scholarly journals High-resolution models of actin-bound myosin from EPR of a bifunctional spin label

2018 ◽  
Author(s):  
Benjamin P. Binder ◽  
Andrew R. Thompson ◽  
David D. Thomas
Keyword(s):  
High Resolution ◽  
Spin Label ◽  
Continuous Wave ◽  
Catalytic Domain ◽  
Structural Models ◽  
Spin Labels ◽  
Paramagnetic Resonance ◽  
Distance Constraints ◽  
Helix Orientation ◽  
Double Electron Electron Resonance

AbstractWe have employed two complementary high-resolution electron paramagnetic resonance (EPR) techniques with a bifunctional spin label (BSL) to test and refine protein structural models based on crystal structures and cryo-EM. We demonstrate this approach by investigating the effects of nucleotide binding on the structure of myosin’s catalytic domain (CD), while myosin is in complex with actin. Unlike conventional spin labels attached to single Cys, BSL reacts with a pair of Cys; in this study, we thoroughly characterize BSL’s rigid, highly stereoselective attachment to protein α-helices, which permits accurate measurements of orientation and distance. Distance constraints were obtained from double electron-electron resonance (DEER) on myosin constructs labeled with BSL specifically at two sites. Constraints for orientation of individual helices were obtained previously from continuous-wave EPR (CW-EPR) of myosin labeled at specific sites with BSL in oriented muscle fibers. We have shown previously that CW-EPR of BSL quantifies helix orientation within actin-bound myosin; here we show that the addition of high-resolution distance constraints by DEER alleviates remaining spatial ambiguity, allowing for direct testing and refinement of atomic structural models. This approach is applicable to any orientable complex (e.g., membranes or filaments) in which site-specific di- Cys mutation is feasible.

scholarly journals High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label

2015 ◽  
Vol 112 (26) ◽  
pp. 7972-7977 ◽  
Author(s):  
Benjamin P. Binder ◽  
Sinziana Cornea ◽  
Andrew R. Thompson ◽  
Rebecca J. Moen ◽  
David D. Thomas
Keyword(s):  
High Resolution ◽  
Spin Label ◽  
Catalytic Domain ◽  
Structural Data ◽  
Structural Elements ◽  
Paramagnetic Resonance ◽  
Filament Axis ◽  
Adp Release ◽  
Helix Orientation ◽  
Electron Paramagnetic

Using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) bound stereospecifically to Dictyostelium myosin II, we determined with high resolution the orientation of individual structural elements in the catalytic domain while myosin is in complex with actin. BSL was attached to a pair of engineered cysteine side chains four residues apart on known α-helical segments, within a construct of the myosin catalytic domain that lacks other reactive cysteines. EPR spectra of BSL-myosin bound to actin in oriented muscle fibers showed sharp three-line spectra, indicating a well-defined orientation relative to the actin filament axis. Spectral analysis indicated that orientation of the spin label can be determined within <2.1° accuracy, and comparison with existing structural data in the absence of nucleotide indicates that helix orientation can also be determined with <4.2° accuracy. We used this approach to examine the crucial ADP release step in myosin’s catalytic cycle and detected reversible rotations of two helices in actin-bound myosin in response to ADP binding and dissociation. One of these rotations has not been observed in myosin-only crystal structures.

scholarly journals High-resolution EPR distance measurements on RNA and DNA with the non-covalent Ǵ spin label

2019 ◽  
Vol 48 (2) ◽  
pp. 924-933 ◽  
Author(s):  
Marcel Heinz ◽  
Nicole Erlenbach ◽  
Lukas S Stelzl ◽  
Grace Thierolf ◽  
Nilesh R Kamble ◽  
...  
Keyword(s):  
High Resolution ◽  
Nucleic Acids ◽  
Spin Label ◽  
Double Resonance ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Spin Labels ◽  
Abasic Site ◽  
Abasic Sites ◽  
Rna And Dna

