HOMOLOGY PREDICTED STRUCTURE AND COMPARISON WITH THE SECONDARY STRUCTURE FROM NMR DATA FOR PLASTOCYANIN FROM THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803

Abstract

Sequence specific 1H NMR resonance assignments are reported here for 95 amino acids of reduced plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803 at pH 5.4. Spin systems of different amino acids were classified and identified using DQF-COSY and TOCSY spectra in D2O and H2O solvents. A variety of temperature conditions and two magnetic fields (400 and 600 MHz) were used to overcome resonance overlap. Sequence specific assignment of the spin systems was made using NOE connectivities between adjacent amide, α and β protons (α1Ni + 1, NiNi + 1 and β1Ni + 1. A selection of βH stereospecific assignments and Ξ1 anges for some buried and surface residues of Synechocytis plastocyanin are presented as well. Analysis of interrrsidue NOE connectivities made possible the characterization of the secondary structure, which is the β-sheet type as is the case in all other plastocyanins. The protein in solution contains eight β-strands, one short α-helix segment, five reverse turns and two loops. However, some differences in local conformation are observed between cyanobacterial, algal and higher plant plastocyanins. Four different homologous proteins as templates, two distinct alignments and two approaches (MODELLER and CONGEN) were used to model the three-dimensional structure of Synechocystis plastocyanin. The lowest energy predicted structures reported herein can be considered of medium range resolution ( ∼ 3A). The detailed analysis of hydrogen bonded amide protons in Synchocystis models, the slowly exchanging amide protons determined by NMR, and comparison with hydrogen bonds in other characterized plastocyanins indicate that the folding of Synechocystis plastocyanin is quite similar to that of other plastocyanins.