Proton detection for signal enhancement in solid-state NMR experiments on mobile species in membrane proteins.
Direct proton detection is becoming an increasingly popular method for enhancing sensitivity in solid-state nuclear magnetic resonance spectroscopy. Generally, these experiments require extensive deuteration of the protein, fast magic angle spinning (MAS), or a combination of both. Here, we implement direct proton detection to selectively observe the mobile entities in fully-protonated membrane proteins at moderate MAS frequencies. We demonstrate this method on two proteins that exhibit different motional regimes. Myelin basic protein is an intrinsically-disordered, peripherally membrane-associated protein that is highly flexible, whereas Anabaena sensory rhodopsin is composed of seven rigid transmembrane α-helices connected by mobile loop regions. In both cases, we observe narrow proton linewidths and, on average, a 10× increase in sensitivity in 2D insensitive nuclear enhancement of polarization transfer-based HSQC experiments when proton detection is compared to carbon detection. We further show that our proton-detected experiments can be easily extended to three dimensions and used to build complete amino acid systems, including sidechain protons, and obtain inter-residue correlations. Additionally, we detect signals which do not correspond to amino acids, but rather to lipids and/or carbohydrates which interact strongly with membrane proteins.
Department of Physics, University of Guelph, Guelph, ON, Canada.|Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada.
Meaghan E
ME
Ward
Department of Physics, University of Guelph, Guelph, ON, Canada.|Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada.
Emily
E
Ritz
Department of Physics, University of Guelph, Guelph, ON, Canada.|Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada.|Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.|The Department of Physics, Faculty of Science, Suez University, Suez, 43533, Egypt.
Mumdooh A M
MAM
Ahmed
Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada.|Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
Vladimir V
VV
Bamm
Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada.|Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
George
G
Harauz
Department of Physics, University of Guelph, Guelph, ON, Canada.|Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada.
Leonid S
LS
Brown
Department of Physics, University of Guelph, Guelph, ON, Canada. vladizha@uoguelph.ca.|Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada. vladizha@uoguelph.ca.
Vladimir
V
Ladizhansky
D017033:Anabaena; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D004676:Myelin Basic Protein / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D011522:Protons; D012243:Rhodopsin / Q000737:chemistry*; D059629:Signal-To-Noise Ratio
D001426:Bacterial Proteins; D004676:Myelin Basic Protein; D011522:Protons; D012243:Rhodopsin
INEPT; J-couplings; Magic angle spinning (MAS); Membrane proteins; Proton detection; Solid-state nuclear magnetic resonance (SSNMR)