The process of the determination of the 1H NMR solution structure of P. hollandica plastocyanin (PC) will include several steps, beginning with the acquisition of two-dimnsional 1H-1H correlated NMR spectra (DQF-COSY, TOCSY, and NOESY) of P. hollandica PC in H2O at different temperatures, by using standard pulse sequences. Bowling Green State University has a 400 MHz Varian NMR spectrometer on site, and additional spectra will be acquired when appropriate from higher field instruments located elsewhere. Water signal suppression will typically be achieved by presaturation or pulse-field gradient. The above mentioned spectra will also be acquired in D2O to identify the slow and fast exchanging amide protons and to identify the ?-protons otherwise saturated by water irradiation. Sequence-specific resonance assignments will be obtained by standard procedures. If required, the ambiguities caused by signal overlaps will be solved by using 1H 3D TOCSY-NOESY spectrum. The distance and dihedral angle constraints will be eveluated from nuclear Overhauser effects and scalar coupling constants. The obtained constraints will be used to generate structures in the program X-PLOR. The structure calculations and the NOESY signal assignments will be performed iteratively. Next, the resulting structures will be refined by CHARM energy minimization or a combination of energy minimization and restrained molecular dynamics methods. The analysis of P. hollandica and other PC structures will be performed using the program QUANTA (Biosym/MSI, San Diego). NMR data analysis, restraints generation and comparison of the experimental NOESY spectra with the back-calculated spectra together with the structure generated will be carried out using FELIX (Biosym/MSI, San Diego).
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