Purification, crystallization and preliminary X-ray diffraction studies of a complex between G protein-coupled receptor kinase 2 and Gbeta1gamma2.

G protein-coupled receptor kinase 2 (GRK2) phosphorylates activated G protein-coupled receptors (GPCRs), which ultimately leads to their desensitization and/or downregulation. The enzyme is recruited to the plasma membrane via the interaction of its carboxyl-terminal pleckstrin-homology (PH) domain with the beta and gamma subunits of heterotrimeric G proteins (Gbetagamma). An improved purification scheme for GRK2 has been developed, conditions under which GRK2 forms a complex with Gbeta(1)gamma(2) have been determined and the complex has been crystallized in CHAPS detergent micelles. Crystals of the GRK2-Gbetagamma complex belong to space group C2 and have unit-cell parameters a = 187.0, b = 72.1, c = 122.0 A, beta = 115.2 degrees. A complete data set has been collected to 3.2 A resolution with Cu Kalpha radiation.

Statistical analysis of crystallization database links protein physico-chemical features with crystallization mechanisms.

X-ray crystallography is the predominant method for obtaining atomic-scale information about biological macromolecules. Despite the success of the technique, obtaining well diffracting crystals still critically limits going from protein to structure. In practice, the crystallization process proceeds through knowledge-informed empiricism. Better physico-chemical understanding remains elusive because of the large number of variables involved, hence little guidance is available to systematically identify solution conditions that promote crystallization. To help determine relationships between macromolecular properties and their crystallization propensity, we have trained statistical models on samples for 182 proteins supplied by the Northeast Structural Genomics consortium. Gaussian processes, which capture trends beyond the reach of linear statistical models, distinguish between two main physico-chemical mechanisms driving crystallization. One is characterized by low levels of side chain entropy and has been extensively reported in the literature. The other identifies specific electrostatic interactions not previously described in the crystallization context. Because evidence for two distinct mechanisms can be gleaned both from crystal contacts and from solution conditions leading to successful crystallization, the model offers future avenues for optimizing crystallization screens based on partial structural information. The availability of crystallization data coupled with structural outcomes analyzed through state-of-the-art statistical models may thus guide macromolecular crystallization toward a more rational basis.

Crystallization and phase transformation behaviour of electroless nickel-phosphorus deposits with low and medium phosphorus contents under continuous heating

Electroless nickel-phosphorus deposits with 5-8 wt% P and 3-5 wt% P were analysed for the effects of continuous heating on the crystallization kinetics and phase transformation behaviour of the deposits. The as-deposited coatings consist of a mixture of amorphous and microcrystalline nickel phases, featuring in their X-ray diffraction patterns. Continuous heating processes to 300C-800C at 20C/min were carried out on the deposits in a differential scanning calorimetric apparatus. The subsequent X-ray diffraction analyses show that the sequence of phase transformation process was: amorphous phase + microcrystalline nickel, f.c.c. nickel + Ni3P stable phases. Preferred orientation of nickel {200} plane developed in the deposits after the heating processes. Differential scanning calorimetry of the deposits indicates that the crystallization temperatures increased with decreasing phosphorus content, and increasing heating rate. Crystallization activation energies of the deposits (230 and 322 kJ/mol, respectively) were calculated using the peak temperatures of crystallization process, from the differential scanning calorimetric curves at the heating rates ranging from 5 to 50C/min. It was found that the deposit with lower phosphorus content has higher activation energy.

Crystallization and preliminary X-ray diffraction analysis of naphthalene dioxygenase from Rhodococcus sp strain NCIMB 12038

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by Rhodococcus sp. strain NCIMB 12038. The terminal oxygenase component (naphthalene 1,2-dioxygenase) that catalyzes this reaction belongs to the aromatic ring hydroxylating dioxygenase family and has been crystallized. These enzymes utilize a mononuclear nonheme iron centre to catalyze the addition of dioxygen to their respective substrates. In this reaction, two electrons, two protons and a dioxygen molecule are consumed. The Rhodococcus enzyme has only 33 and 29% sequence identity to the corresponding alpha- and beta-subunits of the NDO system of Pseudomonas putida NCIMB 9816-4, for which the tertiary structure has been reported. In order to determine the three-dimensional structure of the Rhodococcus NDO, diffraction-quality crystals have been prepared by the hanging-drop method. The crystals belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 144, c = 185.6 Angstrom, alpha = beta = gamma = 90degrees, and diffract to 2.3 Angstrom resolution.