Expanding the horizons for structural analysis of fully protonated protein assemblies by NMR spectroscopy at MAS frequencies above 100 kHz

Abstract

The recent breakthroughs in NMR probe technologies resulted in the development of MAS NMR probes with rotation frequencies exceeding 100 kHz. Herein, we explore dramatic increases in sensitivity and resolution observed at MAS frequencies of 110–111 kHz in a novel 0.7 mm HCND probe that enable structural analysis of fully protonated biological systems. Proton-detected 2D and 3D correlation spectroscopy under such conditions requires only 0.1 – 0.5 mg of sample and a fraction of time compared to conventional 13C-detected experiments. We discuss the performance of several proton- and heteronuclear- (13C-,15N-) based correlation experiments in terms of sensitivity and resolution, using a model microcrystalline fMLF tripeptide. We demonstrate the applications of ultrafast MAS to a large, fully protonated protein assembly of the 231-residue HIV-1 CA capsid protein. Resonance assignments of protons and heteronuclei, as well as 1H-15N dipolar and 1HN CSA tensors are readily obtained from the high sensitivity and resolution proton-detected 3D experiments. The approach demonstrated here is expected to enable the determination of atomic-resolution structures of large protein assemblies, inaccessible by current methodologies.

Nucleotide Binding Modes in a Motor Protein Revealed by 31P‐ and 1H‐Detected MAS Solid‐State NMR Spectroscopy

“…These interactions have been typicallys tudied in the past by high-resolution X-ray crystallography. [14][15][16][17][18][19][20][21][22][23] Proton chemical-shift values are highly sensitivet oh ydrogen bonding [24][25][26][27] as shown in theoretical, [26,27] buta lso in experimental studies. [8] And indeed, solid-state NMR spectroscopy has been used to identify residues at protein-RNA interfaces in smaller proteins.…”

“…[9] Intermolecular information can also be obtained from 31 P-detected, heteronuclear correlation experimentsp robingt he spatial proximity of nucleotide 31 Pa nd protein 15 No r 13 Cn uclei. [14][15][16][17][18][19][20][21][22][23] Proton chemical-shift values are highly sensitivet oh ydrogen bonding [24][25][26][27] as shown in theoretical, [26,27] buta lso in experimental studies. [14][15][16][17][18][19][20][21][22][23] Proton chemical-shift values are highly sensitivet oh ydrogen bonding [24][25][26][27] as shown in theoretical, [26,27] buta lso in experimental studies.…”

Picturing the Membrane‐assisted Choreography of Cytochrome P450 with Lipid Nanodiscs

“…[6] Some of the important discoveries of the last decade in P450 related research have foundations on those pioneering observations. [108][109][110][111][112][113][114][115][116][117][118][119][120][121] We also expect the macro-nanodiscs to be highly valuable for studies using cryoEM and diffraction experiments. [107] Forty-years later, those cautious speculations were ultimately and quantitatively demonstrated by our group using sophisticated tools.…”

High Accuracy Protein Structures from Minimal Sparse Paramagnetic Solid‐State NMR Restraints

“…[1][2][3][4][5] In spite of the latest advances, [6,7] high-throughput protein structure elucidation by conventional ssNMR methods remains challenging due to difficulties in obtaining critical non-local contacts.D uring the past decade,s sNMR measurements of nuclear paramagnetic relaxation enhancements (PREs) have been explored for structural studies of native metalloproteins [8,9] and proteins modified with covalent paramagnetic tags,including nitroxide spin labels [10] and metal chelates. [1][2][3][4][5] In spite of the latest advances, [6,7] high-throughput protein structure elucidation by conventional ssNMR methods remains challenging due to difficulties in obtaining critical non-local contacts.D uring the past decade,s sNMR measurements of nuclear paramagnetic relaxation enhancements (PREs) have been explored for structural studies of native metalloproteins [8,9] and proteins modified with covalent paramagnetic tags,including nitroxide spin labels [10] and metal chelates.…”