scholarly journals Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4143
Author(s):  
Jeannette M. Laugwitz ◽  
Haleh H. Haeri ◽  
Anette Kaiser ◽  
Ulrike Krug ◽  
Dariush Hinderberger ◽  
...  
Keyword(s):  
Continuous Wave ◽  
Conformational Dynamics ◽  
Spin Labeling ◽  
Conformational States ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Pulsed Epr ◽  
Y2 Receptor ◽  
Epr Measurements ◽  
Double Electron Electron Resonance

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor.

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 Combining Site-Directed Spin Labeling in Vivo and In-Cell EPR Distance Determination

2019 ◽  
Author(s):  
Pia Widder ◽  
Julian Schuck ◽  
Daniel Summerer ◽  
Malte Drescher
Keyword(s):  
Spin Labeling ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Distance Determination ◽  
Bioorthogonal Labeling ◽  
In Vitro Measurements ◽  
Site Directed Spin Labeling ◽  
Double Electron Electron Resonance

Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E.coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.<br>

scholarly journals Combining Site-Directed Spin Labeling in Vivo and In-Cell EPR Distance Determination

2019 ◽  
Author(s):  
Pia Widder ◽  
Julian Schuck ◽  
Daniel Summerer ◽  
Malte Drescher
Keyword(s):  
Spin Labeling ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Distance Determination ◽  
Bioorthogonal Labeling ◽  
In Vitro Measurements ◽  
Site Directed Spin Labeling ◽  
Double Electron Electron Resonance

Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E.coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.<br>

scholarly journals Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts

2016 ◽  
Vol 18 (8) ◽  
pp. 5819-5831 ◽  
Author(s):  
Zhanglong Liu ◽  
Thomas M. Casey ◽  
Mandy E. Blackburn ◽  
Xi Huang ◽  
Linh Pham ◽  
...  
Keyword(s):  
Spin Labeling ◽  
Conformational Sampling ◽  
Electron Resonance ◽  
Pulsed Epr ◽  
Double Electron ◽  
Conformational Landscape ◽  
Double Electron Electron Resonance ◽  
Hiv 1

The conformational landscape of HIV-1 protease can be characterized by double electron–electron resonance (DEER) spin-labeling.

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 Pulsed ELDOR Measurement of the Distance Between a Spin-Label and Copper (II) Centre in the Copper Loaded R48C Mutant of N. gonorrhoeae Ferric Binding Protein

2018 ◽  
Author(s):  
Matt Bawn ◽  
Justin Bradley ◽  
Fraser MacMillan
Keyword(s):  
Spin Label ◽  
Binding Protein ◽  
Double Resonance ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Distance Determination ◽  
Pulsed Epr ◽  
Ferric Binding Protein ◽  
Relaxation Experiments ◽  
Electron Paramagnetic

AbstractDistance determination in proteins and biomolecules using pulsed EPR (electron paramagnetic resonance) techniques is becoming an increasingly popular and accessible technique. PELDOR (pulsed electron-electron double resonance), is a technique designed for distance determination over a nanoscopic scale. Here, ferric binding protein (Fbp) is used to demonstrate the practicability of this technique to Cu (II) Metalloproteins. PELDOR is usually applied to bi-radicals or endogenous radicals, and distance determination using pulsed EPR of metal containing centres in biomolecules has been restricted to relaxation experiments. PELDOR distance measurements between a Cu (II) ion and a nitroxide have previously only been reported for model compounds [1, 2].Fbp as the name suggests usually, contains a Fe (III) ion centre. For the purposes of this investigation the Fe (III) ion was removed and replaced by a Cu (II) ion, after a nitroxide spin-label was added to the Fbp using of site directed spin-labelling (SDSL). PELDOR was then applied to measure the distance between the two centres.Simulation methods were then employed to fully investigate these data and allow a quantitative interpretation of the copper nitroxide PELDOR data. The observed PELDOR time traces were analysed using DEER analysis[3].

