Osmolytes modulate conformational exchange in solvent‐exposed regions of membrane proteins

Jiménez, Ricardo H. Flores; Cao, Marie‐Ange Do; Kim, Mi‐Yeon; Cafiso, David S.

doi:10.1002/pro.305

Cited by 32 publications

(29 citation statements)

References 46 publications

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“…19 Because the least solvent-exposed conformation of a protein is the most compact with increased opportunities for interaction of R1, osmotic perturbation generally shifts spectral populations toward more immobilized states. 19,54 …”

Section: Resultsmentioning

confidence: 99%

Mapping Molecular Flexibility of Proteins with Site-Directed Spin Labeling: A Case Study of Myoglobin

2012

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Site directed spin labeling (SDSL) has potential for mapping protein flexibility under physiological conditions. The purpose of the present study was to explore this potential using 38 singly spin-labeled mutants of myoglobin distributed throughout the sequence. Correlation of the EPR spectra with protein structure provides new evidence that the site dependent variation in lineshape, and hence motion of the spin label, is due largely to differences in mobility of the helical backbone in the ns time range. Fluctuations between conformational substates, typically in the μs-ms time range, are slow on the EPR time scale and the spectra provide a snapshot of conformational equilibria frozen in time as revealed by multiple components in the spectra. A recent study showed that osmolyte perturbation can positively identify conformational exchange as the origin of multicomponent spectra (Lopez et al. (2009), Protein Sci. 18, 1637). In the present study this new strategy is employed in combination with lineshape analysis and pulsed-EPR interspin distance measurements to investigate the conformation and flexibility of myoglobin in three folded and partially folded states. The regions identified to be in conformational exchange in the three forms agree remarkably well with those assigned by NMR, but the faster time scale of EPR allows characterization of localized states not detected in NMR. Collectively, the results suggest that SDSL-EPR and osmolyte perturbation provide a facile means for mapping the amplitude of fast backbone fluctuations and for detecting sequences in slow conformational exchange in folded and partially folded protein sequences.

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Section: Resultsmentioning

confidence: 99%

Mapping Molecular Flexibility of Proteins with Site-Directed Spin Labeling: A Case Study of Myoglobin

2012

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“…One approach to distinguish between these two possibilities is to test the spectra for sensitivity to stabilizing osmolytes, such as sucrose or a polyethylene glycol (PEG). 25;27; 28 Osmolytes appear to be preferentially excluded from the protein surface, thereby raising the energy of the protein. 34; 35; 36; 37 In the case where two conformers are in equilibrium, osmolytes will lower the energy of the least hydrated, or more compact, protein form.…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, EPR spectroscopy is one of the best methods to examine equilibria between conformational substates in proteins. 27;28;29 In FecA, EPR spectra from the N-terminal transcriptional domain show that the structure of the domain is not altered by substrate; however, regions of the domain are in conformational exchange between substates. In lipid bilayers, the domain is positioned so that the second β-strand from the N-terminus (β2) may interact with the Ton box.…”

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confidence: 99%

Ligand-Induced Structural Changes in the Escherichia coli Ferric Citrate Transporter Reveal Modes for Regulating Protein–Protein Interactions

Mokdad

Herrick

Kahn

et al. 2012

Journal of Molecular Biology

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Outer-membrane TonB-dependent transporters, such as the Escherichia coli ferric citrate transporter FecA, interact with the inner-membrane protein TonB through an energy-coupling segment termed the Ton box. In FecA, which regulates its own transcription, the Ton box is preceded by an N-terminal extension that interacts with the inner membrane protein FecR. Here, site-directed spin labeling was used to examine the structural basis for transcriptional signaling and Ton box regulation in FecA. EPR spectroscopy indicates that regions of the N-terminal domain are in conformational exchange, consistent with its role as a protein binding element; however, the local fold and dynamics of the domain are not altered by substrate or TonB. Distance restraints derived from pulse EPR were used to generate models for the position of the extension in the apo, substrate- and TonB-bound states. In the apo state, this domain is positioned at the periplasmic surface of FecA, where it interacts with the Ton box and blocks access of the Ton box to the periplasm. Substrate addition rotates the transcriptional domain and exposes the Ton box, leading to a disorder transition in the Ton box that may facilitate interactions with TonB. When a soluble fragment of TonB is bound to FecA, the transcriptional domain is displaced to one edge of the barrel, consistent with a proposed β-strand exchange mechanism. However, neither substrate nor TonB displace the N-terminus further into the periplasm. This result suggests that the intact TonB system mediates both signaling and transport by unfolding portions of the transporter.

