Time-Resolved Detection of Transient Movement of Helices F and G in Doubly Spin-Labeled Bacteriorhodopsin

Radzwill, Nicole; Gerwert, Klaus; Steinhoff, Heinz‐Jürgen

doi:10.1016/s0006-3495(01)76252-2

Cited by 115 publications

(118 citation statements)

References 47 publications

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“…For K55R1 A364R1, the percentage of the fractions with and without dipolar broadening is given in brackets. Distance values estimated from the spectral second moment 20 (denoted with asterisk) indicate the presence of interspin distances <1.2 nm. Distance distribution width was fixed to 0.3 nm for the fittings of the CW EPR spectra.…”

Section: (I)mentioning

confidence: 99%

Conformational heterogeneity of the aspartate transporter GltPh

Hänelt

Wunnicke

Bordignon

et al. 2013

Nat Struct Mol Biol

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104

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Glt Ph is a Pyrococcus horikoshii homotrimeric Na + -coupled aspartate transporter that belongs to the glutamate transporter family. Each protomer consists of a trimerization domain involved in subunit interaction and a transporting domain with the substrate-binding site. Here, we have studied the conformational changes underlying transport by Glt Ph using EPR spectroscopy. The trimerization domains form a rigid scaffold, whereas the transporting domains sample multiple conformations, consistent with large-scale movements during the transport cycle. Binding of substrates changed the occupancies of the different conformational states, but the domains remained heterogeneous. The membrane environment favored conformations different from those observed in detergent micelles, but the transporting domain remained structurally heterogeneous in both environments. We conclude that the transporting domains sample multiple conformational states with substantial occupancy regardless of the presence of substrate and coupling ions, consistent with equilibrium constants close to unity between the observed transporter conformations. npg © 2013 Nature America, Inc. All rights reserved.nature structural & molecular biology VOLUME 20 NUMBER 2 FEBRUARY 2013 2 1 1 a r t i c l e s of 160 K to qualitatively extract information on interspin distances <1.8 nm (ref. 14). We analyzed Glt Ph both solubilized in detergent micelles and reconstituted in proteoliposomes and compared the results from each experiment type. The trimerization domain is a stable scaffoldIntroduction of a spin label at a single position in Glt Ph allows for the measurement of the distance between the protomers of the homotrimeric protein. We constructed two trimerization-domain mutants by replacing Thr166 (located in the cytoplasmic end of transmembrane helix 4) and Val176 (located in the cytoplasmic end of helix 5) with an MTSL-modified cysteine (R1). Simulations on the available crystal structures indicated that these spin labels are >1.8 nm apart and therefore suitable for DEER measurements 15 . We did not observe spectral broadening in the CW EPR measurements, which further supports the notion that the spin labels were >1.8 nm apart (Supplementary Fig. 1). We recorded spectra of the apoprotein and of the protein in the presence of saturating concentrations of Na + alone or Na + and aspartate. In agreement with previous biochemical and structural data 12,13 , the trimerization domain did not show any large-scale conformational changes upon addition of coupling ions or substrate (Fig. 2). We observed two sharp peaks (centered around 2.8 nm and 3.8 nm for T166R1 and 2.6 nm and 3.4 nm for V176R1) in the interspin distance distributions for proteins in the presence and absence of substrates. The measured distances were in agreement with the interprotomer distances calculated on the basis of the crystal structures (Table 1 and Fig. 2).Because Glt Ph is a trimer, each mutation resulted in the presence of three cysteines per protein complex, which could result in a cons...

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Section: (I)mentioning

confidence: 99%

Conformational heterogeneity of the aspartate transporter GltPh

Hänelt

Wunnicke

Bordignon

et al. 2013

Nat Struct Mol Biol

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104

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“…In principle, one could combine measurements of average splitting (i.e., absolute first moment) and second moment to give estimates of both hr 12 i and r r 12 by combining Eqs. (10) and (11). But it is important to do this with the same spectral dataset.…”

Section: Resultsmentioning

confidence: 99%

“…In cases where the deconvoluted powder line shape is relatively featureless, a simplified analysis has been introduced that is based on the average splitting [6,9]. A further simplified approach is to determine the dipolar contribution to the spectral second moment [10,11], for which an analytical solution is available in the manybody case [12].…”

Section: Introductionmentioning

confidence: 99%

Intermediate dipolar distances from spin labels

Marsh

2014

Journal of Magnetic Resonance

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a b s t r a c tMethods for determining inter-spin distances between nitroxide spin labels from dipolar couplings in the intermediate range (r 12 % 1.1-2 nm) by CW-EPR are addressed. For nitroxide powder patterns, the assumption of unlike spins is a better approximation than assuming strong coupling between like spins. Methods that determine the average splitting in dipolar deconvolutions yield the mean value h1=r 3 12 i, and those that determine the dipolar contribution to the spectral second moment correspondingly yield h1=r 6 12 i. To relate these ensemble averages to the mean inter-spin distance hr 12 i requires knowledge of the distribution in r 12 . Values of the inverse roots r eff ;n ¼ h1=rÀ1=n always lie below hr 12 i. Consistent comparisons of literature data on double-labelled a-helical peptides support this view. Evaluations of r eff,n for Gaussian distributions in r 12 yield calibrations to determine hr 12 i for a given distribution width, r r 12 .

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“…For this purpose the spectral line amplitude ratios or the spectral second moment values are calculated, which identifies spin probes in close or distant proximity. This is commonly executed for a series of double-labeled protein [37] as this approach does not allow for the determination of the exact interspin distance values. Interspin distance values below 0.8 nm are not reliable due to the increased partial overlap of the nitrogen π-orbitals of the two interacting spin label side chains [38].…”

Section: Interspin Distance Measurementsmentioning

confidence: 99%

The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins

Wunnicke

Hänelt

2017

Crystals

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Protein structures as provided by structural biology such as X-ray crystallography, cryo-electron microscopy and NMR spectroscopy are key elements to understand the function of a protein on the molecular level. Nonetheless, they might be error-prone due to crystallization artifacts or, in particular in case of membrane-imbedded proteins, a mostly artificial environment. In this review, we will introduce different EPR spectroscopy methods as powerful tools to complement and validate structural data gaining insights in the dynamics of proteins and protein complexes such that functional cycles can be derived. We will highlight the use of EPR spectroscopy on membrane-embedded proteins and protein complexes ranging from receptors to secondary active transporters as structural information is still limited in this field and the lipid environment is a particular challenge.

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Time-Resolved Detection of Transient Movement of Helices F and G in Doubly Spin-Labeled Bacteriorhodopsin

Cited by 115 publications

References 47 publications

Conformational heterogeneity of the aspartate transporter GltPh

Conformational heterogeneity of the aspartate transporter GltPh

Intermediate dipolar distances from spin labels

The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins

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