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Biochemical and structural results on two key mutants, one postulated to be important for the interaction with the partner protein and the other for substrate entry is presented. Trp138, adjacent to one type of the type-1 Cu ligands, is one of the residues surrounding a small depression speculated to be important in complex formation with the physiological redox partners, azurin I and II.

Research reveals that removal of 35 residues from the N-terminus of the rusticyanin renders the protein insoluble above pH 5 and the precipitate in solution exhibits an oligomeric form. Data show that the beta-sructure is retained and exposed residues of the deletion mutant are less acidic.

The high redox potential of the blue copper protein rusticyanin in the oxidized state is probably due to the destabilizing effect of the hydrophobic residues near the ion. The interaction between the ion and the axial methionine is less important.

The methionine ligand (Met148) in rustacyanin was replaced with Leu, Gln, Lys and Glu to examine the effect of these substitutions on redox potential, acid stability and copper site geometry. Further examination of the Met ligand in type 1 copper centers showed that its role was fairly complex. The ligand helps to ensure a stable Cu geometry and efficient conversion between the Cu2+ and Cu+ redox states. This was confirmed when redox potentials changed upon substitution with Gln and Lys.

A study was conducted to characterize rusticyanin in Escherichia coli a different mutants for Ser86. Cells were prepared by centrifugation prior to resuspension in loading buffers. Recombinant rusticyanin was determined a spinvac before N-terminal sequencing. Results indicated that the oxidized form of the mutants supported less acid stability than the wild-type protein. Findings also showed that the denaturation of a portion of Ser86Gln was directly correlated with low pH values.

The detailed structure of the copper center of Pseudomonas aeruginosa Met121Glu azurin mutant was analyzed by hydrogen-ion concentration (pH)-dependent X-ray absorption fine structure (XAFS) analysis. XAFS analysis of the Cu(II) center of Met121Glu indicated the presence of pH-dependent changes on the conformation of the copper site. The copper center forms four strong bonds at pH eight with longer Cu-His bond distances.

Alcaligenes xylosoxidans grown under denitrifying conditions are capable of synthesizing two biochemically distinct azurins, and both of these proteins can donate electrons to purified nitrite reductase. These two azurins crystallize distinctly; azurin I crystallizes as thin needles while azurin II grows as blue elongate rectangular prisms with the tetragonal space group P4122 and unit cell parameters a=b=52.65 angstrom = 100.63 angstrom. X-ray crystallographic and other spectrochemical data for both of these azurins are also presented.

Rusticyanin is in possession of beta-sandwich structure characterized by single blue copper proteins which is often related to their high stability. Molecular modeling reveals that the high stability must rise partly from a more protective nature of the site. Serine's location next to the ligating histidine results in a tighter eu cavity through tighter hydrogen bonding. This is in keeping with comparatively smaller structural changes seen in rusticyanin on reduction compared to azurin and explains the high redox capacity of rusticyanin.

X-ray absorption spectroscopic studies at different pH values of the structure of copper site in oxidized and reduced Rhus vernicifera stellacyanin reveals that the fourth ligand in the oxidized protein is O- or N-donating. This ligand is absent in the inner coordination sphere but shows a distant interaction at 2.7 Angstroms from the copper atom. Reduction of the stellacyanin removes a histidine ligand by nearly 0.2 Angstroms from the copper atom. The reduced protein lacks the S-donating fourth ligand.

The X-ray scattering technique was employed to study the molecular conformation of multimeric proteins in solution. The technique used synchrotron radiation to widen data collection and include medium angles. The solution structure of the dissimilatory nitrite reductase of the enzyme Achromobacter xylosoxidans (AxNiR) was defined. An empirical profile of the X-ray scattering was based on the data of a high-resolution crystal structure of the nitrite reductase. The solution structure of AxNiR was verified by sedimentation equilibrium ultracentrifugation studies.

The structural and functional characteristics of azurin obtained from Pseudomonas aeruginosa and two of its methionine-121 mutants were examined using reduction potential measurements, X-ray absorption fine structure and electron paramagnetic resonance techniques. Results indicate that a single mutation is sufficient to alter the spectral and redox properties of azurin's Cu(II) center, which was found to be sensitive to ligands that replace the methionine ligand.

A study was conducted on methods of constrained and restrained refinement of extended x-ray absorption fine structure (EXAFS) data. In the constrained refinement, the quantity of free parameters is reduced. On the other hand, restrained refinement provides for additional structural data that are used in additional observations in the data analysis. The methods are of great use for biological molecules and are particularly useful in studying ligands.

The studies of the primary and high-pH proton channels in copper nitrite reductase based on high- to atomic-resolution crystal structures are reported. The data obtained indicated that the primary proton channel is not essential for efficient catalysis in Cu nitrite reductase and that the high-pH proton channel is effective for proton transfer and operative at physiological pH values.

