Pulsed EPR Distance Measurements in Soluble Proteins by Site‐Directed Spin Labeling (SDSL)

Vera, Ian Mitchelle S. de; Blackburn, Mandy E.; Galiano, Luis; Fanucci, Gail E.

doi:10.1002/0471140864.ps1717s74

Cited by 21 publications

(46 citation statements)

References 91 publications

(167 reference statements)

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“…Standard DEER methodology requires flash-freezing of a sample, typically in liquid nitrogen, and data acquisition at 50-80 K (25,26,32,33). To use DEER to monitor the structure of high-pressure states, a procedure is introduced here in which the pressurized sample is rapidly frozen in a dry ice/ethanol bath to 200 K. The frozen sample is then depressurized and cooled further to 77 K in liquid nitrogen for subsequent data acquisition at atmospheric pressure.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…NMR investigations demonstrate that the backbone dynamics decrease from the nanosecond to microsecond to millisecond regime upon inhibitor binding (22). In recent years, our laboratory has pioneered the application of site-directed spin labeling (SDSL) with pulsed electron double resonance, also called double electron-electron resonance (DEER) to characterize flap conformational ensembles in HIV-1 PR (14,(23)(24)(25)(26)(27)(28). Other groups have also utilized this methodology to investigate the catalytic mechanism of HIV-1 PR (29,30).…”

Section: Hiv-1 Protease (Hiv-1 Pr)mentioning

confidence: 99%

“…Fig. 5 also shows results from population analyses of the DEER distance profiles (full details of data analysis are given in supplemental material), which proceeds by assuming the distance profile is an ensemble of all conformational states and that this ensemble can be modeled as a linear combination of Gaussian-shaped functions, where each Gaussian-shaped peak (sometimes combined peaks) is taken to correspond to a conformational ensemble of protein/spin label (23)(24)(25)(26)(27)(28). For our HIV-1 PR DEER data, typically four final functions, i.e.…”

Section: Pr5 Hiv-1 Pr Has An Additional Curled Flap Conformation Compmentioning

confidence: 99%

The Role of Select Subtype Polymorphisms on HIV-1 Protease Conformational Sampling and Dynamics

Huang

Britto

Kear-Scott

et al. 2014

Journal of Biological Chemistry

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Background: HIV-1 protease is an essential enzyme for HIV maturation. Results: Select and naturally occurring polymorphisms alter the conformational sampling and backbone dynamics of HIV-1 protease. Conclusion: These mutations lead to an alternative flap ensemble that we suspect is a curled flap orientation. Significance: The mechanism of distal mutations on drug resistance is unclear, but altered dynamics and conformational equilibria likely play key roles.

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“…Coupled with site-directed spin-labeling (SDSL), EPR is oftentimes used to characterize protein and nucleic acid structures and dynamics, conformational changes, molecule folding, macromolecule complexes, and oligomeric structures [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]17]. The majority of biomolecules do not contain unpaired electrons from which one can obtain an EPR signal; therefore, spin-labeling approaches have been developed [15,[18][19][20][21][22][23][24][25] where site-specific persistent radicals or paramagnetic metal-probes are incorporated at specific locations within a biomolecule.…”

Section: Introductionmentioning

confidence: 99%

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

Song

Liu

Kaur

et al. 2016

Journal of Magnetic Resonance

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High-field, high-frequency electron paramagnetic resonance (EPR) spectroscopy at W-(~95 GHz) and D-band (~140 GHz) is important for investigating the conformational dynamics of flexible biological macromolecules because this frequency range has increased spectral sensitivity to nitroxide motion over the 100 ps to 2 ns regime. However, low concentration sensitivity remains a roadblock for studying aqueous samples at high magnetic fields. Here, we examine the sensitivity of a non-resonant thin-layer cylindrical sample holder, coupled to a quasi-optical induction-mode W-band EPR spectrometer (HiPER), for continuous wave (CW) EPR analyses of: (i) the aqueous nitroxide standard, TEMPO; (ii) the unstructured to -helical transition of a model IDP protein; and (iii) the base-stacking transition in a kink-turn motif of a large 232 nt RNA. For sample volumes of ~50 L, concentration sensitivities of 2-20 µM were achieved, representing a ~10-fold enhancement compared to a cylindrical TE 011 resonator on a commercial Bruker W-band spectrometer. These results therefore highlight the sensitivity of the thin-layer sample holders employed in HiPER for spin-labeling studies of biological macromolecules at high fields, where applications can extend to other systems that are facilitated by the modest sample volumes and ease of sample loading and geometry.2

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Pulsed EPR Distance Measurements in Soluble Proteins by Site‐Directed Spin Labeling (SDSL)

Cited by 21 publications

References 91 publications

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

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

The Role of Select Subtype Polymorphisms on HIV-1 Protease Conformational Sampling and Dynamics

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

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