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. 2009 Oct 14;131(40):14138-9.
doi: 10.1021/ja904766g.

One-sample approach to determine the relative orientations of proteins in ternary and binary complexes from residual dipolar coupling measurements

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

One-sample approach to determine the relative orientations of proteins in ternary and binary complexes from residual dipolar coupling measurements

Marco Tonelli et al. J Am Chem Soc. .

Abstract

We present a procedure that supports the acquisition of (1)H-(15)N residual dipolar coupling (RDC) values for individual subunits in binary or ternary protein assemblies from a single experimental sample. Our method relies on asymmetric labeling of each subunit with the following scheme: species A uniformly with (15)N, species B uniformly with (15)N and (13)C, and species C uniformly with (15)N but selectively with (13)C' or (13)C(alpha). Because only a single sample is required, the approach obviates the need for preparing multiple samples and eliminates potential errors introduced from differences in sample conditions. Because numerous biological processes rely on protein assemblies or transient interactions, this method should be well suited for a wide range of future applications.

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Figures

Figure 1
Figure 1
Schematic for the gradient-selected TROSY-based pulse sequences for binary (A) or ternary (B) protein mixtures. A reference spectrum is obtained by applying the 180° 13C pulses (open pulses) at position a; while 13C′ or 13Cα suppression is obtained with pulses position b or c, respectively. Delay durations: Δ = 2.4 ms; δ = 0.11 ms; TNC′ = 16.5 ms, TNCα = 23.5 ms. Further details, including spectral editing to obtain each subunit, are provided in the Supporting Information.
Figure 2
Figure 2
Spectral analysis of the convoluted spectrum containing [U-2H, U-15N]-MBP, [U-15N,13C]-ubiquitin, and [15N-Ser, 13C′-Ala4]- Kemptide. TROSY-detection was used to obtain all 15N resonances in the sample (A). Isotropic J-coupling values were obtained from spectral editing of TROSY and anti-TROSY datasets as shown for MBP (B), ubiquitin (C), and Kemptide (D).
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