Category Archives: Spin Labeling

Site-Directed Spin Labeling EPR for Studying Membrane Proteins #DNPNMR #EPR

Sahu, Indra D., and Gary A. Lorigan. “Site-Directed Spin Labeling EPR for Studying Membrane Proteins.” BioMed Research International 2018 (2018): 1–13.

https://doi.org/10.1155/2018/3248289

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy is a rapidly expanding powerful biophysical technique to study the structural and dynamic properties of membrane proteins in a native environment. Membrane proteins are responsible for performing important functions in a wide variety of complicated biological systems that are responsible for the survival of living organisms. In this review, a brief introduction of the most popular SDSL EPR techniques and illustrations of recent applications for studying pertinent structural and dynamic properties on membrane proteins will be discussed.

[NMR] Postdoctoral position at Miami University

Postdoctoral Position: EPR Spectroscopic Studies of Membrane Proteins

Miami University, Oxford, OH, USA

A Postdoctoral research position is available immediately to study the structural and dynamic properties of integral membrane proteins in the laboratory of Prof. Gary A. Lorigan in the Department of Chemistry and Biochemistry at Miami University in Ohio. The postdoctoral position is funded through a NIH MIRA R35 grant.

Candidates who are interested in studying the structural and dynamic properties of membrane proteins are encouraged to apply. Experience in two of the following areas is desirable: molecular biology and biochemistry of membrane proteins, protein purification, and EPR spectroscopy. 2 pulsed EPR spectrometers (X-band/Q-band) for DEER and ESEEM experiments, 2 CW-EPR spectrometers, and a 500 MHz solid-state NMR instrument are available for this project. Miami University is home to the Ohio Advanced EPR Lab (http://epr.miamioh.edu). Please send a CV and two letters of recommendation to: Professor Gary A. Lorigan, Department of Chemistry and Biochemistry, Miami University gary.lorigan@miamioh.edu. A Ph.D. in Chemistry/Biochemistry or related fields is required. Contact phone is 513-529-3338. 

Miami University, an Equal Opportunity/Affirmative Action employer, encourages applications from minorities, women, protected veterans and individuals with disabilities. Miami University prohibits harassment, discrimination and retaliation on the basis of sex/gender (including sexual harassment, sexual violence, sexual misconduct, domestic violence, dating violence, or stalking), race, color, religion, national origin (ancestry), disability, age (40 years or older), sexual orientation, gender identity, pregnancy, status as a parent or foster parent, military status, or veteran status in its recruitment, selection, and employment practices. Requests for all reasonable accommodations for disabilities related to employment should be directed to ADAFacultyStaff@miamioh.edu or 513-529-3560.

As part of the University’s commitment to maintaining a healthy and safe living, learning, and working environment, we encourage you to read Miami University’s Annual Security & Fire Safety Report at http://www.MiamiOH.edu/campus-safety/annual-report/index.html(http://www.miamioh.edu/campus-safety/annual-report/index.html), which contains information about campus safety, crime statistics, and our drug and alcohol abuse and prevention program designed to prevent the unlawful possession, use, and distribution of drugs and alcohol on campus and at university events and activities. This report also contains information on programs and policies designed to prevent and address sexual violence, domestic violence, dating violence, and stalking. Each year, email notification of this website is made to all faculty, staff, and enrolled students. Written notification is also provided to prospective students and employees. Hard copies of the Annual Security & Fire Safety Report may be obtained from the Miami University Police Department at (513) 529-2225.

Gary A. Lorigan

John W. Steube Professor 

Department of Chemistry and Biochemistry

Miami University

651 E. High St.

Oxford, Ohio 45056

Office: 137 Hughes Laboratories

Phone: (513) 529-3338

Fax: (513) 529-5715

e-mail: gary.lorigan@miamioh.edu

web: www.users.muohio.edu/lorigag/index.html

EPR facility: epr.muohio.edu

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EPR Spectroscopy of Nitroxide Spin Probes #EPR #DNPNMR

Nitroxide spin labels are extensively used in EPR for distance measurements and many polarizing agents are based on nitroxides. More recently they are also used in Overhauser DNP measurements (ODNP) to study surface hydration dynamics of larger (membrane) proteins. Although the article is already a bit older, it is a nice review of spin labels and their use in EPR spectroscopy.

Bordignon, E., EPR Spectroscopy of Nitroxide Spin Probes, in eMagRes. 2017, John Wiley & Sons, Ltd. p. 235-254.

http://dx.doi.org/10.1002/9780470034590.emrstm1513

In this article, we will introduce the main chemical and spectroscopic properties of nitroxides. These paramagnetic non-endogenous probes have been widely used in EPR spectroscopy in the last decade due to their high stability and simple spectral fingerprint, which provides a wealth of qualitative and quantitative information about their microscopic environment under almost unrestricted experimental conditions. Nitroxides can be covalently or noncovalently introduced into a variety of different materials to monitor viscosity, local dynamics, pH, polarity, H-bond networks, transition temperatures, and distances toward other nitroxide probes. In general, these small probes minimally perturb the system under investigation, and being the unique paramagnetic centers in an otherwise diamagnetic sample, they provide unequivocal information. Here we will focus on their exquisite sensitivity to report molecular motions within defined ‘EPR timescales’ and spin-spin interactions via changes in their spectral lineshape. Additionally, we will discuss some methods to monitor polarity and formation of H-bonds in their microenvironment.

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR

Voinov, M.A., et al., Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR. J Phys Chem B, 2015. 119(32): p. 10180-90.

http://www.ncbi.nlm.nih.gov/pubmed/26230514

Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with ~17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins.

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR

Voinov, M.A., et al., Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR. J Phys Chem B, 2015. 119(32): p. 10180-90.

http://www.ncbi.nlm.nih.gov/pubmed/26230514

Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with approximately 17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins.

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