Molecular Motion of Spin Labeled Side Chains in α-Helices:  Analysis by Variation of Side Chain Structure

Columbus, Linda; Kálai, Tamás; Jekő, József; Hideg, Kálmán; Hubbell, Wayne L.

doi:10.1021/bi002645h

Cited by 277 publications

(617 citation statements)

References 32 publications

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“…The characteristic lineshape of the stacked A/U pair has been observed in previous investigations of lipid bilayer [27] and protein [28], and has been shown to originate from a nitroxide undergoing restricted, anisotropic rotation [13,28]. In the TLR studies, the A/U site spectrum has been quantitatively simulated using a "MOMD" (Microscopic Ordered Macroscopic Disordered) model developed by Freed and co-workers [23], which uses an order parameter (S 20 ) and an effective correlation time (τ) to describe diffusive motion of the nitroxide under the restraint of an ordering potential.…”

Section: A Similar Lineshape For Stacked A/u Pairs In Two Rna Moleculessupporting

confidence: 64%

A model system for investigating lineshape/structure correlations in RNA site-directed spin labeling

Qin

Iseri

Oki

2006

Biochemical and Biophysical Research Communications

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In RNA site-directed spin labeling (SDSL) studies, structural and dynamic information at the individual RNA nucleotide level is derived from the observed electron paramagnetic resonance spectrum of a covalently attached nitroxide. A systematic approach for RNA SDSL is to establish a library that categorizes observed spectral lineshapes based on known RNA structures, thus enabling lineshape-based structure identification at any RNA site. To establish the first RNA SDSL library, selective secondary structure elements have been systematically engineered into a model RNA. Nitroxide lineshapes reporting features specific to each element were obtained utilizing a new avidintethering scheme for suppressing spectral effects due to uniform RNA tumbling. The data demonstrated two key features required for a SDSL library with a predicting power: (i) spectral divergence-distinctive lineshape for different elements; and (ii) spectral convergence-similar lineshape for the same element in different contexts. This sets the foundation for further RNA SDSL library development. KeywordsRNA; Site-directed spin labeling; Secondary structure; Lineshape; Library RNA is a versatile molecule both in terms of structure and function. Recent advances in highresolution structure determination of large RNAs [1-3] and RNPs, particularly the ribosome [4-6], have drastically expanded the scope of the available RNA structural information. However, understanding the structure/function relationship in RNA requires information that goes beyond the static structure and tools that provide time-dependent structural information under physiological conditions are needed. Recently, the technique of site-directed spin labeling (SDSL) has been utilized to study RNAs with complex three-dimensional structures (see review [7]). SDSL utilizes a site-specifically attached nitroxide moiety that contains a stable, unpaired electron, and obtains local structural information by analyzing the electron paramagnetic resonance (EPR) spectrum of the nitroxide [8]. It is capable of providing information on high molecular weight assemblies under physiological conditions using a small amount of sample (∼5 μl of 50 μM of sample per measurement), and has been utilized to monitor solution structure and conformational changes at specific sites of RNA molecules [9][10][11][12][13][14][15][16] and to measure distances between two labeled sites within RNAs [17][18][19][20].An important source of information in RNA SDSL is the spectral lineshape of a singly labeled nitroxide [7]. In the conventional X-band measurements, the observed lineshape changes

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Section: A Similar Lineshape For Stacked A/u Pairs In Two Rna Moleculessupporting

confidence: 64%

A model system for investigating lineshape/structure correlations in RNA site-directed spin labeling

Qin

Iseri

Oki

2006

Biochemical and Biophysical Research Communications

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“…The theoretical results are compared with experimental spectra recorded at different temperatures and in different solvents, which are available in the literature. 8,13 A spectrum has been obtained as the sum of contributions, each due to an independent set of conformers, with suitable weights. Each spectral component is calculated as explained in the previous Section.…”

Section: Resultsmentioning

confidence: 99%

“…In the case of X-band (9GHz) ESR experiments, such a condition is satisfied by motions with correlation times shorter than about one nanosecond. If the fast-motional regime can be assumed for the side-chain motion, the ESR spectrum of a nitroxide spin label, in a protein at the orientation Ω D in the laboratory frame, is given by three lines whose positions are determined by the spin Hamiltonian eq 4, partially averaged by the side-chain dynamics: (8) where the upper bar denotes the average over the fast motions.…”

Section: A Superposition Of Fast and Slow Motionsmentioning

confidence: 99%

“…The nitroxide spin probe is identified with the rotor and the constraints deriving from the chain geometry and from the steric hindrance of the environment are taken into account through an orienting potential. The MOMD approach has been used for proteins in high viscosity media and/or low temperature, 8 as well as for very high frequency ESR, 9 but of course it is no longer suitable when the rigid limit cannot be assumed for the protein motions.…”

Section: Introductionmentioning

confidence: 99%

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Modeling the Effects of Structure and Dynamics of the Nitroxide Side Chain on the ESR Spectra of Spin-Labeled Proteins

2006

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In a companion paper (Tombolato, F.; Ferrarini, A.; Freed, J.H., ... ) a study of the conformational dynamics of methanethiosulfonate spin probes linked at a surface exposed α-helix has been presented. Here, on the basis of this analysis, X-band ESR spectra of these spin labels are simulated, within the framework of the Stochastic Liouville Equation methodology. Slow reorientations of the whole protein are superimposed on fast chain motions, which have been identified with conformational jumps and fluctuations in the minima of the chain torsional potential. Fast chain motions are introduced in the SLE for the protein reorientations through partially averaged magnetic tensors and relaxation times calculated according to the motional narrowing theory. The 72R1 and 72R2 mutants of T4 lysozyme, which bear the spin label at a solvent-exposed helix site, have been taken as test systems. For the side-chain of the R2 spin label, only a few non-interconverting conformers are possible, whose mobility is limited to torsional fluctuations, yielding almost identical spectra, typical of slightly mobile nitroxides. In the case of R1, more complex spectra result from the simultaneous presence of constrained and mobile chain conformers, with relative weights which can depend on the local environment. The model provides an explanation for the experimentally observed dependence of the spectral lineshapes on temperature, solvent, and pattern of substituents in the pyrroline ring. The relatively simple methodology presented here allows the introduction of realistic features of the spin probe dynamics into the simulation of ESR spectra of spin labeled proteins; moreover, it provides suggestions for a proper account of such dynamics in more sophisticated approaches.

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“…The label is linked to the protein backbone through five rotatable bonds; however, interactions between the distal sulfur and the Ca proton 20 restrict rotameric conversions to X4 and X5. 21 differences that are seen between spectroscopic and crystallographic methods and suggest that regions where the two crystal structures of BtuB differ represent sites of conformational exchange. The results also indicate that osmolytes such as PEGs when used in combination with SDSL are a powerful tool to explore conformational exchange in membrane proteins.…”

Section: Introductionmentioning

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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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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Molecular Motion of Spin Labeled Side Chains in α-Helices: Analysis by Variation of Side Chain Structure

Cited by 277 publications

References 32 publications

A model system for investigating lineshape/structure correlations in RNA site-directed spin labeling

A model system for investigating lineshape/structure correlations in RNA site-directed spin labeling

Modeling the Effects of Structure and Dynamics of the Nitroxide Side Chain on the ESR Spectra of Spin-Labeled Proteins

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

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