Efficient and precise measurement of H^α–C^α, C^α–C′, C^α–C^β and H^N–N residual dipolar couplings from 2D H^N–N correlation spectra

Abstract

A suite of experiments are presented for the measurement of H^α–C^α, C^α–C′, C^α–C^β and H^N–N couplings from uniformly ¹⁵N, ¹³C labeled proteins. Couplings are obtained from a series of intensity modulated two-dimensional H^N–N spectra equivalent to the common ¹H–¹⁵N–HSQC spectra, alleviating many overlap and assignment issues associated with other techniques. To illustrate the efficiency of this method, H^α–C^α, C^α–C′, and H^N–N isotropic scalar couplings were determined for ubiquitin from data collected in less than 4.5 h, C^α–C^β data collection required 10 h. The resulting couplings were measured with an average error of ±0.06, ±0.05, ±0.04 and ±0.10 Hz, respectively. This study also shows H^α–C^α and C^α–C^β couplings, valuable because they provide orientation of bond vectors outside the peptide plane, can be measured in a uniform and precise way. Superior accuracy and precision to existing 3D measurements for C^α–C′ couplings and increased precision compared to IPAP measurements for H^N–N couplings are demonstrated. Minor modifications allow for acquisition of modulated H^N–C′ 2D spectra, which can yield additional well resolved peaks and significantly increase the number of measured RDCs for proteins with crowded ¹H–¹⁵N resonances.