Protein and Peptide Letters - Volume 11, Issue 4, 2004

The high technical standard of current peptide chemistry, which has evolved over the past three decades, has profoundly facilitated the investigation of proteins and their interactions with other molecules at the level of individual amino acids. Using currently available peptide synthesis methods, sequentially continuous protein binding sites can be readily mapped, characterized, optimized, and used as lead compounds for inhibitors of protein-ligand interactions. The mimicry of sequentially discontinuous protein binding sites, on the other hand, continues to present a challenge for peptide and organic chemists. This mini-review summarizes currently used and emerging, rational and random strategies for the design of synthetic mimetics of discontinuous protein binding sites.

Bactenecin7 (Bac7), a cationic antibacterial peptide, contains a repeating region of Xaa-Pro-Arg- Pro (Xaa = hydrophobic residue). To investigate the structure and property of a Pro / Arg-rich region, we synthesized a series of peptides, Xaa-Pro-Arg-Pro (Xaa = Gly, Arg, Leu, Ile, and Phe) as models and characterized. The conformational preferences of these peptides in water and trifluoroethanol were examined by circular dichroism. The results suggest the presence of largely poly(Pro)-II helical conformation in aqueous and trifluoroethanol solutions. Their antibacterial activity against gram-negative bacteria such as Escherichia coli, Klebsiella Pneumoniae, Pseudomonas aeruginosa, and Escherichia coliHB101, and grampositive bacteria such as Staphylococcus aureus were measured at various peptide concentrations. Two of our synthetic tetrapeptide fragments containing Gly and Arg were efficiently killed the gram-positive bacteria, Staphylococcus aureus, at the concentration level of 200 μg / mL.

The small angle X-ray scattering (SAXS) data of xylanase XYNII (endo-1,4-β-xylan xylanohydrolase EC 3.2.1.8) from Trichoderma longibrachiatum, an enzyme catalysing the reaction of accidental hydrolysis of β-1,4-D-xylosidic linkages of xylan, were recorded for protein solution using synchrotron radiation. The experimental data were compared with those of theoretical scattering calculated on the basis of the known crystal structure. The radius of gyration measured by SAXS (RG=1.7 nm) was about 3.5% larger and the maximum dimension in the distance distribution function about 5 % larger than the corresponding values calculated on the basis of the crystal structure.

Alkaline pH induced conformational changes in different domains of bovine serum albumin were studied by using domain specific ligands: chloroform, bilirubin and diazepam for domains I, II and III respectively. The effect of alkaline pH on the secondary structure of BSA was monitored by far-UV CD in the range 250 nm to 200 nm. The pH profiles of BSA in the alkaline region showed a two-step change, one corresponding to NB transition (pH 7.5 to 9.0) and the other to B (→) U (pH 11.0 to 13.5). Binding of chloroform decreased continuously on increasing pH, whereas binding of diazepam, remained unchanged up to pH 9 and decreased thereafter. In contrast, binding of bilirubin gradually increased up to pH 11.0 and decreased thereafter reaching a value similar to one obtained with native BSA at pH 11.5. Above pH 11.5, bilirubin binding decreased and was abolished completely at pH 12.5. In the pH region 7.5 to 11.0, a continuous decrease in chloroform binding (pH 7.5 to 9.5) and a late decrease in diazepam binding (pH 9.5 to 11.0) suggested major loss of native conformation of domain I followed by domain III during alkaline induced unfolding of BSA. However, a significant increase in bilirubin binding showed a favorable conformational rearrangement in domain II in this pH region (pH7.5 to 11.0). Further, a nearly complete abolishment of bilirubin binding to BSA and significant loss of secondary structure around pH 12.5 indicated that domain II was more resistant to alkaline pH and unfolds only at extreme alkalinity. Taken together, these data suggest that unfolding of three domains of BSA follow the following order of susceptibility towards alkaline denaturation of BSA domain I>domain III>domain II.

Human osteoprotegrin (OPG) and its truncated mutant OPG-280 and lengthened mutant OPG-Fc were constructed and successfully expressed in Trichoplusia ni cells and Bombyx mori larvae. Native SDSPAGE and Western blot analysis revealed that OPG-Fc is present as a homodimer in Tn cells or B. mori larvae compared with OPG and OPG-280. Furthermore, the hypocalcemic effect assay showed that truncation of the C-terminal 100 residues OPG does not abolish the biological activity and Fc can be helpful in forming the OPG homodimer with improved biological activity.

The effect of pressure on the unfolding of the molten globule (MG) state of canine milk lysozyme (CML) was examined using ultraviolet spectroscopy. The volume changes of the MG-unfolded-state transition were observed at pH 2.0 and around 20 to 60°C, but no volume change has been found for bovine α-lactalbumin, which is homologous to CML. Our results suggest that the MG state of CML possesses a tightly packed hydrophobic core.

The binding of Pb2+ to bovine serum albumin (BSA) at neutral pH was studied using lead ion selective electrode. The binding data was treated according to Scatchard Equation. The number of binding classes and the number of binding sites, intrinsic dissociation constants and stepwise binding constants for each class were determined. Two binding classes were found. Four binding sites in the first class and five binding sites in the second class were determined. Binding in the first class was stronger than in the second. Similar binding studies were carried out with heat treated BSA. It was found that not only the number of binding sites but also the strength of binding increases upon heat treatment.

