Julien Roche1, Jinfa Ying1, Yang Shen1, Dennis A Torchia1, Ad Bax1. 1. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract
A new and convenient method, named ARTSY-J, is introduced that permits extraction of the (3)JHNHα couplings in proteins from the relative intensities in a pair of (15)N-(1)H TROSY-HSQC spectra. The pulse scheme includes (3)JHNHα dephasing of the narrower TROSY (1)H(N)-{(15)N} doublet component during a delay, integrated into the regular two-dimensional TROSY-HSQC pulse scheme, and compares the obtained intensity with a reference spectrum where (3)JHNHα dephasing is suppressed. The effect of passive (1)H(α) spin flips downscales the apparent (3)JHNHα coupling by a uniform factor that depends approximately linearly on both the duration of the (3)JHNHα dephasing delay and the (1)H-(1)H cross relaxation rate. Using such a correction factor, which accounts for the effects of both inhomogeneity of the radiofrequency field and (1)H(α) spin flips, agreement between prior and newly measured values for the small model protein GB3 is better than 0.3Hz. Measurement for the HIV-1 protease homodimer (22kDa) yields (3)JHNHα values that agree to better than 0.7Hz with predictions made on the basis of a previously parameterized Karplus equation. Although for Gly residues the two individual (3)JHNHα couplings cannot be extracted from a single set of ARTSY-J spectra, the measurement provides valuable ϕ angle information. Published by Elsevier Inc.
A new and convenient method, named ARTSY-J, is introduced that permits extraction of the (3)JHNHα couplings in proteins from the relative intensities in a pair of (15)N-(1)H TROSY-HSQC spectra. The pulse scheme includes (3)JHNHα dephasing of the narrower TROSY (1)H(N)-{(15)N} doublet component during a delay, integrated into the regular two-dimensional TROSY-HSQC pulse scheme, and compares the obtained intensity with a reference spectrum where (3)JHNHα dephasing is suppressed. The effect of passive (1)H(α) n class="Gene">spin flips downscales the apparent (3)JHNHα coupling by a uniform factor that depends approximately linearly on both the duration of the (3)JHNHα dephasing delay and the (1)H-(1)H cross relaxation rate. Using such a correction factor, which accounts for the effects of both inhomogeneity of the radiofrequency field and (1)H(α) spin flips, agreement between prior and newly measured values for the small model protein GB3 is better than 0.3Hz. Measurement for the HIV-1protease homodimer (22kDa) yields (3)JHNHα values that agree to better than 0.7Hz with predictions made on the basis of a previously parameterized Karplus equation. Although for Gly residues the two individual (3)JHNHα couplings cannot be extracted from a single set of ARTSY-J spectra, the measurement provides valuable ϕ angle information. Published by Elsevier Inc.
Authors: C Ashley Barnes; Yang Shen; Jinfa Ying; Yasuharu Takagi; Dennis A Torchia; James R Sellers; Ad Bax Journal: J Am Chem Soc Date: 2019-05-23 Impact factor: 15.419
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