Locating a Lipid at the Portal to the Lipoxygenase Active Site

Gaffney, Betty J.; Bradshaw, Miles D.; Frausto, Stephen D.; Wu, Fayi; Freed, Jack H.; Borbat, Peter P.

doi:10.1016/j.bpj.2012.10.002

Cited by 56 publications

(91 citation statements)

References 60 publications

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“…The standard deviations of the interspin distances were estimated from the plots of distance distribution assuming that the distributions have a Gaussian profile.bMtsslTrilaterate was run using mtsslWizard-generated labels as input.cResults from the original publication [13]. …”

Section: Resultsmentioning

confidence: 99%

mtsslSuite:In silicospin labelling, trilateration and distance-constrained rigid body docking in PyMOL

et al. 2013

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Nanometer distance measurements based on electron paramagnetic resonance methods in combination with site-directed spin labelling are powerful tools for the structural analysis of macromolecules. The software package mtsslSuite provides scientists with a set of tools for the translation of experimental distance distributions into structural information. The package is based on the previously published mtsslWizard software for in silico spin labelling. The mtsslSuite includes a new version of MtsslWizard that has improved performance and now includes additional types of spin labels. Moreover, it contains applications for the trilateration of paramagnetic centres in biomolecules and for rigid-body docking of subdomains of macromolecular complexes. The mtsslSuite is tested on a number of challenging test cases and its strengths and weaknesses are evaluated.

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Section: Resultsmentioning

confidence: 99%

mtsslSuite:In silicospin labelling, trilateration and distance-constrained rigid body docking in PyMOL

et al. 2013

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“…If the ligand can be spin-labelled or the metal ion is paramagnetic, they can be localized by a distance matrix approach [36] or by an approach akin to the global positioning system (GPS), where the position of an observer can be determined by at least three distance measurements to reference sites [37]. The distance matrix approach tests and relaxes the assumption of a rigid reference domain at the expense of additional measurements between reference points.…”

Section: Model Building With Epr Restraintsmentioning

confidence: 99%

The contribution of modern EPR to structural biology

Jeschke

2018

Emerging Topics in Life Sciences

111

133

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Electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labelling is applicable to biomolecules and their complexes irrespective of system size and in a broad range of environments. Neither short-range nor long-range order is required to obtain structural restraints on accessibility of sites to water or oxygen, on secondary structure, and on distances between sites. Many of the experiments characterize a static ensemble obtained by shock-freezing. Compared with characterizing the dynamic ensemble at ambient temperature, analysis is simplified and information loss due to overlapping timescales of measurement and system dynamics is avoided. The necessity for labelling leads to sparse restraint sets that require integration with data from other methodologies for building models. The double electron-electron resonance experiment provides distance distributions in the nanometre range that carry information not only on the mean conformation but also on the width of the native ensemble. The distribution widths are often inconsistent with Anfinsen's concept that a sequence encodes a single native conformation defined at atomic resolution under physiological conditions.

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“…4 Residues in helix 2, the loop preceding it, and helix 11 are close to the headgroup of that lipid. Now, we substituted a spin-labeled cysteine for 18 of 21 residues in the helix to examine the dynamic state of helix 2.…”

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Fluctuations of an Exposed π-Helix Involved in Lipoxygenase Substrate Recognition

Bradshaw

Gaffney

2014

Biochemistry

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The second helix in lipoxygenases adapts to permit substrate access to the active site, but details of this process are varied and poorly understood. We therefore examined the dynamics of helix 2 in solutions of spin-labeled soybean lipoxygenase-1 and spin relaxation at 60 K of the spin-labels by catalytic iron. Helix 2 in soybean lipoxygenase structures is surface-exposed and contains one turn of π-helix, centrally located. A site-directed spin-label scan of 18 of the 21 helix 2 residues, and electron paramagnetic resonance, showed that the π-helical segment became unusually mobile, on a nanosecond time scale, under conditions favoring substrate binding (pH 9 and lipid addition), while segments before and after had relatively unchanged dynamics. Backbone dynamics of residues in the π-helical segment appeared to be correlated, at pH 9. Samples also were frozen to examine the polarity and proticity of the local environments, the effect of the local environment on intrinsic relaxation, and dipolar relaxation by two symmetries of catalytic iron. The average hyperfine tensor component, Azz, of four π-helix residues decreased by 1.75 G, with an increase in pH from 7 to 9, while it remained unaffected for nearby buried residues. Power saturation data suggested the change in polarity specific to the π-helix altered the intrinsic relaxation rates. Different symmetries of iron contributed to distance-dependent magnetic relaxation. We interpret these data to mean that a π-helix in the second helix of plant lipoxygenases is highly dynamic and is the site where lipid chains penetrate to inner helices that outline the substrate pocket.

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Locating a Lipid at the Portal to the Lipoxygenase Active Site

Cited by 56 publications

References 60 publications

mtsslSuite:In silicospin labelling, trilateration and distance-constrained rigid body docking in PyMOL

mtsslSuite:In silicospin labelling, trilateration and distance-constrained rigid body docking in PyMOL

The contribution of modern EPR to structural biology

Fluctuations of an Exposed π-Helix Involved in Lipoxygenase Substrate Recognition

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