Multiple acquisition/multiple observation separated local field/chemical shift correlation solid-state magic angle spinning NMR spectroscopy

Abstract

Multiple acquisition spectroscopy (MACSY) experiments that enable multiple free induction decays to be recorded during individual experiments are demonstrated. In particular, the experiments incorporate separated local field spectroscopy into homonuclear and heteronuclear correlation spectroscopy. The measured heteronuclear dipolar couplings are valuable in structure determination as well as in enhancing resolution by providing an additional frequency axis. In one example four different three-dimensional spectra are obtained in a single experiment, demonstrating that substantial potential saving in experimental time is available when multiple multi-dimensional spectra are required as part of solid-state NMR studies.

Keywords: CXCR1; Dipolar couplings; Dual acquisition; Dual observation; MACSY; PELF; Protein NMR; R-INEPT.

Figure 1

Diagrams of MACSY pulse sequences that integrate separated local field spectroscopy and chemical shift correlation. A. Single acquisition, dual observation (SADO). B. Dual acquisition, dual observation (DADO). C. Dual acquisition, multiple observation (DAMO). D. Triple acquisition, multiple observation (TAMO). Thin and thick vertical lines represent 90° and 180° pulses, respectively. The thinner lines in Panel B represents small flip angle (35°) pulses. The phase cycles are as follows; ϕ₁= 1, ϕ₂= 1133, ϕ₃= 11113333, ϕ₄= 0022, ϕ₅=3311, ϕ₆= 1133. ϕ_(N)(DCP)= 0022, ϕ_(C)(DCP)= 00002222, ϕ_(rec)= 02202002

Figure 2

Four different three-dimensional spectra obtained from a single DAMO experiment at 700 MHz. The three-dimensional spectra are from a uniformly ¹³C, ¹⁵N labeled sample of polycrystalline methionine-leucine-phenylalanine (MLF) obtained using the pulse sequence diagrammed in Figure 1C. A. ¹H-¹⁵N/N(CA)CX spectrum with ¹³C chemical shift, ¹⁵N chemical shift, and ¹H-¹⁵N heteronuclear dipolar coupling frequency dimensions. B.¹H-¹³C/CXCY spectrum with ¹³C chemical shift, ¹³C chemical shift, and ¹H-¹³C dipolar coupling frequency axes. C. ¹H-¹⁵N/NCO spectrum with ¹³C chemical shift, ¹⁵N chemical shift, and ¹H-¹⁵N dipolar coupling axes. D. ¹HA-¹³CA/CA(N)CO spectrum with ¹³C chemical shift, ¹³C chemical shift, and ¹H-¹³C dipolar coupling frequency axes.

Figure 3

Two-dimensional SLF and HETCOR planes from three-dimensional spectra of a uniformly ¹³C, ¹⁵N labeled sample of polycrystalline methionine-leucine-phenylalanine obtained using the pulse sequence diagrammed in Figure 1A (SADO) at 700 MHz. A. ¹⁵N/¹³C two-dimensional heteronuclear correlation spectrum with ¹³C chemical shift and ¹⁵N chemical shift frequency axes. B. ¹³C/¹³C two-dimensional homonuclear correlation spectrum with ¹³C chemical shift frequency axes. C. ¹H-¹⁵N/¹³C two-dimensional separated local field spectrum with ¹³C chemical shift and ¹H-¹⁵N heteronuclear dipolar coupling frequency axes. (It corresponds to the ¹⁵N chemical shift frequency at 128 ppm marked with an arrow in Panel A.) D. ¹H-¹³C/¹³C two-dimensional separated local field spectrum with ¹³C chemical shift and ¹H-¹³C heteronuclear dipolar coupling frequency axes. (It corresponds to the methionine ¹³C chemical shift frequency at 52 ppm marked with an arrow in Panel B.)

Figure 4

Two-dimensional SLF and HETCOR planes from three-dimensional spectra of a uniformly ¹³C, ¹⁵N labeled sample of polycrystalline N-acetyl leucine obtained using the pulse sequence diagrammed in Figure 1B (DADO) at 700 MHz. A. and B. ¹H/¹³C two-dimensional heteronuclear correlation spectrum with ¹³C chemical shift and ¹H chemical shift frequency axes. C. ¹H-¹⁵N/¹³C two-dimensional separated local field spectrum with ¹³C chemical shift and ¹H-¹⁵N heteronuclear dipolar coupling frequency axes. (It corresponds to the signal marked with an arrow in Panel A.) D. ¹H-¹³C/¹³C two-dimensional separated local field spectrum with ¹³C chemical shift and ¹H-¹³C heteronuclear dipolar coupling frequency axes. (It corresponds to the signal marked with an arrow in Panel B.)

Figure 5

Two-dimensional correlation spectra obtained from a uniformly ¹³C,¹⁵N labeled sample of the 350-residue membrane protein CXCR1 in phospholipid bilayers at 750 MHz. It was obtained with 50% data point coverage NUS using the pulse sequence diagramed in Figure 1D without the dipolar frequency evolution in the third dimension. A. and B. Sections of the same ¹³C/¹³C homonuclear correlation spectrum with ¹³C chemical shift frequency axes. C. and D. Section of ¹⁵N/¹³C heteronuclear correlation spectra with ¹³C chemical shift and ¹⁵N chemical shift axes.