Phase Determination by Multiple-Wavelength X-Ray Diffraction: Crystal Structure of a Basic "Blue" Copper Protein from Cucumbers

Abstract

A novel x-ray diffraction technique, multiple-wavelength anomalous dispersion (MAD) phasing, has been applied to the de novo determination of an unknown protein structure, that of the "blue" copper protein isolated from cucumber seedlings. This method makes use of crystallographic phases determined from measurements made at several wavelengths and has recently been made technically feasible through the use of intense, polychromatic synchrotron radiation together with accurate data collection from multiwire electronic area detectors. In contrast with all of the conventional methods of solving protein structures, which require either multiple isomorphous derivatives or coordinates of a similar structure for molecular replacement, this technique allows direct solution of the classical "phase problem" in x-ray crystallography. MAD phase assignment should be particularly useful for determining structures of small to medium-sized metalloproteins for which isomorphous derivatives are difficult or impossible to make. The structure of this particular protein provides new insights into the spectroscopic and redox properties of blue copper proteins, an important class of metalloproteins widely distributed in nature.

A 1H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

“…This behavior has been previously observed for only one of the NH resonances of Co(II)-substituted azurin [21]. This indicates a larger accessibility of ST in the solvent with respect to azurin, thus resembling the metal site of the cucumber basic protein [12] which has been used as a starting point for constructing a three-dimensional model for ST [13]. This finding could explain the faster electron transfer rate found for ST with respect to azurin [7].…”

Cloning, expression, and spectroscopic characterization of Cucumis sativus stellacyanin in its nonglycosylated form

“…Plantacyanins, also known as cucumber basic protein, CBP, are strongly basic mononuclear blue copper proteins from plants (Markossian et al, 1974). According to the X-ray structural data, they have a methionine as an axial ligand rather than glutamine as in stellacyanin (Guss et al, 1988). The similarity in the pH-dependent behavior of stellacyanins and plantacyanins argues strongly that this change is not attributable to the glutamine ligand in stellacyanin.…”

The refined structure of functional unit h of keyhole limpet hemocyanin (KLH1‐h) reveals disulfide bridges

“…The disulfide bridge in the cupredoxin-like domain is also present in cucumber basic protein and is considered as a distinguishing feature of phytocyanins (31). In cucumber basic protein, the disulfide bridge is in the direct vicinity of the active site with one cysteine being the neighbor of a copper coordinating histidine of the active site (32). In the cupredoxinlike domain of hemocyanin, it seems that this disulfide bridge has been conserved to maintain the integrity of the tertiary (6,20).…”