Abstract Pulsed electron paramagnetic resonance (EPR) experiments, among them most prominently pulsed electron-electron double resonance experiments (PELDOR/DEER), resolve the conformational dynamics of nucleic acids with high resolution. The wide application of these powerful experiments is limited by the synthetic complexity of some of the best-performing spin labels. The recently developed $\bf\acute{G}$ (G-spin) label, an isoindoline-nitroxide derivative of guanine, can be incorporated non-covalently into DNA and RNA duplexes via Watson-Crick base pairing in an abasic site. We used PELDOR and molecular dynamics (MD) simulations to characterize $\bf\acute{G}$, obtaining excellent agreement between experiments and time traces calculated from MD simulations of RNA and DNA double helices with explicitly modeled $\bf\acute{G}$ bound in two abasic sites. The MD simulations reveal stable hydrogen bonds between the spin labels and the paired cytosines. The abasic sites do not significantly perturb the helical structure. $\bf\acute{G}$ remains rigidly bound to helical RNA and DNA. The distance distributions between the two bound $\bf\acute{G}$ labels are not substantially broadened by spin-label motions in the abasic site and agree well between experiment and MD. $\bf\acute{G}$ and similar non-covalently attached spin labels promise high-quality distance and orientation information, also of complexes of nucleic acids and proteins.

scholarly journals Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution

2020 ◽  
Vol 48 (9) ◽  
pp. e49-e49 ◽  
Author(s):  
Shreya Ghosh ◽  
Matthew J Lawless ◽  
Hanna J Brubaker ◽  
Kevin Singewald ◽  
Michael R Kurpiewski ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Conformational Changes ◽  
Continuous Wave ◽  
Md Simulations ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Pulsed Epr ◽  
Distance Distributions ◽  
Dna Backbone ◽  
Double Electron Electron Resonance

Abstract Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2′dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules.

scholarly journals High-Resolution EPR of a Bifunctional Spin Label Reveals an ADP Induced Structural Rearrangement of the Actin-Bound Myosin Catalytic Domain

2011 ◽  
Vol 100 (3) ◽  
pp. 130a
Author(s):  
Ryan N. Mello ◽  
Roman V. Agafonov ◽  
Andrew R. Thompson ◽  
David D. Thomas
Keyword(s):  
High Resolution ◽  
Spin Label ◽  
Catalytic Domain ◽  
Structural Rearrangement

scholarly journals High-Resolution EPR of a Bifunctional Spin Label Reveals Structural Transitions within Myosin's Catalytic Domain

2013 ◽  
Vol 104 (2) ◽  
pp. 648a
Author(s):  
Benjamin Binder ◽  
Ryan Mello ◽  
Rebecca Moen ◽  
David D. Thomas
Keyword(s):  
High Resolution ◽  
Spin Label ◽  
Catalytic Domain ◽  
Structural Transitions

scholarly journals Changes in the fraction of strongly attached cross bridges in mouse atrophic and hypertrophic muscles as revealed by continuous wave electron paramagnetic resonance

2019 ◽  
Vol 316 (5) ◽  
pp. C722-C730
Author(s):  
Laura Galazzo ◽  
Leonardo Nogara ◽  
Francesca LoVerso ◽  
Antonino Polimeno ◽  
Bert Blaauw ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Spin Label ◽  
Structural Changes ◽  
Continuous Wave ◽  
Myosin Head ◽  
Force Generation ◽  
Paramagnetic Resonance ◽  
The Difference ◽  
Cross Bridges ◽  
Electron Paramagnetic

