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. 2008 Jun 25;130(25):7818-9.
doi: 10.1021/ja802701w. Epub 2008 May 31.

Simultaneous detection and deconvolution of congested NMR spectra containing three isotopically labeled species

Larry R Masterson  1 , Marco Tonelli, John L Markley, Gianluigi Veglia
Affiliations

Simultaneous detection and deconvolution of congested NMR spectra containing three isotopically labeled species

Larry R Masterson et al. J Am Chem Soc. .

Abstract

We present a procedure for isolating subspectra corresponding to individual protein or peptide components in a ternary mixture or complex. Each of the three-component species is labeled differently: species A uniformly with 15N, species B uniformly with 15N and 13C, and species C uniformly with 15N but selectively with 13C' or 13Calpha. By using the dual carbon label selective HSQC (DCLS-HSQC) pulse sequence and exploiting differences in 1J 15N-13C coupling patterns to filter selected 15N resonances from detection during a constant time period, a subspectrum from each species can be generated from three spectra acquired from a single sample. Many important biological pathways involve dynamic interactions among members of multicomponent protein assemblies, and this approach offers a powerful way to monitor such processes.

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Figures

Figure 1
Figure 1
(A) The DCLS-HSQC pulse sequence. Optimal suppression of both 13Cα and 13C′ attached NH groups use TNCα and TNC′ delays of 24.5 and 16.4 ms, respectively (CT period of 49 ms). Further pulse sequence details in Supporting Information. (B) Representative spectra obtained at 37 °C on a Varian VNMRS 800 MHz spectrometer equipped with a cryogenic probe: i. full ternary mixture consisting of 1 mm [U–2H, U–15N]MBP, 0.8 mM [U–13C, U–15N]-ubiquitin, and 0.8 mM [13C′]-Ala4, [15N]-Ser5 Kemptide; ii. MBP subspectrum; iii. ubiquitin subspectrum; iv. Kemptide subspectrum.
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