Publication year: 2012 Source:Journal of Magnetic Resonance
Andrin Doll, Enrica Bordignon, Benesh Joseph, René Tschaggelar, Gunnar Jeschke
We demonstrate the application of continuous wave dynamic nuclear polarization (DNP) at 0.35 Tesla for site-specific water accessibility studies on spin-labeled membrane proteins at concentrations in the 10-100 micromolar range. The DNP effects at such low concentrations are weak and the experimentally achievable dynamic nuclear polarizations can be below the equilibrium polarization. This sensitivity problem is solved with an optimized home-built DNP probe head consisting of a dielectric microwave resonator and a saddle coil as close as possible to the sample. The performance of the probe head is demonstrated with both a modified pulsed EPR spectrometer and a dedicated CW EPR spectrometer equipped with a commercial NMR console. In comparison to a commercial pulsed ENDOR resonator, the home-built resonator has an FID detection sensitivity improvement of 2.15 and an electron spin excitation field improvement of 1.2. The reproducibility of the DNP results is tested on the water soluble maltose binding protein MalE of the ABC maltose importer, where we determine a net standard deviation of 9% in the primary DNP data in the concentration range between 10 and 100 micromolar. DNP parameters are measured in a spin-labeled membrane protein, namely the vitamin B 12 importer BtuCD in both detergent-solubilized and reconstituted states. The data obtained in different nucleotide states in the presence and absence of binding protein BtuF reveal the applicability of this technique to qualitatively extract water accessibility changes between different conformations by the ratio of primary DNP parameters ?. The ?-ratio unveils the physiologically relevant transmembrane communication in the transporter in terms of changes in water accessibility at the cytoplasmic gate of the protein induced by both BtuF binding at the periplasmic region of the transporter and ATP binding at the cytoplasmic nucleotide binding domains. Graphical abstract
Graphical abstract Highlights
? home-built X-Band DNP resonator for measurements down to 10 micromolar concentration. ? DNP measurements on spin-labeled membrane proteins at physiological temperatures. ? water accessibility changes in the ABC importer BtuCD-F characterized by DNP. ? interaction of BtuCD with BtuF or ATP changes spin label water accessibility.
Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers
Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers
Abstract Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring 1H-15N dipolar couplings (DC) and 15N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal...
nmrlearner
Journal club
0
10-10-2011 06:27 AM
Water proton spin saturation affects measured protein backboneN spin relaxation rates
Water proton spin saturation affects measured protein backboneN spin relaxation rates
Publication year: 2011
Source: Journal of Magnetic Resonance, Available online 1 October 2011</br>
Kang*Chen, Nico*Tjandra</br>
Protein backboneN NMR spin relaxation rates are useful in characterizing the protein dynamics and structures. To observe the protein nuclear-spin resonances a pulse sequence has to include a water suppression scheme. There are two commonly employed methods, saturating or dephasing the water spins with pulse field gradients and keeping them unperturbed with flip-back pulses....
nmrlearner
Journal club
0
10-02-2011 08:25 AM
[NMR paper] How to prepare membrane proteins for solid-state NMR: A case study on the alpha-helical integral membrane protein diacylglycerol kinase from E. coli.
How to prepare membrane proteins for solid-state NMR: A case study on the alpha-helical integral membrane protein diacylglycerol kinase from E. coli.
Related Articles How to prepare membrane proteins for solid-state NMR: A case study on the alpha-helical integral membrane protein diacylglycerol kinase from E. coli.
Chembiochem. 2005 Sep;6(9):1693-700
Authors: Lorch M, Faham S, Kaiser C, Weber I, Mason AJ, Bowie JU, Glaubitz C
Several studies have demonstrated that it is viable to use microcrystalline preparations of water-soluble proteins as...
nmrlearner
Journal club
0
12-01-2010 06:56 PM
Probing water-accessibility in HET-s(218-289) amyloid fibrils by solid-state NMR.
Probing water-accessibility in HET-s(218-289) amyloid fibrils by solid-state NMR.
Probing water-accessibility in HET-s(218-289) amyloid fibrils by solid-state NMR.
J Mol Biol. 2010 Nov 18;
Authors: Van Melckebeke H, Schanda P, Gath J, Wasmer C, Verel R, Lange A, Meier BH, Böckmann A
Despite its importance in the context of conformational diseases, structural information is still sparse for protein fibrils. Hydrogen/deuterium exchange measurements of backbone amides allow to identify hydrogen-bonding patterns and reveal pertinent information about...
nmrlearner
Journal club
0
11-26-2010 05:32 PM
[NMR paper] Membrane protein topology probed by (1)H spin diffusion from lipids using solid-state
Membrane protein topology probed by (1)H spin diffusion from lipids using solid-state NMR spectroscopy.
Related Articles Membrane protein topology probed by (1)H spin diffusion from lipids using solid-state NMR spectroscopy.
J Am Chem Soc. 2002 Feb 6;124(5):874-83
Authors: Huster D, Yao X, Hong M
We describe a two-dimensional solid-state NMR technique to investigate membrane protein topology under magic-angle spinning conditions. The experiment detects the rate of (1)H spin diffusion from the mobile lipids to the rigid protein. While spin...
nmrlearner
Journal club
0
11-24-2010 08:49 PM
[NMR paper] Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: d
Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: differences in solution and crystal forms of maltodextrin binding protein loaded with beta-cyclodextrin.
Related Articles Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: differences in solution and crystal forms of maltodextrin binding protein loaded with beta-cyclodextrin.
J Mol Biol. 2000 Feb 4;295(5):1265-73
Authors: Skrynnikov NR, Goto NK, Yang D, Choy WY, Tolman JR, Mueller GA, Kay LE
Protein function is often regulated...
nmrlearner
Journal club
0
11-18-2010 09:15 PM
[NMR paper] 31P solid-state NMR measurements used to detect interactions between NADPH and water
31P solid-state NMR measurements used to detect interactions between NADPH and water and to determine the ionisation state of NADPH in a protein-ligand complex subjected to low-level hydration.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www3.interscience.wiley.com-aboutus-images-wiley_interscience_pubmed_logo_FREE_120x27.gif Related Articles 31P solid-state NMR measurements used to detect interactions between NADPH and water and to determine the ionisation state of NADPH in a protein-ligand complex subjected to low-level hydration.
Eur J Biochem. 1996 Jan...
nmrlearner
Journal club
0
08-22-2010 02:27 PM
Solid State NMR of membrane peptides and proteins
Solid State NMR of membrane peptides and proteins
Lecture notes on "Solid State NMR of membrane peptides and proteins" by Dr. SK Straus from Univ. of British Columbia
More...
Liquid State DNP for Water Accessibility Measurements on Spin-labeled Membrane Proteins at Physiological Temperatures
Graphical abstract Highlights 
Linear Mode
