Jarmila Jancarik

Optimum solubility (OS) screening: an efficient method to optimize buffer conditions for homogeneity and crystallization of proteins

One of the most critical steps in the preparation of protein samples for structural studies by X-ray crystallography is to obtain biochemically pure and conformationally homogenous protein samples. Very often, the purified sample does not meet these qualifications and therefore does not crystallize. A screening method, Optimum Solubility Screen, has been developed that consists of two steps. The first step selects a better buffer than that used during purification. 24 different buffers ranging from pH 3 to pH 10 are screened using a vapor-diffusion method and very small amounts of protein. The solubility of the protein is first determined by visual examination using a light microscope and those drops that remain clear after 24 h are further evaluated using dynamic light scattering. If the results from the first step are still not satisfactory, a second step explores a variety of chemical additives in order to improve the monodispersity of the protein sample. In 64% of the cases, crystallization was successful from proteins that had initially shown high levels of aggregation. This screen can be configured to perform in an automated high-throughput mode and can be expanded for additional buffers and additives.

Crystallization and preliminary X-ray diffraction study of the ligand-binding domain of the bacterial chemotaxis-mediating aspartate receptor of Salmonella typhimurium

The periplasmic domain of the aspartate chemotaxis receptor from Salmonella typhimurium has been crystallized in the presence and absence of bound aspartate. Both crystal forms were grown by precipitation with lithium sulfate and diffract to 1.8 A resolution. The aspartate receptor structure is believed to be prototypical of a large class of receptors including those for polypeptide growth factor hormones as well as those for small chemotaxis-affector molecules such as aspartate and serine.

Structural characterization of an iron-sulfur cluster assembly protein IscU in a zinc-bound form.

Introduction. Iron–sulfur (Fe-S) clusters are simple inorganic prosthetic groups widely distributed in nature. Proteins that contain Fe-S clusters play essential roles in diverse biological processes including electron transfer, gene regulation, environmental sensing, and substrate activation. Although it is possible to assemble Fe-S clusters into proteins from inorganic sulfide and iron in vitro, the biogenesis of Fe-S cluster in vivo, however, appears to be facilitated by proteins rather than spontaneous formation. Recent studies have led to the discovery that a highly conserved gene cluster iscSUA-hscBA-fdx is essential for the biogenesis of Fe-S cluster proteins in bacteria. Proteins encoded by this gene cluster include: IscS, IscU, IscA, HscA, HscB, and Ferredoxin (Fd). Homologies of these proteins have also been identified in eukaryotic organisms, indicating a conserved mechanism for the biogenesis of Fe-S proteins. Further biochemical studies have revealed detailed roles of these Fe-S cluster proteins. IscS, a homolog of NifS, is a homodimeric pyridoxal phosphate-dependent cysteine desulfurase. It catalyzes the desulfurization of L-cysteine to L-alanine and provides S to IscU for Fe-S cluster assembly. IscU provides a scaffold for the assembly of a nascent iron– sulfur cluster prior to its delivery to apo Fe-S proteins. IscA is proposed to function as an alternative scaffold for Fe-S cluster assembly, but the exact role of this protein is still not determined. HscA and HscB are molecular chaperones that can selectively bind IscU and assist in the biogenesis of Fe-S proteins. The detailed roles of these molecular chaperones are also not clear. Sequence comparisons suggest IscU is a highly conserved protein and is homologous to the N-terminal domain of NifU (nNifU), an essential protein for nitrogen fixation. IscU/nNifU contains three strictly conserved cysteine residues. Site-directed mutagenesis data suggest that all three cysteine residues are essential for the function of IscU/nNifU proteins. Biochemical assay showed IscS can form a covalent complex with IscU through residue Cys328 in IscS and residue Cys63 in IscU in Escherichia coli. Characterization of Fe-S cluster assembly on IscU or ISU (eukaryotic IscU) from several organisms including Azotobacter vinelandii, E. coli, Thermotoga maritima (Tm) and human have been carried out. In presence of IscS/NifS, D-cysteine, Fe and reducing agent, IscU/nNifU is able to assemble a transient [Fe2S2] 2

Structure of O67745_AQUAE, a hypothetical protein from Aquifex aeolicus.

Using single-wavelength anomalous dispersion data obtained from a gold-derivatized crystal, the X-ray crystal structure of the protein 067745_AQUAE from the prokaryotic organism Aquifex aeolicus has been determined to a resolution of 2.0 A. Amino-acid residues 1-371 of the 44 kDa protein were identified by Pfam as an HD domain and a member of the metal-dependent phosphohydrolase superfamily (accession No. PF01966). Although three families from this large and diverse group of enzymatic proteins are represented in the PDB, the structure of 067745_AQUAE reveals a unique fold that is unlike the others and that is likely to represent a new subfamily, further organizing the families and characterizing the proteins. Data are presented that provide the first insights into the structural organization of the proteins within this clan and a distal alternative GDP-binding domain outside the metal-binding active site is proposed.

Crystallization of human c-H-ras oncogene products

There is compelling evidence that cancer develops as a consequence of genetic changes (probably multiple) in some members of a selected set of cellular genes. DNA isolated from a variety of tumors, but not normal tissues, possesses the ability to malignantly transform non-tumorigenic cells. Many oncogenes responsible for such transformation have been isolated from transformed cell lines and animal and human tumors induced spontaneously, by virus, by chemical, or by radiation. The most commonly found transforming genes isolated from human tumor cells by DNA transfection assay are the ras gene family (c-H-ras, c-K-ras and N-ras). We report crystallization of several human c-H-ras oncogene proteins.