Site-specific analysis of heteronuclear Overhauser effects in microcrystalline proteins
 

Site-specific analysis of heteronuclear Overhauser effects in microcrystalline proteins

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.



Source: Journal of Biomolecular NMR