Osmolyte perturbation reveals conformational equilibria in spin‐labeled proteins

López, Carlos J.; Fleissner, Mark R.; Guo, Zhefeng; Kusnetzow, Ana Karin; Hubbell, Wayne L.

doi:10.1002/pro.180

Cited by 100 publications

(209 citation statements)

References 93 publications

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“…At X-band, the spectroscopic timescale is sufficiently fast to allow different protein states to be resolved in an EPR spectrum; as a result, SDSL can be used to examine the sensitivity to osmolyte and conformational equilibria in select regions of proteins. 24 In this study, conformational exchange is demonstrated at the N-terminus of BtuB and in the Ton box of FecA. At least two motional components are found in the EPR spectra from R1 placed within the N-terminal five residues of BtuB.…”

Section: Discussionmentioning

confidence: 69%

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

Jiménez¹,

Cao²,

Kim³

et al. 2010

Protein Science

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Site-directed spin labeling (SDSL) was used to investigate local structure and conformational exchange in two bacterial outer-membrane TonB-dependent transporters, BtuB and FecA. Protecting osmolytes, such as polyethylene glycols (PEGs) are known to modulate a substrate-dependent conformational equilibrium in the energy coupling motif (Ton box) of BtuB. Here, we demonstrate that a segment that is N-terminal to the Ton box in BtuB, is in conformational exchange between ordered and disordered states with or without substrate. Protecting osmolytes shift this equilibrium to favor the more ordered, folded state. However, a segment of BtuB that is C-terminal to the Ton box that is not solvent exposed is insensitive to PEGs. Protecting osmolytes also modulate a conformational equilibrium in the Ton box of FecA, with larger molecular weight PEGs producing the largest shifts in the conformational free energy. These data indicate that solvent-exposed regions of these transporters undergo conformational exchange and that regions of these transporters that are involved in protein-protein interactions sample multiple conformational substates. The sensitivity to solute provides an explanation for differences seen between two high-resolution structures of BtuB, which each likely represent one conformation from a subset of states that are normally sampled by the protein. This work also illustrates how SDSL and osmolytes may be used to characterize and quantitate conformational equilibria in membrane proteins.

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Section: Discussionmentioning

confidence: 69%

“…Protecting osmolytes are observed to have little effect upon rotameric states of R1, probably because of the small differences in solvent-exposed areas for different rotamers. 24 As a result, sensitivity to PEGs indicates that the populations in an EPR spectrum result from a protein conformational equilibrium.…”

Section: Resultsmentioning

confidence: 99%

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

Jiménez¹,

Cao²,

Kim³

et al. 2010

Protein Science

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“…1A shows a model of the L121A/L133A mutant based on the crystal structure, along with the location of R1 sensor sites and the spectra compared with those of the WT′. The spectra for the WT′ protein have been published previously and interpreted in terms of the protein structure and dynamics (16)(17)(18)(19)(20); the basic principles of spectral analysis are outlined in SI Materials and Methods. The spectra of 131R1, 132R1, 140R1, and 151R1 in the WT′ reflect a simple anisotropic motion characteristic of R1 at solvent-exposed sites in relatively rigid helical segments, whereas the spectra of 48R1, 109R1, 123R1, 128R1, and 135R1 reflect higher mobility of the nitroxide consistent with their location near or at the helix termini (16).…”

Section: Experimental Strategy and Resultsmentioning

confidence: 99%

“…1A, Inset) was introduced at solvent-exposed sites to serve as a sensor of local structure. At such sites, R1 typically has weak or no interactions with the protein, as reflected by a singlecomponent electron paramagnetic resonance (EPR) spectrum, and introduces little structural perturbation (16)(17)(18)(19). The R1 sensor Significance Analysis of protein packing reveals the prevalence of cavities, some of which are evolutionarily conserved.…”

