Protein and Peptide Letters - Volume 13, Issue 9, 2006

Here we developed small molecule inhibitors of SKI-1/S1P enzyme of the Proprotein Convertase family following two approaches. One involves the assembly of multi-branch peptides while the other utilizes the insertion of alkyloxy pseudo peptide bond at P1-P1' cleavage position. In first approach, 2 and 4-branch peptides were designed based on the human (h) SKI-1128-137 sequence, located N-terminal to its secondary activation site (K137fl⇓ L). The 4-branch peptide exhibited the highest SKI-1 inhibitory property (IC50 = 0.9 μM) with ∼8.6 and 1.3-fold more potency than the corresponding single and 2-branch peptides, respectively. In the second strategy, an oxymethylene containing unnatural amino acid such as aminooxy-acetic acid (Aoaa) or 8-amino-3, 6 dioxa-octanoic acid (Adoa) was introduced substituting P1, P1' or both residues of hSKI-1183-190 and hSKI-1178-190 segments. These domains contain the same primary hSKI-1 activation site L186 R. Among those tested, P7-Tyr mutant [178GRYSSRRL(Adoa)AIP190] exhibited higher SKI-1 inhibitory activity (Ki in low μM). Circular dichroism (CD) spectra of SKI-1 inhibitors showed interactions of varying degrees between the enzyme and the inhibitor consistent with the observed inhibition profile. A 3D-homology model structure of SKI-1 catalytic domain indicated a broad catalytic pocket.

In order to identify proteins that bind to the PDZ domain of Erbin, we tested the C-termini of several proteins in a yeast two-hybrid assay. ErbB2, APC, β-catenin, c-Rel and HTLV-1 Tax were identified as ligands of the PDZ domain of Erbin. The interactions were verified by co-immunoprecipitation experiments. These findings demonstrate the promiscuity of the PDZ domain of Erbin.

Deciphering the code that determines the three-dimensional structure of proteins and the ability to predict the final folded form of a protein is still elusive to molecular biophysists. In the case of several proteins a similar tertiary structure is not accompanied by any significant sequence similarity. The question now remains whether a code beyond the genetic code that describes the arrangement of the amino acid within a three dimensional protein structure. The available data undoubtedly demonstrates that the redundancy of this code must be tremendous. Several techniques such as nuclear magnetic resonance spectroscopy and laser detection techniques, coupled with fast initiation of the folding reaction, can now probe the folding events in milliseconds or even faster and provide highly relevant information. The thermodynamic analysis of the folding process and of kinetic intermediates opens whole new avenue of understanding. Breaking the protein folding code would enable scientists to look at a gene whose function is unknown and predict the three-dimensional structure of the protein it encodes. This would give them a very good idea of what the gene does. In this review we hope to bring together the information available about protein folding with particular emphasis on folding intermediate(s). Additionally, the practical consequences of the solution of the protein folding problem in medicine and biotechnology are also discussed.

Stereo-structures of protonated L-phenylalanine (L-Phe), L-tyrosine (L-Tyr) and L-tryptophan (L-Trp) containing homodipeptides (L-Tyr-L-Tyr, L-Phe-L-Phe, L-Trp-L-Trp) are carried out by ab initio calculations. The obtained data in gas phase are compared with experimental ones, received by linear-dichroic infrared (IR-LD) spectroscopy of solids, oriented as suspension in nematic mesophase. An observation of a good correlation between theoretical and spectroscopic geometry parameters established and illustrated the possibilities of this complex study approach for the prediction of the stereostructures in compounds in solid state. The protonation leads to little variance in the bond lengths and angles, expecting the COO- fragment, where a distortion of equalized COO- bond lengths, stabilizing a C=O double bond and C-O(H) one is established. Significant deviations of the dihedral angles as a result of the protonation are obtained in the skeletal aliphatic and amide- fragments. In L-Tyr-L-Tyr and L-Phe-L-Phe, a deviation of O=C-N-H torsion angle about 10-140 is predicted. The calculations show a trans-amide configuration in L-Trp-L-Trp and a cis-one after its protonation.

A lectin from the seeds of Amaranthus viridis Linn has been purified by affinity chromatography on asialofetuin- linked amino activated silica. Amaranthus viridis lectin (AVL) has a native molecular mass of 67 kDa. It is a homodimer composed of two 36.6 kDa subunits. The lectin gave a single band in non-denaturing PAGE at pH 4.5 and pH 8.3 and a single peak on HPLC size exclusion and cation exchange columns. The purified lectin was specific for both Tantigen and N-acetyl-D-lactosamine, markers for various carcinomas, in addition to N-acetyl-D-galactosamine, asialofetuin and fetuin. This lectin reacted strongly with red blood cells (RBCs) from human ABO blood groups and rat. It also reacted with rabbit, sheep, goat and guinea pig RBCs. The lectin is a glycoprotein having no metal ion requirement for its activity. Denaturing agents such as urea, thiourea and guanidine-HCl had no effect on its activity when treated for 15 minutes. AVL showed significant antiproliferative activity towards HB98 and P388D1 murine cancer cell lines. It also exerted antifungal activity against phytopathogenic fungi Botrytis cincerea and Fusarium oxysporum but not against Rhizoctonia solani, Trichoderma reesei, Alternaria solani and Fusarium graminearum.

