectra provide crucial information about quaternary structure within the amyloid fibril (i.e., intermolecular contact sites and packing of individual protein monomers).…”

Section: Resultsmentioning

confidence: 99%

“…Thus, if a single label is present on each protein molecule, EPR spectra provide crucial information about quaternary structure within the amyloid fibril (i.e., intermolecular contact sites and packing of individual protein monomers). Typically, spin-spin interactions between nitroxide labels in proteins are dominated by dipolar effects that result in a broadening of EPR spectra, providing distance information in the range of Ϸ8-25 Å (23,24). A second type of interaction, spin exchange, occurs at much smaller distances, when on the EPR time scale (Ͻ10 Ϫ7 s) multiple nitroxide labels are in sufficient proximity to allow orbital overlap (25)(26)(27).…”

Section: Resultsmentioning

confidence: 99%

“…This is evidenced by multiple features of the central line, as well as a sharp component present in the downfield peak. Multiple nitroxide populations complicate precise distance measurements by standard deconvolution methods (23,24). Nevertheless, it is clear that sites outside the Ϸ160-220 core region do not possess the tight packing and high degree of order required for efficient spin exchange, and for all of them interspin distances are calculated to be Ͼ13 Å (for sites N-terminal to 118 above 17 Å).…”

Section: Resultsmentioning

confidence: 99%

“…Field modulation was varied from 1 to 2 G depending on the degree of spin label immobilization. When applicable, spectra in the dipolar broadening regime were analyzed to obtain interspin distance estimates using a previously described methodology (23). Spectra within individual panels were normalized to the same number of spins.…”

Section: Methodsmentioning

confidence: 99%

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Molecular architecture of human prion protein amyloid: A parallel, in-register β-structure

Cobb

Sönnichsen

Mchaourab³

et al. 2007

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

303

342

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Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative diseases that are associated with conformational conversion of the normally monomeric and ␣-helical prion protein, PrP C , to the ␤-sheet-rich PrP Sc . This latter conformer is believed to constitute the main component of the infectious TSE agent. In contrast to high-resolution data for the PrP C monomer, structures of the pathogenic PrP Sc or synthetic PrP Sc -like aggregates remain elusive. Here we have used sitedirected spin labeling and EPR spectroscopy to probe the molecular architecture of the recombinant PrP amyloid, a misfolded form recently reported to induce transmissible disease in mice overexpressing an N-terminally truncated form of PrP C . Our data show that, in contrast to earlier, largely theoretical models, the conformational conversion of PrP C involves major refolding of the C-terminal ␣-helical region. The core of the amyloid maps to C-terminal residues from Ϸ160 -220, and these residues form single-molecule layers that stack on top of one another with parallel, in-register alignment of ␤-strands. This structural insight has important implications for understanding the molecular basis of prion propagation, as well as hereditary prion diseases, most of which are associated with point mutations in the region found to undergo a refolding to ␤-structure.EPR ͉ spin labeling ͉ transmissible spongiform encephalopathy

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Molecular architecture of human prion protein amyloid: A parallel, in-register β-structure

Cobb

Sönnichsen

Mchaourab³

et al. 2007

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

303

342

View full text Add to dashboard Cite

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“…In previous SDSL studies of rhodopsin directed at detecting conformational changes via distance measurements (13,24,25), the short range (Ͻ20 Å) of the continuous-wave (CW) dipolar interaction method used (26,27) imposed constraints on the experimental design that could limit the effective resolution. In particular, it was necessary to place spin labels at buried sites in the molecule in order for them to be sufficiently close for interaction, raising the possibility that spin-label perturbation and/or rearrangement of the spin-label side chain itself contributed to the distance changes observed upon activation.…”

mentioning

confidence: 99%

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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428

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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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Pulsed EPR Distance Measurements in Soluble Proteins by Site‐Directed Spin Labeling (SDSL)

et al. 2013

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The resurgence of pulsed electron paramagnetic resonance (EPR) in structural biology centers on recent improvements in distance measurements using the double electron-electron resonance (DEER) technique. This unit focuses on EPR-based distance measurements by site-directed spin-labeling (SDSL) of engineered cysteine residues in soluble proteins, with HIV-1 protease used as a model. To elucidate conformational changes in proteins, experimental protocols were optimized and existing data analysis programs were employed to derive distance distribution profiles. Experimental considerations, sample preparation and error analysis for artifact suppression are also outlined here.

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Estimation of Inter-Residue Distances in Spin Labeled Proteins at Physiological Temperatures: Experimental Strategies and Practical Limitations

Cited by 238 publications

References 29 publications

Molecular architecture of human prion protein amyloid: A parallel, in-register β-structure

Molecular architecture of human prion protein amyloid: A parallel, in-register β-structure

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

Pulsed EPR Distance Measurements in Soluble Proteins by Site‐Directed Spin Labeling (SDSL)

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