Carbon-detected Experiments for Assignment of Proline Rich Proteins
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| Year of publication | 2010 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | The determination of 3D structures of proteins by NMR requires a complete assignment of resonances. For that purpose, suites of experiments allowing both sequential and side-chain assignments have been developed. Standard heteronuclear NMR techniques for the backbone assignment such as HNCA, CBCA(CO)NH, and CBCANH experiments rely on correlating the HN chemical shift of each residue with the C(alpha) and C(beta) chemical shifts of the same and preceding residues. Because proline lacks the HN, these experiments cannot be used to sequentially connect prolines to the preceding residues or to assign the stretches of prolines. Therefore, proline assignment has to be based either on the observation of NOEs to neighboring residues or on the detection of H(alpha) resonances. As the H(alpha) proton resonances often fall close to the resonance of water, their detection often fails. Both the problem of the missing amide proton and the interference of the water signal can be solved using carbon-detection. An extensive set of "protonless" experiments have been proposed for the assignment of fully deuterated proteins and protein complexes with paramagnetic ions. We have applied (H)CbCaCON, (H)CbCaNCO and CON experiments to assign the backbone of proline stretches in the 16 kDa PsbQ protein, which is a part of Photosystem II. Though all detected nuclei are 13C and 15N, the (H)CbCaCON and (H)CbCaNCO start from 1H polarization to increase the sensitivity. The CON experiment was performed also in a proline-selective manner to reduce the crowding of the spectrum and to help to identify the proline residues. With the carbon-detected experiments, we were able to assign 12 out of 13 proline residues in the PsbQ protein using the sample concentration of about 0.3 mM. The experiments were performed on a 600 MHz spectrometer equipped with a TCI cryoprobe. |
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