Atomic resolution protein structure determination by three-dimensional transferred echo double resonance solid-state nuclear magnetic resonance spectroscopy

Abstract

We show that quantitative internuclear (15)N-(13)C distances can be obtained in sufficient quantity to determine a complete, high-resolution structure of a moderately sized protein by magic-angle spinning solid-state NMR spectroscopy. The three-dimensional ZF-TEDOR pulse sequence is employed in combination with sparse labeling of (13)C sites in the beta1 domain of the immunoglobulin binding protein G (GB1), as obtained by bacterial expression with 1,3-(13)C or 2-(13)C-glycerol as the (13)C source. Quantitative dipolar trajectories are extracted from two-dimensional (15)N-(13)C planes, in which approximately 750 cross peaks are resolved. The experimental data are fit to exact theoretical trajectories for spin clusters (consisting of one (13)C and several (15)N each), yielding quantitative precision as good as 0.1 A for approximately 350 sites, better than 0.3 A for another 150, and approximately 1.0 A for 150 distances in the range of 5-8 A. Along with isotropic chemical shift-based (TALOS) dihedral angle restraints, the distance restraints are incorporated into simulated annealing calculations to yield a highly precise structure (backbone RMSD of 0.25+/-0.09 A), which also demonstrates excellent agreement with the most closely related crystal structure of GB1 (2QMT, bbRMSD 0.79+/-0.03 A). Moreover, side chain heavy atoms are well restrained (0.76+/-0.06 A total heavy atom RMSD). These results demonstrate for the first time that quantitative internuclear distances can be measured throughout an entire solid protein to yield an atomic-resolution structure.

Heteronuclear proton assisted recoupling

“…Contrary to the 15 N-13 C CP case in (c) where the long distance transfer is essentially quenched in presence of the directly bonded carbon, the heteronuclear TSAR mechanism suffers only a small reduction of the long distance polarization transfer in the same situation. For δp 0 PAIN-CP (a) and σ p 3 PAIN-CP (b), we can still transfer about 10% of the initial 15 N magnetization in 15 ms of irradiation. We remark that 15 N-13 C CP experiment (performed with very high power proton decoupling) remains the best option for one bond NC transfer with more than 50% efficient transfer with ∼2 ms of irradiation.…”

“…7, which shows experimental results obtained on the tripeptide [U-13 C, 15 N]-N-f-MLF-OH at ω 0H /2π = 750 MHz with various rf power levels and offsets. These data illustrate the great potential of the PAIN-CP experiment to perform protein resonance assignments using sequential contacts as well as protein structure determination using long distance contacts.…”

“…This is the basic approach employed in the development of the majority of heteronuclear recoupling sequences including rotational echo double resonance (REDOR), 7 transferred echo double resonance (TEDOR), 8 frequency selective (FS)-REDOR, 9 z-filtered (ZF-) and band-selective (BASE-)TEDOR, 9 and frequency selective (FS-)TEDOR 16 which enable accurate N-C distance measurements, and have been especially important in the determination of high resolution 3D structures. 15,17 Nevertheless, efficient heteronuclear recoupling in larger biomolecular systems remains challenging.…”

“…This protocol has yielded several MAS NMR structures with a precision ≤1 Å. 3,[12][13][14][15] Most heteronuclear experiments require 15 N-13 C dipolar couplings to be reintroduced with a train of π pulses at the 13 C and/or 15 N frequencies while simultaneously decoupling the 1 H spins from the 15 N-13 C spin dynamics. This is the basic approach employed in the development of the majority of heteronuclear recoupling sequences including rotational echo double resonance (REDOR), 7 transferred echo double resonance (TEDOR), 8 frequency selective (FS)-REDOR, 9 z-filtered (ZF-) and band-selective (BASE-)TEDOR, 9 and frequency selective (FS-)TEDOR 16 which enable accurate N-C distance measurements, and have been especially important in the determination of high resolution 3D structures.…”

Zero-quantum stochastic dipolar recoupling in solid state nuclear magnetic resonance

“…6 shows examples of 2D 13 C-13 C NMR spectra of protein GB1, a 56-residue model protein 37 that has been the subject of many previous solid state NMR studies, [42][43][44][45][46][47] obtained with the pulse sequence in Fig. 2(c).…”