Protein and Peptide Letters - Volume 11, Issue 1, 2004

Phaseolus vulgaris leucoagglutinin is a homotetrameric legume lectin possessing the canonical dimeric structure common to other legume lectins. In order to gain insight into the stability of the protein in an acidic environment, it was characterized by CD and fluorescence studies at pH 2.5. This was then compared with the native protein at physiological pH (7.2). The extinction coefficient of the native protein was calculated to be 3.58x104 from its UV absorption spectra. The far- and near-UV CD spectra of the protein at pH 2.5 showed very little difference even though the protein was found to exist as a dimer at pH 2.5. The fluorescence emission maxima of the protein upon excitation at 280 nm were found to shift only from 331 nm at pH 7.2 to 333 nm at pH 2.5. Based on the above observation it was concluded that the protein exhibits extreme pH stability especially in the acidic range. The secondary and tertiary structure of the protein is lost only when it is incubated for two days in 6 M GdnHCl at pH 2.5. At pH 7.2 it could be denatured in 6 M GdnHCl after one week of incubation.

The N-terminal domain of the GLP-1 receptor binds the putative helical region of the peptide agonists, GLP-1 and exendin-4. Here we demonstrate that this interaction also determines the magnitude of a separate interaction between the N-terminus of these peptides and the receptor's core domain. Enhancing the pre-formation of the C-terminal Trp-Cage motif of exendin-4, a motif critical for high-affinity binding, results in no improvement in receptor affinity, suggesting that this motif forms after the initial peptidereceptor binding event.

A mutagenesis study to systematically analyse residues spanning the first extracellular loop of the GLP-1 receptor identified a double mutant, Met-204 / Tyr-205-Ala / Ala, which displayed: markedly reduced affinity for the natural agonist GLP-1; slightly reduced affinity for its analogue exendin-4; and unaltered affinity for several N-terminally truncated analogues of GLP-1 and exendin-4. This suggests that the locus is important for the formation of the binding site for the N-terminal residues of peptide agonists.

The NMR studies of the prionogenic peptide derived from Sup35 are presented. The peptide molecules were dissolved in the half-aqueous solution to prevent severe aggregation, and were found to be in an extended conformation from the chemical shift and the coupling constant data. They could form higher order multimers by making intermolecular hydrogen bonds, judging from the observation that the NMR sample became a gel-like state at lower temperatures. This work reports the first structural information in the solution state about the prionogenic peptide mimicking the state of amyloid fibrils, and provides a solid foundation for further structural analysis of peptide molecules forming insoluble aggregates.

It has been recently shown that insulin retains its biological activity after receptor-directed internalization and it may affect the cell metabolism by interaction with cytosolic insulin-binding proteins (CIBPs). Using affinity chromatography combined with SDS-PAGE and MALDI-TOF mass-spectrometry we have identified 7 proteins from mouse liver cells that specifically bind to the insulin, including adenylate kinase 2 (25.6 kD), kinesin superfamily protein 20B (26.0 kD), hepatic arginase 1 (34.8 kD), fructosebisphosphate aldolase B (39.5 kD), 4-hydroxyphenylpyruvate dioxygenase (45.1 kD), betainehomocysteine methyl-transferase (45.0 kD) and KRIT1 (83.4 kD).

Pleurocidin (Ple), a 25-residue α-helical antimicrobial peptide, isolated from skin mucosa of the winter flounder, shows potent bacterial cell selectivity. In this study, the effect of two glycine residues in positions 13 and 17 of Ple on structure and bacterial cell selectivity was investigated by Gly®Ala substitution. Ala-substitution (Gly13, 17→Ala, Gly13→ Ala and Gly17→ Ala) in positions 13 and 17 of Ple did not induce a significant change in antibacterial activity, but increased hemolytic activity. Both Gly13→ Ala and Gly17→ Ala substitution did not cause a remarkable change in α-helical content in SDS micelles, while Gly13,17→Ala substitution caused a drastic increase in α-helical content. These results suggest that the hinge region from Gly13 to Gly17 of Ple is assumed to provide its conformational flexibility and bacterial cell selectivity.

The interactions between an oligomeric heat-shock protein, α-crystallin, and its individual subunits with unfolded proteins were monitored by surface plasmon resonance. Immobilization at the sensor chip allowed us for the first time to study isolated α-crystallin subunits under physiological conditions. We observe that these subunits, in contrast to α-crystallin oligomers, do not bind unfolded protein. Our data indicate that quaternary structure of α-crystallin is necessary for its chaperone-like activity.

A fusion protein (scu-PA:AV) was expressed in the baculovirus expression system and secreted from Sf9 cells lead by signal peptides, 2mel and 3egt. The scu-PA:AV displays both the urokinase activity and membrane binding activity of its parental components. Our work indicated that it is possible to be developed as a thrombus-targeting drug.

A method for seed proteome analysis using MALDI-TOF mass spectrometry is described. The data were used to estimate the genetic diversity degree among twelve genotypes of pepper (Capsicum). The resulting spectra were converted into a binary matrix consisting of 23 protein data sets, and genetic similarity values were calculated with the FreeTree software and Jaccard's coefficient of similarity. We have also been able to identify the presence of certain proteins in the extracts, by checking their masses on online databases.

we constructed a mouse PC6B truncated mutant and introduced a tag of 6 consecutive histidines at its carboxyl terminus for simple purification. Using the baculovirus expression system and standard enzymatic assay, we obtained recombinant mouse PC6B protein and with enzymatic activity.

In the sequence analyses of peptides from nucleoprotein in influenza virus, it was very difficult to obtain the sufficient numbers of fragment ions using the post-source decay (PSD) method in MALDITOF- MS. Fluorescent modification of the N-terminal amino group in the target peptides was introduced for the PSD measurement. The fluorescently-labelled peptides gave sufficient fragment ions in the PSD spectrum, which leads to the complete sequence analysis of peptide.

Crystals of a new inhibitor present in rye seeds active against alpha-amylases from crop pests Acanthoscelides obtectus and Zabrotes subfasciatus have been obtained. A native dataset was collected at 2.21 Å resolution with 99.3 % completeness at CPr beamline at LNLS. The crystals belong to the trigonal system, space group P3121 with a = b = 78.21 Å, and c = 59.61 Å. The crystal calculated solvent content is compatible with one dimer per asymmetric unit.

Insect lysozyme from Manduca sexta (MS-lys) was overexpressed in E. coli and refolded to obtain active protein. Recombinant MS-lys presented a globular structure, with an alpha-helical content of 57% as assessed by circular dichroism spectroscopy. Light scattering studies showed that in solution MSlys has a quasi-monodisperse size distribution, with a rod-like structure similar to nucleation clusters reported in egg lysozyme pre-crystallization stages. These results show that MS-lys is an excellent candidate for crystallization, folding and denaturation studies.

Hexadecaheme high molecular weight cytochrome c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and crystallized. X-ray diffraction data have been collected by the multiple wavelength anomalous dispersion method. The crystal belongs to the space group P212121 with unit-cell parameters a = 60.42, b = 84.29 and c = 144.16 Å and contains one molecule per asymmetric unit.