Measurement of eight scalar and dipolar couplings for methine–methylene pairs in proteins and nucleic acids

Abstract

A new 3D, spin-state-selective coherence transfer NMR experiment is described that yields accurate measurements for eight scalar or dipolar couplings within a spin system composed of a methylene adjacent to a methine group. Implementations of the experiment have been optimized for proteins and for nucleic acids. The experiments are demonstrated for C^β–C^α moieties of the third IgG-binding domain from Streptococcal Protein G (GB3) and for C$^{5^{\prime}}$–C$^{4^{\prime}}$ groups in a 24-nt RNA oligomer. Chemical shifts of C^α, C^β and H^β (respectively C$^{4^{\prime}}$, C$^{5^{\prime}}$ and H$^{5^{\prime}}$) are dispersed in the three orthogonal dimensions, and the absence of heteronuclear decoupling leads to distinct and well-resolved E.COSY multiplet patterns. In an isotropic sample, the E.COSY displacements correspond to ¹J_CαHα, ²J_CαHβ2+²J_CαHβ3, ²J_CβHα, ¹J_CβHβ2+¹J_CβHβ3, ¹J_CβHβ2−²J_Hβ2Hβ3, ¹J_CβHβ3−²J_Hβ2Hβ3, ³J_HαHβ2 and ³J_HαHβ3 for proteins, and ¹J$_{{{\rm C}4^{\prime}}{{\rm H}4^{\prime}}}$, ²J$_{{\rm C}4^{\prime}{{\rm H}5^{\prime}}}+^{2}$J$_{{{\rm C}4^{\prime}}{{\rm H}5^{\prime\prime}}}$, ²J$_{{{\rm C}5^{\prime}}{{\rm H}4^{\prime}}}$, ¹J$_{{{\rm C}5^{\prime}}{{\rm H}5^{\prime}}}$+¹J$_{{\rm C}5^{\prime}{{\rm H}5^{\prime\prime}}}$, ¹J$_{{\rm C}5^{\prime}{\rm H}5^{\prime}}-^{2}$J$_{{\rm H}5^{\prime}{\rm H}5^{\prime\prime}}$, ¹J$_{{\rm C}5^{\prime}{{\rm H}5^{\prime\prime}}}-^{2}$J$_{{\rm H}5^{\prime}{\rm H}5^{\prime\prime}}$, ³J$_{{\rm H}4^{\prime}{\rm H}5^{\prime}}$ and ³J$_{{\rm H}4^{\prime}{{\rm H}5^{\prime\prime}}}$ in nucleic acids. The experiment, based on relaxation-optimized spectroscopy, yields best results when applied to residues where the methine–methylene group corresponds to a reasonably isolated spin system, as applies for C, F, Y, W, D, N and H residues in proteins, or the C$^{5^{\prime}}$–C$^{4^{\prime}}$ groups in nucleic acids. Splittings can be measured under either isotropic or weakly aligned conditions, yielding valuable structural information both through the ³J couplings and the one-, two- and three-bond dipolar interactions. Dipolar couplings for 10 out of 13 sidechains in GB3 are found to be in excellent agreement with its X-ray structure, whereas one residue adopts a different backbone geometry, and two residues are subject to extensive χ₁ rotamer averaging. The abundance of dipolar couplings can also yield stereospecific assignments of the non-equivalent methylene protons. For the RNA oligomer, dipolar data yielded stereospecific assignments for six out of the eight C$^{5^{\prime}}$H₂ groups in the loop region of the oligomer, in all cases confirmed by ¹J$_{{\rm C}5^{\prime}{{\rm H}5^{\prime}}} > ^{1}$J$_{{\rm C}5^{\prime}{\rm H}5^{\prime\prime}}$, and H$^{5^{\prime}}$ resonating downfield of H$^{5^{\prime\prime}}$.

Electronic supplementary material