The determination of pair distance distributions by pulsed ESR using Tikhonov regularization

Chiang, Yun‐Wei; Borbat, Peter P.; Freed, Jack H.

doi:10.1016/j.jmr.2004.10.012

Cited by 368 publications

(431 citation statements)

References 55 publications

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“…For long distances (>50 Å), dipolar evolution times can be insufficient, resulting in some uncertainty in the widths of the distance distributions. Echo decays were background-corrected and fit with the DEER Analysis 2011 program (39) using Tikhonov regularization (40) to obtain distance distributions. Aggregated protein, resulting from concentration and validated by gel electrophoresis, appears in some samples as a nonspecific peak near 50 Å.…”

Section: Methodsmentioning

confidence: 99%

Conformational cycle and ion-coupling mechanism of the Na ⁺ /hydantoin transporter Mhp1

Kazmier

Sharma

Islam

et al. 2014

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

141

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Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuTfold has been captured in outward-facing, occluded, and inwardfacing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na + -coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion-and substrate-dependent conformational equilibria. In contrast to the Na + /leucine transporter LeuT, our results suggest that Na + binding at the conserved second Na + binding site does not change the energetics of the inward-and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.EPR | DEER | Na + -coupled symport | conformational dynamics | transport mechanism

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Section: Methodsmentioning

confidence: 99%

Conformational cycle and ion-coupling mechanism of the Na ⁺ /hydantoin transporter Mhp1

Kazmier

Sharma

Islam

et al. 2014

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

141

View full text Add to dashboard Cite

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“…The fibril pellet was collected into a quartz capillary (1.5-mm inner diameter, 1.8-mm outer diameter) and flashfrozen before DEER data were acquired at 78 K. Four-pulse DEER experiments were performed using a Bruker ELEXSYS E580 X-band pulse EPR spectrometer fitted with a 3-mm split ring (MS-3) resonator, a continuous flow helium cryostat (CF935), and a temperature controller (ITC503S, all Oxford Instruments). Data were fit using Gaussian distributions (23) in the DEERAnalysis2008 package (24).…”

Section: Methodsmentioning

confidence: 99%

Fibril Structure of Human Islet Amyloid Polypeptide

Bedrood

Isas

et al. 2012

Journal of Biological Chemistry

142

171

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Background: Human islet amyloid polypeptide (hIAPP) fibrils of unknown structure are formed in type 2 diabetes. Results: A hIAPP fibril structure was derived from EPR data, electron microscopy, and computer modeling. Conclusion:The fibril is a left-handed helix that contains hIAPP monomers in a staggered conformation. Significance: The results provide the basis for therapeutic prevention of fibril formation and growth.

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“…28,29 Inter-subunit distances (<50 Å) can be measured using Double Electron-Electron Resonance (DEER) methods. [30][31][32][33][34] DEER data are collected in detergent samples or reconstituted in nanodiscs because of the limitations associated with these measurements in liposomes 54 . For spinlabeled samples in detergent (~100 μM) in Buffer A with 0.5 mM DDM, 30% (weight/volume) glycerol is used for cryoprotection.…”

Section: Determining Distance Distribution From Eprmentioning

confidence: 99%

“…The pulse lengths for (π/2)mw1 and (π)mw1 are 10 and 20 nsec, respectively, and 40 nsec for (π)mw2. DEER signals are then background corrected assuming a 3D homogeneous background and analyzed by the Tikhonov regularization 31,56 to determine average distances and distributions in distance.…”

Section: Determining Distance Distribution From Eprmentioning

confidence: 99%

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Basak

Chatterjee

Chakrapani

2016

JoVE

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Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics. Video LinkThe video component of this article can be found at

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The determination of pair distance distributions by pulsed ESR using Tikhonov regularization

Cited by 368 publications

References 55 publications

Conformational cycle and ion-coupling mechanism of the Na ⁺ /hydantoin transporter Mhp1

Conformational cycle and ion-coupling mechanism of the Na ⁺ /hydantoin transporter Mhp1

Fibril Structure of Human Islet Amyloid Polypeptide

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

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The determination of pair distance distributions by pulsed ESR using Tikhonov regularization

Cited by 368 publications

References 55 publications

Conformational cycle and ion-coupling mechanism of the Na + /hydantoin transporter Mhp1

Conformational cycle and ion-coupling mechanism of the Na + /hydantoin transporter Mhp1

Fibril Structure of Human Islet Amyloid Polypeptide

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Contact Info

Product

Resources

About

Conformational cycle and ion-coupling mechanism of the Na ⁺ /hydantoin transporter Mhp1

Conformational cycle and ion-coupling mechanism of the Na ⁺ /hydantoin transporter Mhp1