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Very large residual dipolar couplings from deuterated ubiquitin

Journal of Biomolecular NMR volume 54, pages 53–67 (2012)Cite this article

Abstract

Main-chain 1HN–15N residual dipolar couplings (RDCs) ranging from approximately −200 to 200 Hz have been measured for ubiquitin under strong alignment conditions in Pf1 phage. This represents a ten-fold increase in the degree of alignment over the typical weakly aligned samples. The measurements are made possible by extensive proton-dilution of the sample, achieved by deuteration of the protein with partial back-substitution of labile protons from 25 % H2O / 75 % D2O buffer. The spectral quality is further improved by application of deuterium decoupling. Since standard experiments using fixed-delay INEPT elements cannot accommodate a broad range of couplings, the measurements were conducted using J-resolved and J-modulated versions of the HSQC and TROSY sequences. Due to unusually large variations in dipolar couplings, the trosy (sharp) and anti-trosy (broad) signals are often found to be interchanged in the TROSY spectra. To distinguish between the two, we have relied on their respective 15N linewidths. This strategy ultimately allowed us to determine the signs of RDCs. The fitting of the measured RDC values to the crystallographic coordinates of ubiquitin yields the quality factor Q = 0.16, which confirms the perturbation-free character of the Pf1 alignment. Our results demonstrate that RDC data can be successfully acquired not only in dilute liquid crystals, but also in more concentrated ones. As a general rule, the increase in liquid crystal concentration improves the stability of alignment media and makes them more tolerant to variations in sample conditions. The technical ability to measure RDCs under moderately strong alignment conditions may open the door for development of alternative alignment media, including new types of media that mimic biologically relevant systems.

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Notes

  1. In this case strong alignment is achieved due to low ionic strength of the solvent.

  2. Because of the wide scatter in the values of Σ and the rapid loss of magnetization during the transfer, we cannot separate the transfer stage from the modulation stage, as has been done in other sequences (Tjandra et al. 1996; Kelly et al. 1996). For the same reasons we did not use the correction strategies such as J-mismatch compensation (Schanda et al. 2007; Nielsen et al. 1989).

  3. The difference between 15N linewidths of the HSQC and TROSY signals is modest—for most residues it does not exceed 5 Hz. Because cross-correlated relaxation rates in ubiquitin are relatively small, the experiments that rely on cross-correlated relaxation transfer such as CRINEPT (Riek et al. 1999) do not perform well. This fact—which has been verified experimentally—underscores the difference between the current situation and the spectroscopy of large proteins in solution.

  4. Keep in mind, however, that the difference between HSQC and TROSY linewidths is not limited to CSA-dipolar cross-correlation, but also includes a small term originating from dipolar NH auto-relaxation.

  5. The generalized degree of order is 1.11 × 10−2 (Tolman et al. 2001).

  6. Although certain details may vary, e.g. phage particles may form dimers or higher order multimers in a concentration-dependent manner (Zweckstetter and Bax 2001).

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Acknowledgments

We thank Konstantin Pervushin for bringing to our attention the problem of exchange between solution- and solid-like environments. We would also like to acknowledge Nils-Alexander Lakomek and Korvin Walter who provided to us their spectral assignment data. We are thankful to Lewis Kay and Yi Xue for critical reading of the manuscript. This work was supported by NSF awards MCB 0445643 and 105814.

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  1. Joshua M. Ward

    Present address: Chemistry Department, University of Oulu, POB 3000, 90014, Oulu, Finland

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  1. Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN, 47907-2084, USA

    Joshua M. Ward & Nikolai R. Skrynnikov

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Ward, J.M., Skrynnikov, N.R. Very large residual dipolar couplings from deuterated ubiquitin. J Biomol NMR 54, 53–67 (2012). https://doi.org/10.1007/s10858-012-9651-4

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Keywords

  • Residual dipolar couplings
  • Alignment media
  • Deuteration
  • J-resolved and J-modulated spectroscopy
  • HSQC
  • TROSY
  • Differential line broadening
  • Ubiquitin
  • Pf1 phage