Human β-defensin-2 (hBD2) is a small antimicrobial peptide with potential as a therapeutic agent. The effect of codon usage on the expression of hBD2 in Escherichia coli was studied. Two coding sequences encoding the same hBD2 precursor were both expressed as fusion protein with thioredoxin in E. coli BL21 (DE3). One is the wild-type human cDNA and the other is a gene synthesized by a PCR-based method in which rare codons were altered to those frequently used in E. coli. The expression level of recombinant hBD2 was over 50% of the total cellular protein when the synthetic gene with preferential codons was employed which was a 9-fold enhancement over the wild-type cDNA. The result shows the codon bias of the host was a major barrier in high-level expression of recombinant hBD2 and suggests a similar approach may be used in the expression of other defensins in E. coli.

Pearl millet (Pennisetum glaucum L.), a species of the Poaceae family, is an important food crop in Africa, Asia and South America. Its nutritional value is due to storage prolamins accumulated in the seeds. In other species of the same family, the expression of the genes coding for storage prolamins is mediated by the regulatory protein opaque-2. In this paper we show that an opaque-2 -like protein is present in pearl millet too and is expressed during the early stages of seed development. The organization of the gene coding for this protein is similar to that of orthologous genes in other Poaceae species, i.e. six exons separated by five introns. A comparison of amino acid homologies with other described opaque-2 proteins is presented.

Hemoglobin cross-linked with small molecular modifiers turns out to be more stable. Modifications of proteins with polyethylene glycol (PEG) have been proven to enlarge the molecular size of proteins, to prolong their retention time in the circulation as well as blunt immune reactions. In the present study, the optimal conditions for porcine hemoglobin (pHb) modification with bis (3, 5-dibromosalicyl) fumarate (DBBF) and PEG were evaluated. The derivative of DBBF cross-linked pHb (DBBF-pHb) showed improved oxygen affinity and the ability to resist the dissociation of the α2β2 tetramer compared with the natural protein. DBBF-pHb was then bound to the activated PEG. The results indicated that the pHb modified with DBBF and PEG had more stable tetrameric conformation with a molecular weight of 107000. Their oxygen half-saturation pressure (P50) is around 3.33 kPa, which approximates the physiological P50 of human red blood cells. Both routine and reinforced immunizing methods were adopted to study the immunogenicity of modified products and the results showed that the products had very low immunogenicity evaluated by enzyme-linked immunoadsordent assay (ELISA). Somewhat beneficial effects were shown in the treatment of hemorrhagic shock where modified hemoglobin solutions were used as resuscitation fluids in the hemorrhagic shock Sprague-Dawley (SD) rats model.

Statistical analysis of protein-protein interfaces in a database of pure peptide crystals shows that the distribution of the contact area contains two components: a major exponential distribution and a minor flat distribution. Analysis of two sub-databases provides evidence that the two components represent specific and non-specific contacts, respectively. The probability of an interface with a given area being specific can be estimated. A scaled quantity (contact ratio) is introduced that is more useful than contact area for discriminating specific and non-specific contacts in protein crystals.

Application of complementary B and T cell epitopes in inducing anti-idiotypic and anticlonotypic antibodies capable of regulating or suppressing the autoimmune responses in experimental autoimmune myasthenia gravis (EAMG), allergic neuritis (EAN) and allergic encephalomyelitis (EAE) has been the stimulus of many research efforts. Studies on the idiotypic / anti-idiotypic network of anti-La / SSB positive sera from patients with Sjogren's Syndrome (SS) and Systemic Lupus Erythematosus (SLE) and on animals immunized with the complementary epitopes are presented.

The phenomenon of “difficult sequence” has long frustrated chemists in their efforts to assemble peptides that contain such sequences by solid phase synthesis methods. A variety of remedial measures are available to minimize or even abolish the negative impact of these sequences during synthesis. These include the use of elevated temperatures and stronger acylating reagents. Amyloid-β, a fragment of the amyloid precursor protein, contains 40-43 residues and possesses a C-terminal sequence that is particularly resistant to ready solid phase synthesis making it a “difficult sequence” peptide. This review focuses on approaches to successfully assemble the peptide by both Boc- and Fmoc solid phase synthesis.

MALDI-TOF MS and N-terminal amino acid sequencing allowed us to identify several fragments of the C-terminal peptide of Influenza A hemagglutinin (HA) containing transmembrane domains (TMD). These fragments were detected in the organic phase of chloroform-methanol extracts from bromelain-treated virus particles. Heterogeneous fatty acylation of the C-terminus was revealed. Tritium bombardment technique might open an opportunity for 3D structural investigation of the HA TMD in situ.

Two different natural protease inhibitors, the squash inhibitor MCEI III and the third domain of turkey ovomucoid inhibitor OMTKY3, were crystallized in complexes with porcine pancreatic elastase (PPE). About 700 conditions were screened altogether. Crystals of the complex between MCEI III and PPE were grown in citrate buffer with and without ammonium acetate. X-ray diffraction data were collected to 1.9 Å resolution at room temperature using synchrotron radiation. The crystals belong to space group P21, with unit-cell parameters a = 49.17, β = 44.59, c = 67.08 Å, β = 110.97° . Crystals of the OMTKY3 / PPE complex were obtained in the presence of ammonium sulfate, MES buffer and polyethylene glycol monomethylether (PEG). These crystals of this complex diffracted to 2.1 Å resolution and belongs to space group I222, with unit-cell parameters a = 84.58, b = 84.61, c = 89.92 Å and diffracted to 2.2 Å resolution. The diffraction data were collected using a conventional rotating anode X-ray generator at room temperature. In both cases the presence of inhibitor in the crystals was confirmed by crystallography.

We report here on crystallization and preliminary X-ray analysis of plant class I chitinase from rice (OsChia1b). Similar single crystals of full-length OsChia1b were obtained under two independent conditions. The crystals grown under these conditions diffracted up to 2.1 and 2.5 Å resolution, respectively, at a synchrotron beamline, and were found to belong to the tetragonal space group P43212.