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Site-specific analysis of heteronuclear Overhauser effects in microcrystalline proteins |
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| Authors: | Juan Miguel Lopez del Amo Vipin Agarwal Riddhiman Sarkar Justin Porter Sam Asami Martin Rübbelke Uwe Fink Yi Xue Oliver F. Lange Bernd Reif |
| Affiliation: | 1. Munich Center for Integrated Protein Science (CIPS-M) at Department Chemie, Technische Universit?t München (TUM), Lichtenbergstr. 4, 85747, Garching, Germany 2. Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit und Umwelt, Ingolst?dter Landstr. 1, 85764, Neuherberg, Germany 3. Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-R?ssle-Str. 10, 13125, Berlin, Germany 5. CIC Energigne, Albert Einstein 48, 0150, Mi?ano, Spain 6. ETH Zurich, Wolfgang-Pauli-Str. 10, 8093, Zuerich, Switzerland 4. Department of Chemistry, Purdue University, West Lafayette, IN, 47907-2084, USA 7. Department of Biochemistry, Duke University Medical Center, Durham, NC, 27710, USA |
| Abstract: | Relaxation parameters such as longitudinal relaxation are susceptible to artifacts such as spin diffusion, and can be affected by paramagnetic impurities as e.g. oxygen, which make a quantitative interpretation difficult. We present here the site-specific measurement of [1H]13C and [1H]15N heteronuclear rates in an immobilized protein. For methyls, a strong effect is expected due to the three-fold rotation of the methyl group. Quantification of the [1H]13C heteronuclear NOE in combination with 13C-R 1 can yield a more accurate analysis of side chain motional parameters. The observation of significant [1H]15N heteronuclear NOEs for certain backbone amides, as well as for specific asparagine/glutamine sidechain amides is consistent with MD simulations. The measurement of site-specific heteronuclear NOEs is enabled by the use of highly deuterated microcrystalline protein samples in which spin diffusion is reduced in comparison to protonated samples. |
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