Incorporating 1H-chemical shift Determination into 13C-direct detected spectroscopy of intrinsically disordered proteins in solution.
Intrinsically disordered proteins have emerged as critical components of cellular systems, contributing to cell signalling and disease. We seek to implement new NMR-based methodologies for quantifying the structure and dynamics of IDPs with the same level of rigor currently attainable for co-operatively folded proteins. One general factor limiting the quality of IDP spectra is a lack of amide proton chemical shift dispersion, which results in crowded 1H,15N-HSQC spectra. With proper equipment, such as the three TCI cryoprobes we have access to in our NMR facility, this can be relieved by measuring 13C-direct detected spectra. Compare the 13C,15N-CON spectrum of FCP1 below (left) to the traditional amide detected 1H,15N-HSQC (right). The carbonyl carbon retains relatively high chemical shift dispersion in the disordered state.
Recently, we have published a pair of carbon detected NMR experiments that facilitate complete aliphatic 1H resonance assignment in IDPs. The first, which we have named the H(CC)CO-IPAP (panel A in the figure below) correlates the aliphatic hydrogens of a residue with the alpha carbon and the carbonyl carbon of the same residue. The second, named the H(CC)CON-IPAP (panel B in the figure below) correlates the aliphatic hydrogens of a residue with the amide nitrogen of the next residue in the primary sequence and the carbonyl carbon of the same residue as the hydrogens.
The completeness of aliphatic resonance assignment in IDPs obtained by using these experiments is comparable to the level of completeness obtained for folded proteins using traditional 1H-detected spectra such as the HCCCONH and 15N-TOCSY-HSQC. This can be seen in panel A below for Leu 953 of FCP1, which is well enough resolved even in the 1H-detected spectra to provide direct comparison.
In addition, these spectra suffer no loss of completeness near Pro residues because they do not rely on the presence of a backbone 1H-amide in a residue in order for it to be detected. (This also provides an advantage in sites undergoing rapid proton exchange with solvent.) The ease of assigning through a Pro residue using our experiments is illustrated for the neighborhood of FCP1 residue Pro 902 in panel B below.
For experimental details and full resolution images, please see:
O'Hare, B., Benesi, A.J., & Showalter, S.A. (2009) "Incorporating 1H chemical shift determination into 13C-detected spectroscopy of intrinsically disordered proteins in solution." J. Mag. Reson., 200, 354-358.
These experiments have been used along with previously published "protonless" NMR experiments to fully assign the resonances of the intrinsically disordered C-terminus of human FCP1 in the unbound state:
Showalter, S.A. (2009) “NMR Assignment of the Intrinsically Disordered C-terminal Region of Homo sapiens FCP1 in the Unbound State.” Biomol. NMR Assign., 3, 179-181.