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NMR detection of multiple transitions to low-populated states in azurin

¹ Swedish NMR Centre, Göteborg University, 40530 Göteborg, Sweden
² Department of Molecular Biotechnology, Chalmers University of Technology, 40530 Göteborg, Sweden
³ Biochemistry and Biophysics, Göteborg University, 40530 Göteborg, Sweden

Reprint requests to: Martin Billeter, Biochemistry and Biophysics, Göteborg University, Box 462, 40530 Göteborg, Sweden; e-mail: martin.billeter{at}bcbp.gu.se; fax: +46 31 773 3910.

Transitions to conformational states with very low populations were detected for the reduced blue copper protein azurin from Pseudomonas aeruginosa by applying constant relaxation time CPMG measurements to the backbone ¹⁵N nuclei at three magnetic fields (11.7, 14.1, and 18.8 T) and three temperatures (25.7, 35.4, and 44.8°C). Two exchange processes with different rate constants could be discriminated despite populations of the excited states below 1% and spatial neighborhood of the two processes. The group of ¹⁵N nuclei involved in the faster process exhibits at 44.8°C a forward rate constant of 11.7±2.4 s^-1 and a population of the exited state of 0.39±0.07%. They surround the aromatic ring of histidine 35 whose protonation state is coupled to the flipping of a neighboring peptide plane. For the slower process, the forward rate constant and population of the exited state at 44.8°C are 4.1±0.1 s^-1 and 0.45±0.02%, respectively. The residues involved cluster nearby the copper ion, which is separated from the protonation site of histidine 35 by about 8 Å, indicating conformational rearrangements involving the copper coordinating loops. The dependence of the equilibrium constant on the temperature is consistent with an enthalpy-dominated transition around the copper, but an entropy-controlled transition near histidine 35. The detection by nuclear magnetic resonance of millisecond to second conformational transitions near the copper ion suggests a low energy-cost rearrangement of the copper-binding site that may be necessary for efficient electron transfer.

Keywords: Blue copper proteins; dynamics; NMR relaxation; CPMG; conformational exchange

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