DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data

Jeschke, Gunnar; Chechik, Victor; Ioniță, Petre; Godt, Adelheid; Zimmermann, Herbert; Banham, Janet E.; Timmel, Christiane R.; Hilger, Daniel; Jung, Hyun Suk

doi:10.1007/bf03166213

Cited by 940 publications

(1,449 citation statements)

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“…3 A and B, gray trace), expected to adopt a random coiled conformation with some ␣-helical structure (8), yielded broad distance distributions covering the range of 1.5-6 nm. The individual peaks in these distributions may not be significant but rather result from noise-related distortions introduced during conversion of primary DEER data to distance distributions (21). The fully folded and pigmented proteins showed significant narrowing of their distance distributions and a change in the mean distance.…”

Section: Resultsmentioning

confidence: 99%

Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR

Dockter

Volkov

Bauer

et al. 2009

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

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The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in plants self-organizes in vitro. The recombinant apoprotein, denatured in dodecyl sulfate, spontaneously folds when it is mixed with its pigments, chlorophylls, and carotenoids in detergent solution, and assembles into structurally authentic LHCII in the course of several minutes. Pulse EPR techniques, specifically double-electron-electron resonance (DEER), have been used to analyze protein folding during this process. Pairs of nitroxide labels were introduced site-specifically into recombinant LHCII and shown not to affect the stability and function of the pigment-protein complex. Interspin distance distributions between two spin pairs were measured at various time points, one pair located on either end of the second transmembrane helix (helix 3), the other one located near the luminal ends of the intertwined transmembrane helices 1 and 4. In the dodecyl sulfatesolubilized apoprotein, both distance distributions were consistent with a random-coil protein structure. A rapid freeze-quench experiment on the latter spin pair indicated that 1 s after initiating reconstitution the protein structure is virtually unchanged. Subsequently, both distance distributions monitored protein folding in the same time range in which the assembly of chlorophylls into the complex had been observed. The positioning of the spin pair spanning the hydrophobic core of LHCII clearly preceded the juxtaposition of the spin pair on the luminal side of the complex. This indicates that superhelix formation of helices 1 and 4 is a late step in LHCII assembly.assembly kinetics ͉ DEER ͉ LHCII T he major light-harvesting complex LHCII largely increases the efficiency of the photosynthesis process by collecting light energy and conducting it to a photosynthetic reaction center where light-driven charge separation takes place. The apoprotein of LHCII is one of the most abundant membrane proteins on Earth, but even so, our knowledge is fragmentary of how the LHCII apoprotein folds and assembles with pigments. For studying these questions, LHCII offers several advantages. Its crystal structure is known (1), its apoprotein can be recombinantly expressed in Escherichia coli (2), and the protein spontaneously folds and assembles with pigments in detergent solution (2, 3). This spontaneous self-organization can be triggered by mixing the apoprotein solubilized in the ionic detergent dodecyl sulfate with a non-ionic detergent solution of the pigments. The assembly process can then easily be monitored by time-resolved f luorescence spectroscopy using the Chls as built-in fluorescence labels. Such experiments showed that protein folding is dependent on the binding of pigments, and that LHCII formation in vitro occurred in at least two apparent phases, a faster one in the range of some tens of seconds and a slower one taking several minutes (4, 5). The faster step could be assigned to the binding of mostly Chl a, whereas the slower one represents Chl b binding exclus...

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Section: Resultsmentioning

confidence: 99%

Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR

Dockter

Volkov

Bauer

et al. 2009

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

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“…Interspin distance distributions were derived by fitting the background-corrected dipolar evolution function using Tikhonov regularization as implemented in DEERAnalysis2011 (ref. 25). Validation of distance distributions were carried out with the included validation tool.…”

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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“…2 A). Both time traces were analyzed with the DEER Analysis 2016 software (18), leading to the distance distributions in Fig. 2 B.…”

Section: Building a Model Of Substrate-bound Vcsiapmentioning

confidence: 99%

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

Glaenzer

Peter

Thomas

et al. 2017

Biophysical Journal

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The tripartite ATP-independent periplasmic (TRAP) transporters are a widespread class of membrane transporters in bacteria and archaea. Typical substrates for TRAP transporters are organic acids including the sialic acid N-acetylneuraminic acid. The substrate binding proteins (SBP) of TRAP transporters are the best studied component and are responsible for initial high-affinity substrate binding. To better understand the dynamics of the ligand binding process, pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy was applied to study the conformational changes in the N-acetylneuraminic acid-specific SBP VcSiaP. The protein is the SBP of VcSiaPQM, a sialic acid TRAP transporter from Vibrio cholerae. Spin-labeled double-cysteine mutants of VcSiaP were analyzed in the substrate-bound and -free state and the measured distances were compared to available crystal structures. The data were compatible with two clear states only, which are consistent with the open and closed forms seen in TRAP SBP crystal structures. Substrate titration experiments demonstrated the transition of the population from one state to the other with no other observed forms. Mutants of key residues involved in ligand binding and/or proposed to be involved in domain closure were produced and the corresponding PELDOR experiments reveal important insights into the open-closed transition. The results are in excellent agreement with previous in vivo sialylation experiments. The structure of the spin-labeled Q54R1/L173R1 R125A mutant was solved at 2.1 Å resolution, revealing no significant changes in the protein structure. Thus, the loss of domain closure appears to be solely due to loss of binding. In conclusion, these data are consistent with TRAP SBPs undergoing a simple two-state transition from an openunliganded to closed-liganded state during the transport cycle.

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DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data

Cited by 940 publications

References 50 publications

Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR

Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR

Conformational heterogeneity of the aspartate transporter GltPh

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

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