Selective excitation for spectral editing and assignment in separated local field experiments of oriented membrane proteins.
Spectroscopic assignment of NMR spectra for oriented uniformly labeled membrane proteins embedded in their native-like bilayer environment is essential for their structure determination. However, sequence-specific assignment in oriented-sample (OS) NMR is often complicated by insufficient resolution and spectral crowding. Therefore, the assignment process is usually done by a laborious and expensive "shotgun" method involving multiple selective labeling of amino acid residues. Presented here is a strategy to overcome poor spectral resolution in crowded regions of 2D spectra by selecting resolved "seed" residues via soft Gaussian pulses inserted into spin-exchange separated local-field experiments. The Gaussian pulse places the selected polarization along the z-axis while dephasing the other signals before the evolution of the1H-15N dipolar couplings. The transfer of magnetization is accomplished via mismatched Hartmann-Hahn conditions to the nearest-neighbor peaks via the proton bath. By optimizing the length and amplitude of the Gaussian pulse, one can also achieve a phase inversion of the closest peaks, thus providing an additional phase contrast. From the superposition of the selective spin-exchanged SAMPI4 onto the fully excited SAMPI4 spectrum, the15N sites that are directly adjacent to the selectively excited residues can be easily identified, thereby providing a straightforward method for initiating the assignment process in oriented membrane proteins.
Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC 27695-8204, USA.
Sophie N
SN
Koroloff
Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC 27695-8204, USA. Electronic address: alex_nevzorov@ncsu.edu.
Alexander A
AA
Nevzorov
D000465:Algorithms; D000596:Amino Acids / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D007930:Leucine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D008279:Magnetic Resonance Imaging; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D009587:Nitrogen Isotopes; D016011:Normal Distribution; D019906:Nuclear Magnetic Resonance, Biomolecular; D013113:Spin Labels
D000596:Amino Acids; D008565:Membrane Proteins; D008823:Micelles; D009587:Nitrogen Isotopes; D013113:Spin Labels; D007930:Leucine; C088117:acetylleucine
Membrane proteins; Oriented samples; SAMPI4; Selective excitation; Separated local field experiments; Soft Gaussian pulses; Solid-state NMR; Spectral editing