Determination of interspin distances between spin labels attached to insulin: comparison of electron paramagnetic resonance data with the X-ray structure

Steinhoff, Heinz‐Jürgen; Radzwill, Nicole; Thevis, W.; Lenz, V.; Brandenburg, Dietrich; Antson, A.A.; Dodson, G.G.; Wollmer, Axel

doi:10.1016/s0006-3495(97)78353-x

Cited by 161 publications

(184 citation statements)

References 29 publications

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“…Simulated dipolar broadened EPR spectra were fitted to experimental low temperature CW EPR spectra considering a Gaussian distribution of interspin distances using DipFit 24 . Best-fit parameters for interspin distance distributions were determined.…”

Section: Methodsmentioning

confidence: 99%

Conformational heterogeneity of the aspartate transporter GltPh

Hänelt

Wunnicke

Bordignon

et al. 2013

Nat Struct Mol Biol

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: Methodsmentioning

confidence: 99%

Conformational heterogeneity of the aspartate transporter GltPh

Hänelt

Wunnicke

Bordignon

et al. 2013

Nat Struct Mol Biol

104

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“…Fitting of Simulated EPR Spectra-Fitting of simulated dipolar broadened EPR powder spectra to the experimental ones detected at 160 K revealed the average inter-spin distance according to the method described previously (33). To account for a range of distances expected to arise from different spin label side chain orientations, a Gaussian distribution of interspin distances with a distribution width of 0.2 nm is assumed.…”

mentioning

confidence: 99%

Salt-driven Equilibrium between Two Conformations in the HAMP Domain from Natronomonas pharaonis

Doebber

Bordignon

Klare

et al. 2008

Journal of Biological Chemistry

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HAMP domains (conserved in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) perform their putative function as signal transducing units in diversified environments in a variety of protein families. Here the conformational changes induced by environmental agents, namely salt and temperature, on the structure and function of a HAMP domain of the phototransducer from Natronomonas pharaonis (NpHtrII) in complex with sensory rhodopsin II (NpSRII) were investigated by site-directed spin labeling electron paramagnetic resonance. A series of spin labeled mutants were engineered in NpHtrII 157 , a truncated analog containing only the first HAMP domain following the transmembrane helix 2. This truncated transducer is shown to be a valid model system for a signal transduction domain anchored to the transmembrane light sensor NpSRII. The HAMP domain is found to be engaged in a "two-state" equilibrium between a highly dynamic (dHAMP) and a more compact (cHAMP) conformation. The structural properties of the cHAMP as proven by mobility, accessibility, and intra-transducer-dimer distance data are in agreement with the four helical bundle NMR model of the HAMP domain from Archaeoglobus fulgidus.

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“…The resulting interspin distances determined for crystallized insulins in the R6 and T6 structure agreed well with structural data obtained by X-ray crystallography and by modeling of the spin-labeled samples. The EPR experiments revealed differences between crystal and frozen solution structures of the B-chain amino termini in the R6 and T6 states of hexameric insulins (Steinhoff et al, 1997). This study of interspin distances between attached spin labels applied to proteins is a nice example how to obtain structural information on proteins under conditions when other methods like two-dimensional NMR spectroscopy or X-ray crystallography are not applicable.…”

Section: Phosphorylation Processes Drove the Disassembly Of The Vimenmentioning

confidence: 88%

“…The interspin distances of two or more nitroxide spin labels attached to specific sites in insulins were determined for different conformations with application of EPR by the line broadening due to dipolar interaction (Steinhoff et al, 1997). The procedure was carried out by fitting simulated EPR powder spectra to experimental data, measured at temperatures below 200 K to freeze the protein motion.…”

Section: Phosphorylation Processes Drove the Disassembly Of The Vimenmentioning

confidence: 99%

Forty Years of the d1/d Parameter

Кокорин¹

2012

Nitroxides - Theory, Experiment and Applications

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Determination of interspin distances between spin labels attached to insulin: comparison of electron paramagnetic resonance data with the X-ray structure

Cited by 161 publications

References 29 publications

Conformational heterogeneity of the aspartate transporter GltPh

Conformational heterogeneity of the aspartate transporter GltPh

Salt-driven Equilibrium between Two Conformations in the HAMP Domain from Natronomonas pharaonis

Forty Years of the d1/d Parameter

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