BEST-HNN and 2D-(HN)NH experiments for rapid backbone assignment in proteins

Abstract

HNN has proven to be an extremely valuable experiment for rapid and unambiguous backbone (H^N, ¹⁵N) assignment in (¹³C, ¹⁵N) labeled proteins. However, low sensitivity of the experiment is often a limiting factor, especially when the transverse relaxation times (T₂) are short. We show here that BEST modification Schanda et al. (2006) increases the sensitivity per unit time by more than a factor of 2.0 and thus substantially increases the speed of data collection; good 3D data can be collected in 8-10 h. Next, we present a simple method for amino-acid type identification based on simple 2D versions of the HNN experiment, labeled here as 2D-(HN)NH. Each of these experiments which produce anchor points for Gly, Ala, Ser/Thr residues, can be recorded in less than an hour. These enable rapid data acquisition, rapid analysis, and consequently rapid assignment of backbone (H^N, ¹⁵N) resonances. The 2D-(HN)NH experiment does not involve aliphatic/aromatic protons and hence can be applied to deuterated protein samples as well, which is an additional advantage. The experiments have been demonstrated with human ubiquitin (76 aa) and acetic-acid denatured HIV-1 protease (99 aa), as representatives of folded and unfolded protein systems, respectively.