Rapid Sample-Mixing Technique for Transient NMR and Photo-CIDNP Spectroscopy:  Applications to Real-Time Protein Folding

Mok, K Hun, Nagashima, Toshio, Day, Iain J, Jones, Jonathan A, Jones, Charles J V, Dobson, Christopher M and Hore, P J (2003) Rapid Sample-Mixing Technique for Transient NMR and Photo-CIDNP Spectroscopy:  Applications to Real-Time Protein Folding. Journal of the American Chemical Society, 125 (41). pp. 12484-12492. ISSN 0002-7863

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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 H2O 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.

Item Type: Article
Additional Information: ID contributed to the development of the rapid mixing device, control experiments, the acquisition, analysis and interpretation of all experimental data, and drafting of the manuscript. This injection device is one of the best performing devices for NMR spectroscopy, currently being used by 4 research groups in Europe.
Schools and Departments: School of Life Sciences > Chemistry
Depositing User: Iain Day
Date Deposited: 06 Feb 2012 19:21
Last Modified: 22 Mar 2012 15:39
URI: http://sro.sussex.ac.uk/id/eprint/20230
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