Electron paramagnetic resonance (EPR), coupled with site-directed spin labeling, has been proven to be a particularly suitable technique to extract information on the fraction of myosin heads strongly bound to actin upon muscle contraction. The approach can be used to investigate possible structural changes occurring in myosin of fiber s altered by diseases and aging. In this work, we labeled myosin at position Cys707, located in the SH1-SH2 helix in the myosin head cleft, with iodoacetamide spin label, a spin label that is sensitive to the reorientational motion of this protein during the ATPase cycle and characterized the biochemical states of the labeled myosin head by means of continuous wave EPR. After checking the sensitivity and the power of the technique on different muscles and species, we investigated whether changes in the fraction of strongly bound myosin heads might explain the contractile alterations observed in atrophic and hypertrophic murine muscles. In both conditions, the difference in contractile force could not be justified simply by the difference in muscle mass. Our results showed that in atrophic muscles the decrease in force generation was attributable to a lower fraction of strongly bound cross bridges during maximal activation. In contrast in hypertrophic muscles, the increase in force generation was likely due to several factors, as pointed out by the comparison of the EPR experiments with the tension measurements on single skinned fibers.

scholarly journals Probing iso-1-cytochrome c structure by site-directed spin labeling and electron paramagnetic resonance techniques.

1999 ◽  
Vol 46 (4) ◽  
pp. 889-899 ◽  
Author(s):  
J Pyka ◽  
A Osyczka ◽  
B Turyna ◽  
W Blicharski ◽  
W Froncisz
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Cytochrome C ◽  
Spin Label ◽  
Continuous Wave ◽  
Spin Labeling ◽  
Local Dynamic ◽  
Epr Spectrum ◽  
Paramagnetic Resonance ◽  
Site Directed Spin Labeling ◽  
Electron Paramagnetic

A cysteine-specific methanethiosulfonate spin label was introduced into yeast iso-1-cytochrome c at three different positions. The modified forms of cytochrome c included: the wild-type protein labeled at naturally occurring C102, and two mutated proteins, S47C and L85C, labeled at positions 47 and 85, respectively (both S47C and L85C derived from the protein in which C102 had been replaced by threonine). All three spin-labeled protein derivatives were characterized using electron paramagnetic resonance (EPR) techniques. The continuous wave (CW) EPR spectrum of spin label attached to L85C differed from those recorded for spin label attached to C102 or S47C, indicating that spin label at position 85 was more immobilized and exhibited more complex tumbling than spin label at two other positions. The temperature dependence of the CW EPR spectra and CW EPR power saturation revealed further differences of spin-labeled L85C. The results were discussed in terms of application of the site-directed spin labeling technique in probing the local dynamic structure of iso-1-cytochrome c.

scholarly journals Exploring the pH-Induced Functional Phase Space of Human Serum Albumin by EPR Spectroscopy

2018 ◽  
Vol 4 (4) ◽  
pp. 47 ◽  
Author(s):  
Jörg Reichenwallner ◽  
Marie-T. Oehmichen ◽  
Christian Schmelzer ◽  
Till Hauenschild ◽  
Andreas Kerth ◽  
...  
Keyword(s):  
Phase Space ◽  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Continuous Wave ◽  
Molten Globule ◽  
Paramagnetic Resonance ◽  
Ph Range ◽  
Double Electron Electron Resonance ◽  
Electron Paramagnetic

A systematic study on the self-assembled solution system of human serum albumin (HSA) and paramagnetic doxyl stearic acid (5-DSA and 16-DSA) ligands is reported covering the broad pH range 0.7–12.9, mainly using electron paramagnetic resonance (EPR) methods. It is tested to which extent the pH-induced conformational isomers of HSA reveal themselves in continuous wave (CW) EPR spectra from this spin probing approach in comparison to an established spin-labeling strategy utilizing 3-maleimido proxyl (5-MSL). Most analyses are conducted on empirical levels with robust strategies that allow for the detection of dynamic changes of ligand, as well as protein. Special emphasis has been placed on the EPR spectroscopic detection of a molten globule (MG) state of HSA that is typically found by the fluorescent probe 8-Anilino- naphthalene-1-sulfonic acid (ANS). Moreover, four-pulse double electron-electron resonance (DEER) experiments are conducted and substantiated with dynamic light scattering (DLS) data to determine changes in the solution shape of HSA with pH. All results are ultimately combined in a detailed scheme that describes the pH-induced functional phase space of HSA.

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