Resolving Conformational and Rotameric Exchange in Spin-Labeled Proteins Using Saturation Recovery EPR

Bridges, Michael D.; Hideg, Kálmán; Hubbell, Wayne L.

doi:10.1007/s00723-009-0079-2

Cited by 63 publications

(112 citation statements)

References 76 publications

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“…The appearance of two-component spectra in the cavity mutants clearly signals a structural change in the protein, but the origin of the two components could either be due to two rotamers of the R1 side chain in a single new conformation or to two conformations of the protein. These cases can apparently be distinguished by SR EPR methods, as discussed in detail elsewhere (21,24) and outlined in SI Materials and Methods. Briefly, SR measures the recovery of the EPR signal following a saturating microwave pulse applied to the maximum absorbance of the nitroxide M I = 0 resonance ( Fig.…”

Section: Experimental Strategy and Resultsmentioning

confidence: 96%

“…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). The spectra of 109R1 and 116R1 have two components; for 109R1, the two components apparently arise from different rotamers of R1 (21). Residue 130R1 is unique among the sites investigated in that it is located at a contact site between helices H and J, where the nitroxide interacts with the protein, as revealed by the presence of a second spectral component reflecting immobilization (arrows, Fig.…”

Section: Experimental Strategy and Resultsmentioning

confidence: 97%

“…On the other hand, for slow protein conformational exchange, the relaxation is biexponential and SR data in the presence of various concentrations of a relaxation reagent nickel ethylenediaminediacetate (NiEDDA) can be analyzed in terms of the exchange lifetime in the range of 1-70 μs. In addition, the data provide a measure of the residue solvent accessibility (21,25).…”

Section: Experimental Strategy and Resultsmentioning

confidence: 99%

“…S3). SR gives a monoexponential recovery curve with a spin lattice relaxation time (T 1 ) for 130R1 of 6.6 μs (Table S2), which is characteristic of immobile nitroxides at buried sites in proteins (21). Moreover, the T 1 is insensitive to NiEDDA up to 1.5 mM, which shows that residue 130R1 is solvent-inaccessible (SI Materials and Methods and Table S2).…”

Section: Deer Spectroscopy Reveals Structural Differences Between Thementioning

confidence: 99%

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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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Section: Experimental Strategy and Resultsmentioning

confidence: 96%

Section: Experimental Strategy and Resultsmentioning

confidence: 97%

Section: Experimental Strategy and Resultsmentioning

confidence: 99%

Section: Deer Spectroscopy Reveals Structural Differences Between Thementioning

confidence: 99%

See 2 more Smart Citations

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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“…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).…”

mentioning

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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Detection of drug‐induced conformational change of a transmembrane protein in lipid bilayers using site‐directed spin labeling

et al. 2012

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As a target of antiviral drugs, the influenza A M2 protein has been the focus of numerous structural studies and has been extensively explored as a model ion channel. In this study, we capitalize on the expanding body of high-resolution structural data available for the M2 protein to design and interpret site-directed spin-labeling electron paramagnetic resonance spectroscopy experiments on drug-induced conformational changes of the M2 protein embedded in lipid bilayers. We obtained data in the presence of adamantane drugs for two different M2 constructs (M2TM 22-46 and M2TMC 23-60). M2TM peptides were spin labeled at the N-terminal end of the transmembrane domain. M2TMC peptides were spin labeled site specifically at cysteine residues substituted for amino acids within the transmembrane domain (L36, I39, I42, and L43) and the C-terminal amphipathic helix (L46, F47, F48, C50, I51, Y52, R53, F54, F55, and E56). Addition of adamantane drugs brought about significant changes in measured electron paramagnetic resonance spectroscopy environmental parameters consistent with narrowing of the transmembrane channel pore and closer packing of the C-terminal amphipathic helices.

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Resolving Conformational and Rotameric Exchange in Spin-Labeled Proteins Using Saturation Recovery EPR

Cited by 63 publications

References 76 publications

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

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

Detection of drug‐induced conformational change of a transmembrane protein in lipid bilayers using site‐directed spin labeling

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