Phosphorylation of serine 51 residue on the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) inhibits the guanine nucleotide exchange (GNE) activity of eIF2B, presumably, by forming a tight complex with eIF2B. Inhibition of the GNE activity of eIF2B leads to impairment in eIF2 recycling and protein synthesis. We have partially purified the wild-type (wt) and mutants of eIF2alpha in which the serine 51 residue was replaced with alanine (51A mutant) or aspartic acid (51D mutant) in the baculovirus system. Analysis of these mutants has provided novel insight into the role of 51 serine in the interaction between eIF2 and eIF2B. Neither mutant was phosphorylated in vitro. Both mutants decreased eIF2alpha phosphorylation occurring in hemin and poly(IC)-treated reticulocyte lysates due to the activation of double-stranded RNA-dependent protein kinase (PKR). However, addition of 51D, but not 51A mutant eIF2alpha protein promoted inhibition of the GNE activity of eIF2B in hemin-supplemented rabbit reticulocyte lysates in which relatively little or no endogenous eIF2alpha phosphorylation occurred. The 51D mutant enhanced the inhibition in GNE activity of eIF2B that occurred in hemin and poly(IC)-treated reticulocyte lysates where PKR is active. Our results show that the increased interaction between eIF2 and eIF2B protein, occurring in reticulocyte lysates due to increased eIF2alpha phosphorylation, is decreased significantly by the addition of mutant 51A protein but not 51D. Consistent with the idea that mutant 51D protein behaves like a phosphorylated eIF2alpha, addition of this partially purified recombinant subunit, but not 51A or wt eIF2alpha, increases the interaction between eIF2 and 2B proteins in actively translating hemin-supplemented lysates. These findings support the idea that phosphorylation of the serine 51 residue in eIF2alpha promotes complex formation between eIF2alpha(P) and eIF2B and thereby inhibits the GNE activity of eIF2B.

Phosphorylation of the serine 51 residue in the alpha-subunit of translational initiation factor 2 in eukaryotes (eIF2 alpha) impairs protein synthesis presumably by sequestering eIF2B, a rate-limiting pentameric guanine nucleotide exchange protein which catalyzes the exchange of GTP for GDP in the eIF2-GDP binary complex. To further understand the importance of eIF2 alpha phosphorylation in the interaction between eIF2 alpha(P) and eIF2B proteins and thereby the regulation of eIF2B activity, we expressed the wild type (wt) and a mutant eIF2 alpha in which the serine 48 residue was replaced with alanine (48A mutant) in the baculovirus system. The findings reveal that the expression of both of these recombinant subunits was very efficient (15-20% of the total protein) and both proteins were recognized by an eIF2 alpha monoclonal antibody and were phosphorylated to the same extent by reticulocyte eIF2 alpha kinases. However, partially purified recombinant subunits (wt or 48A mutant) were not phosphorylated as efficiently as the eIF2 alpha subunit present in the purified reticulocyte trimeric eIF2 complex and were also found to inhibit the phosphorylation of eIF2 alpha of the trimeric complex. Furthermore, the extents of inhibition of eIF2B activity and formation of the eIF2 alpha(P)-eIF2B complex that occurs due to eIF2 alpha phosphorylation in poly(IC)-treated rabbit reticulocyte lysates were decreased significantly in the presence of insect cell extracts expressing the 48A mutant eIF2 alpha compared to those for wt. These findings support the hypothesis that the serine 48 residue is required for high-affinity interaction between eIF2 alpha(P) and eIF2B.

E-64, 1-(L-trans-epoxysuccinylleucylamino)-4-guanidinobutane, is a potent and highly selective irreversible inhibitor of cysteine proteases. The crystal structure of a complex of actinidin and E-64 has been determined at 1.86-A resolution by using the difference Fourier method and refined to an R-factor of 14.5%. The electron density map clearly shows that the C2 atom of the E-64 epoxide ring is covalently bonded to the S atom of the active-site cysteine 25. The charged carboxyl group of E-64 forms four H-bonds with the protein and thus may play an important role in favorably positioning the inhibitor molecule for nucleophilic attack by the active-site thiolate anion. The interaction features between E-64 and actinidin are very similar to those seen in the papain-E-64 complex; however, the amino-4-guanidinobutane group orients differently. The crystals of the actinidin-E-64 complex diffracted much better than the papain-E-64 complex, and consequently the present study provides more precise geometrical information on the binding of the inhibitor. Moreover, this study provides yet another confirmation that the binding of E-64 is at the S subsites and not at the S' subsites as has been previously proposed. The original actinidin structure has been revised using the new cDNA sequence information.

Serum transferrin is a metal-binding glycoprotein, molecular weight ca. 80,000, whose primary function is the transport of iron in the plasma of vertebrates. The X-ray crystallographic structure of diferric rabbit serum transferrin has been determined to a resolution of 3.3 A. The molecule has a beta alpha structure of similar topology to human lactoferrin and is composed of two homologous lobes that each bind a single ferric ion. Each lobe is further divided into two dissimilar domains, and the iron-binding site is located within the interdomain cleft. The iron is bound by two tyrosines, a histidine, and an aspartic acid residue. The location of the 19 disulfide bridges is described, and their possible structural roles are discussed in relation to the transferrin family of proteins. Mapping of the intron/exon splice junctions onto the molecule provides some topological evidence in support of the putative secondary role for transferrin in stimulating cell proliferation.