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confidence: 99%

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants

López

Yang

Altenbach

et al. 2013

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

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The studies presented here explore the relationship between protein packing and molecular flexibility using ligand-binding cavity mutants of T4 lysozyme. Although previously reported crystal structures of the mutants investigated show single conformations that are similar to the WT protein, site-directed spin labeling in solution reveals additional conformational substates in equilibrium exchange with a WT-like population. Remarkably, binding of ligands, including the general anesthetic halothane shifts the population to the WT-like state, consistent with a conformational selection model of ligand binding, but structural adaptation to the ligand is also apparent in one mutant. Distance mapping with double electron-electron resonance spectroscopy and the absence of ligand binding suggest that the new substates induced by the cavity-creating mutations represent alternate packing modes in which the protein fills or partially fills the cavity with side chains, including the spin label in one case; external ligands compete with the side chains for the cavity space, stabilizing the WT conformation. The results have implications for mechanisms of anesthesia, the response of proteins to hydrostatic pressure, and protein engineering.EPR | site-directed spin labeling | DEER | benzene | saturation recovery G lobular proteins have overall packing densities similar to those of crystalline solids (1) but nevertheless contain cavities and pockets that can range from a few to hundreds of cubic angstroms (2, 3). These native packing defects are generally destabilizing (4, 5), and improving the packing of the protein interior may be a general way to increase protein stability. Indeed, small-to-large mutations that fill native cavities can increase stability (6-8), but with a concomitant loss of function (7,8). Thus, cavities in the protein interior can play a critical role in function. One role of cavities may be to allow alternative packing arrangements of the core. The resulting ensemble of conformational substates could give rise to promiscuity in protein-protein interactions (9) and allosteric behavior (7,8,10). In addition, large-to-small mutations that generate cavities may have played an important role in the evolution of protein function (11).Considering the potentially important role of cavities in sculpting the energy landscape of proteins, the present study was undertaken to investigate, in solution, the structural and dynamical response of a protein to engineered cavities introduced by large-tosmall mutations. T4 lysozyme (T4L) was selected for study because of the large database of crystal structures of ligand-binding cavity mutations and the corresponding thermodynamic characterization from Matthews and coworkers (4,(12)(13)(14)(15). Remarkably, comparison of the WT and mutant structures showed very little difference or limited local relaxation near the cavity, although the mutations were strongly destabilizing (4, 15).In the present study, we examined the cavity-creating mutants L121A/L133A, L133G, and W138A in sol...

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“…Instead, the presence of two conformations in equilibrium will, for particular locations of R1, give rise to two components in the EPR spectrum, each corresponding to one of the conformations (17,18) and of intensity proportional to the population, permitting the direct determination of the equilibrium constant. However, two-component EPR spectra can also arise from equilibrium between two rotameric states of R1 that place the nitroxide in distinct environments (19,20).…”

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confidence: 99%

High-pressure EPR reveals conformational equilibria and volumetric properties of spin-labeled proteins

McCoy

Hubbell

2011

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

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Identifying equilibrium conformational exchange and characterizing conformational substates is essential for elucidating mechanisms of function in proteins. Site-directed spin labeling has previously been employed to detect conformational changes triggered by some event, but verifying conformational exchange at equilibrium is more challenging. Conformational exchange (microsecond-millisecond) is slow on the EPR time scale, and this proves to be an advantage in directly revealing the presence of multiple substates as distinguishable components in the EPR spectrum, allowing the direct determination of equilibrium constants and free energy differences. However, rotameric exchange of the spin label side chain can also give rise to multiple components in the EPR spectrum. Using spin-labeled mutants of T4 lysozyme, it is shown that high-pressure EPR can be used to: (i) demonstrate equilibrium between spectrally resolved states, (ii) aid in distinguishing conformational from rotameric exchange as the origin of the resolved states, and (iii) determine the relative partial molar volume (ΔV o ) and isothermal compressibility (Δβ T ) of conformational substates in two-component equilibria from the pressure dependence of the equilibrium constant. These volumetric properties provide insight into the structure of the substates. Finally, the pressure dependence of internal side-chain motion is interpreted in terms of volume fluctuations on the nanosecond time scale, the magnitude of which may reflect local backbone flexibility.P roteins undergo structural fluctuations that span a wide range of time scales. Among these motions are fast backbone fluctuations on the picosecond-nanosecond time scale and slower conformational fluctuations on the microsecond and longer time scale (1-3). Molecular flexibility on these time scales plays a central role in protein function (4). For example, in recognitionbinding sequences, dynamic disorder on the nanosecond-microsecond time scale may increase the rate of protein-protein interactions via a "fly casting" mechanism (5). An emerging disorder-to-order paradigm for interaction (6) can also give rise to promiscuity in binding that increases the size of the "interactome."Regulation of protein function is often linked to a conformational switch triggered by an interaction with a chemical or physical signal. One mechanistic interpretation of this event is provided by a "preequilibrium" model, which posits that all possible conformations of a protein exist at equilibrium with populations proportional to their relative energies (7). The exchange ("hopping") event between different conformers is characterized by lifetimes in the microsecond-millisecond range (1, 2, 8). In this model, a conformational switch is viewed as a shift in the relative populations of existing conformational states rather than the creation of a new state.To evaluate the above models and elucidate molecular mechanisms of protein function, it is essential to have experimental means for identifying dynamically disordered sequenc...

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Osmolyte perturbation reveals conformational equilibria in spin‐labeled proteins

Cited by 100 publications

References 93 publications

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

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

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants

High-pressure EPR reveals conformational equilibria and volumetric properties of spin-labeled proteins

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