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A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T1 relaxation enhancement.

ABSTRACT: Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has become an important tool for measuring distances in proteins on the order of a few nm. For this purpose pairs of spin labels, most commonly nitroxides, are site-selectively introduced into the protein. Recent efforts to develop new spin labels are focused on tailoring the intrinsic properties of the label to either extend the upper limit of measurable distances at physiological temperature, or to provide a unique spectral lineshape so that selective pairwise distances can be measured in a protein or complex containing multiple spin label species. Triarylmethyl (TAM) radicals are the foundation for a new class of spin labels that promise to provide both capabilities. Here we report a new methanethiosulfonate derivative of a TAM radical that reacts rapidly and selectively with an engineered cysteine residue to generate a TAM containing side chain (TAM1) in high yield. With a TAM1 residue and Cu(2+) bound to an engineered Cu(2+) binding site, enhanced T1 relaxation of TAM should enable measurement of interspin distances up to 50Å at physiological temperature. To achieve favorable TAM1-labeled protein concentrations without aggregation, proteins are tethered to a solid support either site-selectively using an unnatural amino acid or via native lysine residues. The methodology is general and readily extendable to complex systems, including membrane proteins.

SUBMITTER: Yang Z

PROVIDER: S-EPMC4958593 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T1 relaxation enhancement.

Yang Zhongyu Z Bridges Michael D MD López Carlos J CJ Rogozhnikova Olga Yu OY Trukhin Dmitry V DV Brooks Evan K EK Tormyshev Victor V Halpern Howard J HJ Hubbell Wayne L WL

Journal of magnetic resonance (San Diego, Calif. : 1997) 20160510

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has become an important tool for measuring distances in proteins on the order of a few nm. For this purpose pairs of spin labels, most commonly nitroxides, are site-selectively introduced into the protein. Recent efforts to develop new spin labels are focused on tailoring the intrinsic properties of the label to either extend the upper limit of measurable distances at physiological temperatu ...[more]

PMID: 27214582

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Project description:Longitudinal (Γ1) and transverse (Γ2) solvent paramagnetic relaxation enhancement (sPRE) yields field-dependent information in the form of spectral densities that provides unique information related to cosolute-protein interactions and electrostatics. A typical protein sPRE data set can only sample a few points on the spectral density curve, J(ω), within a narrow frequency window (500 MHz to ∼1 GHz). However, complex interactions and dynamics of paramagnetic cosolutes around a protein make it difficult to directly interpret the few experimentally accessible points of J(ω). In this paper, we show that it is possible to significantly extend the experimentally accessible frequency range (corresponding to a range from ∼270 MHz to 1.8 GHz) by acquiring a series of sPRE experiments at different temperatures. This approach is based on the scaling property of J(ω) originally proposed by Melchior and Fries for small molecules. Here, we demonstrate that the same scaling property also holds for geometrically far more complex systems such as proteins. Using the extended spectral densities derived from the scaling property as the reference dataset, we demonstrate that our previous approach that makes use of a non-Lorentzian Ansatz spectral density function to fit only J(0) and one to two J(ω) points allows one to obtain accurate values for the concentration-normalized equilibrium average of the electron-proton interspin separation ⟨r-6⟩norm and the correlation time τC, which provide quantitative information on the energetics and timescale, respectively, of local cosolute-protein interactions. We also show that effective near-surface potentials, ϕENS, obtained from ⟨r-6⟩norm provide a reliable and quantitative measure of intermolecular interactions including electrostatics, while ϕENS values obtained from only Γ1 or Γ2 sPRE rates can have significant artifacts as a consequence of potential variations and changes in the diffusive properties of the cosolute around the protein surface. Finally, we discuss the experimental feasibility and limitations of extracting the high-frequency limit of J(ω) that is related to ⟨r-8⟩norm and report on the extremely local intermolecular potential.

Dataset Information

A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T1 relaxation enhancement.

Publications

A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T1 relaxation enhancement.

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