A method of isolation of hydrophobic membrane-bound C-terminal domain of influenza virus A hemagglutinin (HA) is suggested. The method is based on the insertion of HA into octylglucoside micelles followed by pepsin or thermolysin hydrolysis. Subsequent treatment of proteolytic digests with chloroform-hexafluoroisopropanol mixture resulted in the extraction of a few hydrophobic peptides into organic phase. Mass-spectrometry (MALDI-TOF) analysis revealed that the peptides with ion masses corresponding to the anchoring C-terminal domain with or without modifications predominated in the organic solution. The data obtained confirmed our speculation on the possibility of the suggested isolation scheme following from the strong interactions of anchoring domains in compact trimeric structure of HA spikes combined with micelle protection effect. Several appropriate peptides presence in the organic phase apparently arises from the presence of a few accessible proteolytic sites in HA transmembrane region.

We report the successful expression and detection of a phosphorylated form of human T cell tyrosine kinase, Lck, in Saccharomyes cerevisiae, which leads to growth suppression of the yeast cells. Expression of an inactive Lck mutant resulted in no phosphorylation and no growth suppression, indicating that cell growth inhibition by Lck is due to the activity of the kinase, consistent with the observed tyrosine-phosphorylation of the Lck and yeast host cell proteins. The addition of a known inhibitor of Lck to the cell culture resulted in recovery of cell growth expressing the active Lck, suggesting that the growth inhibition by lck gene expression can be used to screen inhibitors for the gene product. We have extended such approach to Tob, another potential therapeutic target.

Arginine has been used to suppress aggregation of proteins during refolding and purification. We have further studied in this paper the aggregation-suppressive effects of arginine on two commercially important proteins, i.e., interleukine- 6 (IL-6) and a monoclonal antibody (mAb). These proteins show extensive aggregation in aqueous buffers when subjected to thermal unfolding. Arginine suppresses aggregation concentration-dependently during thermal unfolding. However, this effect was not specific to arginine, as guanidine hydrochloride (GdnHCl) at identical concentrations also was effective. While equally effective in aggregation suppression during thermal unfolding, arginine and GdnHCl differed in their effects on the structure of the native proteins. Arginine showed no apparent adverse effects on the native protein, while GdnHCl induced conformational changes at room temperature, i.e., below the melting temperature. These additives affected the melting temperature of IL-6 as well; arginine increased it concentration-dependently, while GdnHCl increased it at low concentration but decreased at higher concentration. These results clearly demonstrate that arginine suppresses aggregation via different mechanism from that conferred by GdnHCl.

Solving the three dimensional structure of a protein-DNA complex is a prerequisite to understand, at the atomic level, the interactions between DNA-binding proteins and their target DNA sequences. Arranging these complexes into an ordered and repetitive network (a crystal, suitable for X-Ray analysis) is a time-limiting empirical step. Although it has been suggested that the crystallization space for protein-DNA complexes is probably smaller than that of non-complexed proteins, a study presenting a detailed and updated analysis of this space is still missing. Here, we analyze the successful crystallization conditions of several hundred protein-DNA complexes and present a bias-free statistical analysis of 15 crystallization parameters that include concentration, temperature, pH, precipitants, salts, divalent cations and polyamines. Our analysis shows that some crystallization parameters are interestingly restricted into narrow intervals. These restrictions could be very helpful in the design of sparse-matrix crystallization screens that target exclusively protein-DNA complexes.

Monoclonal antibody (mAb) MN423 recognizes Alzheimer's disease specific conformation of tau protein assembled into paired helical filaments (PHF). Since the three-dimensional structure of PHF is currently unavailable, the structure of MN423 binding site could provide important information about PHF conformation with the consequences for the Alzheimer's disease prevention and cure. Fab fragment of MN423 was prepared and purified. We have identified two different conditions for crystallization of the Fab fragment that yielded two crystal forms. They diffracted to 3.0 and 1.6 Å resolution with four and one molecule in the asymmetric unit, respectively. Both crystal forms belonged to the space group P21 with unit cell parameters a = 76.4 Å, b = 138.4 Å, c = 92.4 Å, β = 101.9∞, and a = 71.5 Å, b = 36.8 Å, c = 85.5 Å , β = 113.9∞.

Regulator of G-protein signaling 5 (RGS5), an inhibitor of Gq and Gi activation, is a member of the small RGS protein subfamily. However, despite significant process in the investigation of RGS5, no structure is yet available. In order to elucidate the mechanism of the RGS5 in G protein signaling pathway, we have overexpressed the RGS5 and G_i3 from human in Escherichia coli and crystallized the complex of RGS5 and Gαi3 proteins with GDP/Mg2+/AlF4 - at 3.0 Å resolution using a synchrotron radiation source. The complex crystals belong to the tetragonal space group P41212 or P43212, with unit cell parameters a=b=95.9 Å, and c=138.8 Å. Assuming one complex protein in the crystallographic asymmetric unit, the calculated Matthews parameter (VM) is 2.57 Å3/Da and solvent content is 52.2 %.

We report here on crystallization and preliminary X-ray analysis of the catalytic domain of chitinase D from Bacillus circulans WL-12. The native crystals of this domain were found to belong to the orthorhombic space group P212121. To elucidate the structure of the catalytic domain by the multiple isomorphous replacement method, 30 kinds of derivatized crystals were prepared by soaking the native crystals into a mother liquor containing salts of heavy metal atoms. Difference Patterson maps calculated for four derivatives showed strong peaks in the Harker sections.

Human HSCARG has been annotated as a possible cancer related protein. Amino acid homology, although at a low percentage, suggested that HSCARG contains NmrA domain and might be a member of short chain dehydrogenase reductase superfamily. In order to investigate its structure and function, HSCARG gene has been successfully expressed and purified in E. coli. HSCARG was crystallized and diffracted to a resolution of 2.4 Å on Mar225 CCD Detector at SER-CAT 22BM synchrotron source. The crystals belong to F23 space group, with unit cell parameters a=b=c=223.30Å, α=β=γ=90°. There are two molecules per asymmetry unit.