Category Archives: Membrane Proteins

DNP Solid-state NMR of Biological Membranes #DNPNMR

Bechinger, Burkhard. “DNP Solid-State NMR of Biological Membranes” 7 (2018): 10. 

https://doi.org/10.1002/9780470034590.emrstm1558.

While solid-state NMR/DNP has become a well-established technique to significantly increase the signals of molecules embedded in homogeneous glassy matrices, the enhancement factors observed in heterogeneous and/or matrix-free samples lag somewhat behind. The possible reasons for such differences, present limitations, and future prospects of solid-state NMR/DNP are discussed in the context of membrane protein investigations. Membrane polypeptides and lipids are studied by MAS as well as oriented sample solid-state NMR approaches. Notably, even the more modest DNP signal enhancements obtained in such samples augment the signal intensities by 1–2 orders of magnitude, thus opening up new territory in structural biology by allowing the detection of new conformers, so far invisible intermediate states, or the acquisition of smaller quantities of membrane-associated polypeptides in much less time. New sample preparation protocols, dedicated instrumental hardware, and specifically designed biradicals have much improved the application of DNP to membranes using MAS and/or oriented solid-state NMR technologies.

Postdoctoral Position: EPR Spectroscopic Studies of Membrane Proteins #EPR

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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NMR web database:

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Hydration Dynamics of a Peripheral Membrane Protein #DNPNMR

Fisette, O., et al., Hydration Dynamics of a Peripheral Membrane Protein. J Am Chem Soc, 2016. 138(36): p. 11526-35.

https://www.ncbi.nlm.nih.gov/pubmed/27548572

Water dynamics in the hydration shell of the peripheral membrane protein annexin B12 were studied using MD simulations and Overhauser DNP-enhanced NMR. We show that retardation of water motions near phospholipid bilayers is extended by the presence of a membrane-bound protein, up to around 10 A above that protein. Near the membrane surface, electrostatic interactions with the lipid head groups strongly slow down water dynamics, whereas protein-induced water retardation is weaker and dominates only at distances beyond 10 A from the membrane surface. The results can be understood from a simple model based on additive contributions from the membrane and the protein to the activation free energy barriers of water diffusion next to the biomolecular surfaces. Furthermore, analysis of the intermolecular vibrations of the water network reveals that retarded water motions near the membrane shift the vibrational modes to higher frequencies, which we used to identify an entropy gradient from the membrane surface toward the bulk water. Our results have implications for processes that take place at lipid membrane surfaces, including molecular recognition, binding, and protein-protein interactions.

Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location

Liao, S.Y., et al., Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location. J Biomol NMR, 2016: p. 1-15.

http://www.ncbi.nlm.nih.gov/pubmed/26873390

Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~fourfold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105-160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 A from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes.

Characterization of Membrane Proteins in Isolated Native Cellular Membranes by Dynamic Nuclear Polarization Solid-State NMR Spectroscopy without Purification and Reconstitution

Jacso, T., et al., Characterization of Membrane Proteins in Isolated Native Cellular Membranes by Dynamic Nuclear Polarization Solid-State NMR Spectroscopy without Purification and Reconstitution. Angewandte Chemie, 2012. 124(2): p. 447-450.

http://dx.doi.org/10.1002/ange.201104987

Structural information is key for understanding biological processes. Insoluble proteins, like membrane proteins and amyloid fibrils, are a large class of proteins that are underrepresented in the protein data bank (PDB). As of today, only 7% of all entries in the PDB refer to either a membrane protein or an amyloid fibril structure (membrane protein: 4994 entries; amyloid fibril: 67 entries; total number of entries: 70,303; http://www.rcsb.org/pdb/home/home.do). Given the fact that many drugs target membrane proteins, involved in signal transduction, [1] structural information is highly desirable for a better understanding of the underlying biochemical mechanisms.

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

Yamamoto, K., et al., Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization. Biochimica et Biophysica Acta (BBA) – Biomembranes, 2015. 1848(1, Part B): p. 342-349.

http://dx.doi.org/10.1016/j.bbamem.2014.07.008

While an increasing number of structural biology studies successfully demonstrate the power of high-resolution structures and dynamics of membrane proteins in fully understanding their function, there is considerable interest in developing NMR approaches to obtain such information in a cellular setting. As long as the proteins inside the living cell tumble rapidly in the NMR timescale, recently developed in-cell solution NMR approaches can provide 3D structural information. However, there are numerous challenges to study membrane proteins inside a cell. Research in our laboratory is focused on developing a combination of solid-state NMR and biological approaches to overcome these challenges in order to obtain high-resolution structural insights into electron transfer processes mediated by membrane-bound proteins like mammalian cytochrome-b5, cytochrome-P450 and cytochrome-P450-reductase. In this study, we demonstrate the feasibility of using dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR spectroscopy for in-cell studies on a membrane-anchored protein. Our experimental results obtained from 13C-labeled membrane-anchored cytochrome-b5 in native Escherichia coli cells show a ~ 16-fold DNP signal enhancement. Further, results obtained from a 2D 13C/13C chemical shift correlation MAS experiment demonstrate the feasibility of suppressing the background signals from other cellular contents for high-resolution structural studies on membrane proteins. We believe that this study would pave new avenues for high-resolution structural studies on a variety of membrane-associated proteins and their complexes in the cellular context to fully understand their functional roles in physiological processes. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky.