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“…In addition to the cryoprotective function mentioned above, glycerol is a stabilizing osmolyte that favors folded states of proteins (54)(55)(56)(57), and the effect of glycerol on the structure and conformational equilibria of Mb must be considered. A systematic SDSL-EPR study of the osmolyte effect on holoMb, apoMb, and the MG state of apoMb has been carried out for each of the labeled R1 sites used in the present experiments with sucrose as the osmolyte.…”

Section: Discussionmentioning

confidence: 99%

Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron–electron resonance

Lerch

Yang²,

Brooks³

et al. 2014

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

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The dominance of a single native state for most proteins under ambient conditions belies the functional importance of higherenergy conformational states (excited states), which often are too sparsely populated to allow spectroscopic investigation. Application of high hydrostatic pressure increases the population of excited states for study, but structural characterization is not trivial because of the multiplicity of states in the ensemble and rapid (microsecond to millisecond) exchange between them. Sitedirected spin labeling in combination with double electron-electron resonance (DEER) provides long-range (20-80 Å) distance distributions with angstrom-level resolution and thus is ideally suited to resolve conformational heterogeneity in an excited state populated under high pressure. DEER currently is performed at cryogenic temperatures. Therefore, a method was developed for rapidly freezing spin-labeled proteins under pressure to kinetically trap the highpressure conformational ensemble for subsequent DEER data collection at atmospheric pressure. The methodology was evaluated using seven doubly-labeled mutants of myoglobin designed to monitor selected interhelical distances. For holomyoglobin, the distance distributions are narrow and relatively insensitive to pressure. In apomyoglobin, on the other hand, the distributions reveal a striking conformational heterogeneity involving specific helices in the pressure range of 0-3 kbar, where a molten globule state is formed. The data directly reveal the amplitude of helical fluctuations, information unique to the DEER method that complements previous rate determinations. Comparison of the distance distributions for pressure-and pH-populated molten globules shows them to be remarkably similar despite a lower helical content in the latter.EPR | dipolar spectroscopy | compressibility A lthough a well-defined native state is dominant for most proteins under physiological conditions, excited states involving large (multiangstrom) conformational changes relative to the native state have been shown to play critical roles in biological function (1-3). Even for low-lying excited states with relative energies on the order of a few kilocalories (1 kcal = 4.184 kJ) per mole, the equilibrium population is too small to be characterized by most spectroscopic methods, but such states can be reversibly populated by hydrostatic pressure (4-9). It is generally agreed that the mechanism for population of excited states by pressure is hydration of cavities in the excited state relative to the ground state (10-12), but other mechanisms are possible (13). The pressures required to populate low-lying excited states are typically a few kilobars, corresponding to perturbation energies of a few kilocalories per mole. At these low energies, pressure should not strongly remodel the energy landscape itself but simply shift the relative population of preexisting conformers (14). Thus, pressure should be a powerful perturbation technique for studying functional excited states at equilibrium (15)....

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Osmolytes modulate conformational exchange in solvent‐exposed regions of membrane proteins

Cited by 32 publications

References 46 publications

Mapping Molecular Flexibility of Proteins with Site-Directed Spin Labeling: A Case Study of Myoglobin

Mapping Molecular Flexibility of Proteins with Site-Directed Spin Labeling: A Case Study of Myoglobin

Ligand-Induced Structural Changes in the Escherichia coli Ferric Citrate Transporter Reveal Modes for Regulating Protein–Protein Interactions

Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron–electron resonance

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