Quantitative evaluation of positive ϕ angle propensity in flexible regions of proteins from three-bond J couplings

Abstract

³ J _HNHα and ³J_C′C′ couplings can be readily measured in isotopically enriched proteins and were shown to contain precise information on the backbone torsion angles, ϕ, sampled in disordered regions of proteins. However, quantitative interpretation of these couplings required the population of conformers with positive ϕ angles to be very small. Here, we demonstrate that this restriction can be removed by measurement of ³J_C′Hα values. Even though the functional forms of the ³J_C′Hα and ³J_HNHα Karplus equations are the same, large differences in their coefficients enable accurate determination of the fraction of time that positive ϕ angles are sampled. A four-dimensional triple resonance HACANH[C′] E.COSY experiment is introduced to simultaneously measure ³J_C′Hα and ³J_HNC′ in the typically very congested spectra of disordered proteins. High resolution in these spectra is obtained by non-uniform sampling (in the 0.1–0.5% range). Application to the intrinsically disordered protein α-synuclein shows that while most residues have close-to-zero positive ϕ angle populations, up to 16% positive ϕ population is observed for Asn residues. Positive ϕ angle populations determined with the new approach agree closely with consensus values from protein coil libraries and prior analysis of a large set of other NMR parameters. The combination of ³J_HNC′ and ³J_C′C′ provides information about the amplitude of ϕ angle dynamics.