A method for dynamic nuclear polarization enhancement of membrane proteins

Smith, A.N., et al., A method for dynamic nuclear polarization enhancement of membrane proteins. Angew Chem Int Ed Engl, 2015. 54(5): p. 1542-6.

http://www.ncbi.nlm.nih.gov/pubmed/25504310

Dynamic nuclear polarization (DNP) magic-angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy has the potential to enhance NMR signals by orders of magnitude and to enable NMR characterization of proteins which are inherently dilute, such as membrane proteins. In this work spin-labeled lipid molecules (SL-lipids), when used as polarizing agents, lead to large and relatively homogeneous DNP enhancements throughout the lipid bilayer and to an embedded lung surfactant mimetic peptide, KL4 . Specifically, DNP MAS ssNMR experiments at 600 MHz/395 GHz on KL4 reconstituted in liposomes containing SL-lipids reveal DNP enhancement values over two times larger for KL4 compared to liposome suspensions containing the biradical TOTAPOL. These findings suggest an alternative sample preparation strategy for DNP MAS ssNMR studies of lipid membranes and integral membrane proteins.

Recent advances in magic angle spinning solid state NMR of membrane proteins

Wang, S. and V. Ladizhansky, Recent advances in magic angle spinning solid state NMR of membrane proteins. Prog. NMR. Spec., 2014. 82(0): p. 1-26.

http://www.sciencedirect.com/science/article/pii/S0079656514000478

Membrane proteins mediate many critical functions in cells. Determining their three-dimensional structures in the native lipid environment has been one of the main objectives in structural biology. There are two major NMR methodologies that allow this objective to be accomplished. Oriented sample NMR, which can be applied to membrane proteins that are uniformly aligned in the magnetic field, has been successful in determining the backbone structures of a handful of membrane proteins. Owing to methodological and technological developments, Magic Angle Spinning (MAS) solid-state NMR (ssNMR) spectroscopy has emerged as another major technique for the complete characterization of the structure and dynamics of membrane proteins. First developed on peptides and small microcrystalline proteins, MAS ssNMR has recently been successfully applied to large membrane proteins. In this review we describe recent progress in MAS ssNMR methodologies, which are now available for studies of membrane protein structure determination, and outline a few examples, which highlight the broad capability of ssNMR spectroscopy.

Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature

Solid-state DNP-NMR are typically performed at cryogenic temperatures and samples, especially bio-macromolecules often require cryo-protection. This is a recent review about sample preparation and cryo-protecting samples to preserve the spectral resolution.

Lee, M. and M. Hong, Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature. J Biomol NMR, 2014. 59(4): p. 263-277.

http://www.ncbi.nlm.nih.gov/pubmed/25015530

Solid-state NMR spectra of membrane proteins often show significant line broadening at cryogenic temperatures. Here we investigate the effects of several cryoprotectants to preserve the spectral resolution of lipid membranes and membrane peptides at temperatures down to ~200 K. Trehalose, glycerol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), and polyethylene glycol (PEG), were chosen. These compounds are commonly used in protein crystallography and cryobiology. 13C and 1H magic-angle-spinning spectra of several types of lipid membranes show that DMSO provides the best resolution enhancement over unprotected membranes and also best retards ice formation at low temperature. DMF and PEG-400 show slightly weaker cryoprotection, while glycerol and trehalose neither prevent membrane line broadening nor prevent ice formation under the conditions of our study. Neutral saturated-chain phospholipids are the most amenable to cryoprotection, whereas negatively charged and unsaturated lipids attenuate cryoprotection. 13C-1H dipolar couplings and 31P chemical shift anisotropies indicate that high spectral resolution at low temperature is correlated with stronger immobilization of the lipids at high temperature, indicating that line narrowing results from reduction of the conformational space sampled by the lipid molecules at high temperature. DMSO selectively narrowed the linewidths of the most disordered residues in the influenza M2 transmembrane peptide, while residues that exhibit narrow linewidths in the unprotected membrane are less impacted. A relatively rigid beta-hairpin antimicrobial peptide, PG-1, showed a linewidth increase of ~0.5 ppm over a ~70 K temperature drop both with and without cryoprotection. Finally, a short-chain saturated lipid, DLPE, exhibits excellent linewidths, suggesting that it may be a good medium for membrane protein structure determination. The three best cryoprotectants found in this work-DMSO, PEG, and DMF-should be useful for low-temperature membrane-protein structural studies by SSNMR without compromising spectral resolution.

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