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Review
. 2012:992:35-62.
doi: 10.1007/978-94-007-4954-2_3.

Isotope labeling for solution and solid-state NMR spectroscopy of membrane proteins

Raffaello Verardi  1 , Nathaniel J Traaseth, Larry R Masterson, Vitaly V Vostrikov, Gianluigi Veglia
Affiliations
Review

Isotope labeling for solution and solid-state NMR spectroscopy of membrane proteins

Raffaello Verardi et al. Adv Exp Med Biol. 2012.

Abstract

In this chapter, we summarize the isotopic labeling strategies used to obtain high-quality solution and solid-state NMR spectra of biological samples, with emphasis on integral membrane proteins (IMPs). While solution NMR is used to study IMPs under fast tumbling conditions, such as in the presence of detergent micelles or isotropic bicelles, solid-state NMR is used to study the structure and orientation of IMPs in lipid vesicles and bilayers. In spite of the tremendous progress in biomolecular NMR spectroscopy, the homogeneity and overall quality of the sample is still a substantial obstacle to overcome. Isotopic labeling is a major avenue to simplify overlapped spectra by either diluting the NMR active nuclei or allowing the resonances to be separated in multiple dimensions. In the following we will discuss isotopic labeling approaches that have been successfully used in the study of IMPs by solution and solid-state NMR spectroscopy.

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Figures

Figure 1
Figure 1
Production of isotopic labeled membrane proteins for NMR spectroscopy
Figure 2
Figure 2
Examples of 15N uniform and selective labeling of the membrane protein PLN. A) 15N-1H HSQC of [U-15N] recombinant PLN in 300 mM DPC. B–C) Selective 15N-Ile and 15N Met labeled recombinant PLN. Notice the absence of isotopic scrambling. D) An attempt to label PLN at Gln and Glu residues using 15N-Gln and 15N-Glu labeled amino acids in the growth medium resulted in significant isotopic scrambling. E) Labeling of Glu and Gln in PLN using the reverse labeling approach. No isotopic scrambling is present. F) PLN selective labeled at Q22–Q23 produced by peptide synthesis.
Figure 3
Figure 3
Selective 13C enrichment of methyl containing amino acids using different precursors in the presence of glucose. Carbons derived from the precursors are indicated in red. Note that these precursors lead to very high 13C incorporation for all sites (>90%). We did not include other carbon sources (such as 13C-pyruvate) that lead to lower enrichment levels at the methyl
Figure 4
Figure 4
MAS N-CA 2D correlation spectra of PLN in lipid vesicles. A) uniformly labeled, [U-13C,15N] PLN. B) Selective Leu and Val labeled PLN obtained by addition of [Ile-13C,15N] to the growth medium. Notice the severe overlap in both dimensions. C) PLN labeled with 13C,15N at residues Asn30-Leu31-Phe32-Ile33 produced by peptide synthesis.
Figure 5
Figure 5
Expected 13C distribution using 2-13C-glycerol or 1,3-13C-glycerol as the sole carbon source and E.coli BL21(DE3) strain. 13C labeled carbons are indicated in red. Two studies [193, 194] reported different results using 2-13C-glycerol therefore both are indicated in the labeling pattern for each amino acid.
Figure 6
Figure 6
A–B) CCLS-HSQC. A) Schematic of the CCLS-HSQC pulse sequence. The reference spectrum is obtained by executing the pulse sequence with the 180° 13C′ pulse (open rectangle) at position a; the 13C′ suppressed spectrum is obtained with this pulse at position b. B) Examples of…. C–D) Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR. C) Pulse sequence used to obtain 2D FDR-15N–13Cα. D) FDR-15N–13Cα spectra for N-acetyl-valyl-leucine. Spectra were acquired with (FDR – red spectrum) and without 13C 90° pulses (reference – black spectrum) (Reproduced with permission from Traaseth and Veglia [195].
Figure 7
Figure 7
Asymmetric labeling scheme for the detection of inter-protomer contacts in homo-oligomeric membrane proteins using solution and solid-state NMR. A) 2D planes from 3D [1H, 1H, 15N]-NOESY-HSQC (400 ms mixing time) on a mixed PLN sample with 1:1 ratio of [2H-15N] and [2H-14N-13CH3-Ileδ1] PLN. B) 2D planes from 3D [1H, 13C, 13C]-HSQC-NOESY-HSQC experiment performed on a sample containing 1:1 ratio [2H-14N-13CH3-Ileδ1] and [2H-14N-13CH3-Leuδ1/Valγ1] PLN. C) 2D-DARR experiments (200 ms mixing time) on a 50% [U13C]-Leu/ 50% [U13C]-Ile PLN sample. Intra-residue and interprotomer cross-peaks are labeled in black and blue, respectively (Reproduced with permission from Verardi et al. [90].
Figure 8
Figure 8
Cysteine methylation of SERCA1a by methyl methanethiosulfonate (MMTS) reaction. A) 1H–13C HSQC spectrum of 13C methylthiocysteine in 100 mM 2H dodecylphosphocholine acquired at 14.1 T field strength. B) MAS one-dimensional cross-polarization of 13C methylthiocysteine labeled SERCA1a in 2H DMPC lipid vesicles run at −20°C and spinning rate of 8000Hz acquired at 14.1 T field strength. Dashed lines indicate the peak corresponding to the labeled cysteines.
Figure 9
Figure 9
Expected 13C distribution using pyruvate and sodium bicarbonate as the sole carbon sources in E.coli BL21(DE3). A) 1,2-13C-pyruvate and NaH13CO3 and B) 1-13C-pyruvate and NaH13CO3
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