[NMR paper] Rapid sample-mixing technique for transient NMR and photo-CIDNP spectroscopy: applica

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Rapid sample-mixing technique for transient NMR and photo-CIDNP spectroscopy: applications to real-time protein folding.

Related Articles Rapid sample-mixing technique for transient NMR and photo-CIDNP spectroscopy: applications to real-time protein folding.

J Am Chem Soc. 2003 Oct 15;125(41):12484-92

Authors: Mok KH, Nagashima T, Day IJ, Jones JA, Jones CJ, Dobson CM, Hore PJ

We describe the development and application of a novel rapid sample-mixing technique for real-time NMR (nuclear magnetic resonance) spectroscopy. The apparatus consists of an insert inside a conventional NMR tube coupled to a rapid injection syringe outside the NMR magnet. Efficient and homogeneous mixing of solutions in the NMR tube is achieved with a dead time of tens of milliseconds, without modification of the NMR probe or additional hardware inside the magnet. Provision is made for the inclusion of an optical fiber to allow in situ laser irradiation of samples, for example to generate photo-CIDNP (chemically induced dynamic nuclear polarization). An NMR water suppression method has been implemented to allow experiments in H(2)O as well as in deuterated solvents. The performance of the device has been tested and optimized by a variety of methods, including sensitive detection of residual pH gradients and the use of NMR imaging to monitor the extent of mixing in real time. The potential utility of this device, in conjunction with the sensitivity and selectivity of photo-CIDNP, is demonstrated by experiments on the protein hen lysozyme. These measurements involve the direct detection of spectra during real-time refolding, and the use of CIDNP pulse labeling to study a partially unfolded state of the protein under equilibrium conditions. Magnetization transfer from this disordered state to the well-characterized native state provides evidence for the remarkable persistence of nativelike elements of structure under conditions in which the protein is partially denatured and aggregation prone.

PMID: 14531692 [PubMed - indexed for MEDLINE]



Source: PubMed