Very large residual dipolar couplings from deuterated ubiquitin
Abstract Main-chain 1HNâ??15N residual dipolar couplings (RDCs) ranging from approximately â??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.
- Content Type Journal Article
- Category Article
- Pages 1-15
- DOI 10.1007/s10858-012-9651-4
- Authors
- Joshua M. Ward, Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907-2084, USA
- Nikolai R. Skrynnikov, Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907-2084, USA
Source: Journal of Biomolecular NMR