Probing structure and functional dynamics of (large) proteins with aromatic rings: L-GFT-TROSY (4,3)D HCCH NMR spectroscopy.
NMR assignment of aromatic rings in proteins is a prerequisite for obtaining high-quality solution structures of proteins and for studying the dynamics and folding of their molecular cores. Here we present sensitive PFG-PEP L-GFT-(TROSY) (4,3)D HCCH NMR for identification of aromatic spin systems based on four-dimensional (4D) spectral information which can be rapidly obtained with high digital resolution. The G-matrix Fourier Transform (GFT) experiment relies on newly introduced longitudinal relaxation (L-)optimization for aromatic protons and is optimally suited for both sensitivity and sampling limited data collection, making it particularly attractive for NMR-based structural genomics. Applications are presented for 21 and 13 kDa proteins HR41 and MaR11, targets of the Northeast Structural Genomics Consortium for which data collection is, respectively, sensitivity and sampling limited. Complete assignment of aromatic rings enabled high-quality NMR structure determination, and nearly complete analysis of aromatic proton line widths allowed one to assess the flipping of most rings in HR41. Specifically, the ring of Tyr90 flips very slowly on the seconds time scale, thereby proving the absence of fast larger-amplitude motional modes which could allow the ring to flip. This indicates remarkable rigidity of the substructure in which the ring is embedded. Tyr90 is conserved among ubiquitin-conjugating enzymes E2, to which HR41 belongs, and is located in spatial proximity to the interface between E2 and ubiquitin protein ligase E3. Hence, the conformational rigidity and/or the slow motional mode probed by the ring might be of functional importance.
Eletsky
Alexander
A
Department of Chemistry, State University of New York at Buffalo, New York 14260, USA.
Atreya
Hanudatta S
HS
Liu
Gaohua
G
Szyperski
Thomas
T
D002247:Carbon Isotopes; D003198:Computer Simulation; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D012680:Sensitivity and Specificity
D002247:Carbon Isotopes; D011506:Proteins; D011522:Protons