The Desulfovibrio vulgaris Hildenborough ORP: isolation, cluster reconstitution and NMR resonance assignment

Abstract

DVU2108 is a protein belonging to the Orange Protein (ORP) family that in Desulfovibrio vulgaris Hildenborough forms a protein complex named the Orange Protein Complex. ORP is a conserved protein in anaerobic microorganisms and in Desulfovibrio gigas the homologous protein was isolated with a novel Mo-Cu cluster non-covalently attached to the polypeptide chain. The protein sample isolated by homologous expression of StrepDVU2108 in Desulfovibrio vulgaris Hildenborough displayed an absorption spectra similar to an iron-sulfur containing protein. Nevertheless, the Apo form of DVU2108 (obtained by heterologous expression in E.coli) was able to incorporate a MoCu or a WCu cluster, when in the presence of tetrathiomolybdate or tetrathiotungstate and copper chloride, with a metal to metal ratio of 1Cu:2.3Mo ± 1.2 and 1Cu:1.49W ± 0.03, which is close to the preferable metal ratio for this protein found when titrating Mo or W to a equimolar solution of Apo-DVU2108 and copper (1Cu:2Mo/2W). The same metal stoichiometry was found in Desulfovibrio gigas ORP. The molar extinction coefficients for MoCu-DVU2108 are 10500 ± 280 M-1cm-1 at 334 nm and 5570 ± 150 M-1cm-1 at 483 nm, per molybdenum atom. For WCu-DVU2108 the extinction coefficients are 14560 ± 80 M-1cm-1 at 295 nm and 6990 ± 40 M-1cm-1 at 407 nm, per tungsten atom. The reconstituted proteins, MoCu-DVU2018 and WCu-DVU2108 showed higher stability with higher melting temperatures than the Apo form, 72.1 ± 0.6 ºC, 72.1 ± 0.7 ºC and 65.6 ± 0.2 ºC, respectively. In a second part of this work, the resonance assignment of Apo-DVU2108 was accomplished at 94 %. The 1H-15N HSQC of this protein presented 118 resonances that were assigned to 101 of the total number of amides (113), including 9 side chains.