scholarly journals Protein Conformational Dynamics upon Association with the Surfaces of Lipid Membranes and Engineered Nanoparticles: Insights from Electron Paramagnetic Resonance Spectroscopy

Molecules ◽  
2020 ◽  
Vol 25 (22) ◽  
pp. 5393
Author(s):  
Elka R. Georgieva
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Continuous Wave ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Conformational Dynamics ◽  
Engineered Nanoparticles ◽  
Resonance Spectroscopy ◽  
Functional Protein ◽  
Paramagnetic Resonance ◽  
Conformational Rearrangements ◽  
Electron Paramagnetic

Detailed study of conformational rearrangements and dynamics of proteins is central to our understanding of their physiological functions and the loss of function. This review outlines the applications of the electron paramagnetic resonance (EPR) technique to study the structural aspects of proteins transitioning from a solution environment to the states in which they are associated with the surfaces of biological membranes or engineered nanoobjects. In the former case these structural transitions generally underlie functional protein states. The latter case is mostly relevant to the application of protein immobilization in biotechnological industries, developing methods for protein purification, etc. Therefore, evaluating the stability of the protein functional state is particularly important. EPR spectroscopy in the form of continuous-wave EPR or pulse EPR distance measurements in conjunction with protein spin labeling provides highly versatile and sensitive tools to characterize the changes in protein local dynamics as well as large conformational rearrangements. The technique can be widely utilized in studies of both protein-membrane and engineered nanoobject-protein complexes.

Site-directed spin labeling EPR spectroscopy in protein research

2013 ◽  
Vol 394 (10) ◽  
pp. 1281-1300 ◽  
Author(s):  
Johann P. Klare
Keyword(s):  
Nucleic Acids ◽  
Epr Spectroscopy ◽  
Protein Function ◽  
Conformational Dynamics ◽  
Spin Labeling ◽  
Paramagnetic Resonance ◽  
Protein Research ◽  
Site Directed Spin Labeling ◽  
Electron Paramagnetic ◽  
Natronomonas Pharaonis

Abstract Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has emerged as an efficient tool to elucidate the structure and the conformational dynamics of proteins under conditions close to the native state. This review article summarizes the basics as well as the recent progress in SDSL and EPR methods, especially for investigations on protein structure, protein function, and interaction of proteins with other proteins or nucleic acids. Labeling techniques as well as EPR methods are introduced and exemplified with applications to systems that have been studied in the author’s laboratory in the past 15 years, headmost the sensory rhodopsin-transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis. Further examples underline the application of SDSL EPR spectroscopy to answer specific questions about the system under investigation, such as the nature and influence of interactions of proteins with other proteins or nucleic acids. Finally, it is discussed how SDSL EPR can be combined with other biophysical techniques to combine the strengths of the different methodologies.

scholarly journals Accessing the distance range of interest in biomolecules: Site-directed spin labeling and DEER spectroscopy

2010 ◽  
Vol 24 (3-4) ◽  
pp. 283-288 ◽  
Author(s):  
Sabine Böhme ◽  
Heinz-Jürgen Steinhoff ◽  
Johann P. Klare
Keyword(s):  
Conformational Changes ◽  
Protein Interactions ◽  
Spin Labeling ◽  
Paramagnetic Resonance ◽  
Topological Information ◽  
Pulsed Epr ◽  
Site Directed Spin Labeling ◽  
Intermolecular Distances ◽  
And Function ◽  
Electron Paramagnetic

Investigations on the structure and function of biomolecules often depend on the availability of topological information to build up structural models or to characterize conformational changes during function. Electron paramagnetic resonance (EPR) spectroscopy in combination with site–directed spin labeling (SDSL) allow to determine intra- and intermolecular distances in the range from 4–70 Å, covering the range of interest for biomolecules. The approach does not require crystalline samples and is well suited also for molecules exhibiting intrinsic flexibility. This article is intended to give an overview on pulsed EPR in conjunction with SDSL to study protein interactions as well as conformational changes, exemplified on the tRNA modifying enzyme MnmE.

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