[Analysis of the effects of pH and salt on the conformation of the sericin particles by DLS and TEM measurements].

The particles conformation of the sericin protein extracted from silkworm Bombyx mori was studied under the conditions of different pH and salt concentrations by infrared spectroscopy (IR), dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. The IR spectrum of sericin protein arises predominantly from C=O stretching vibration around the amide I region of 1 700-1 600 cm(-1). A strong trend of aggregation of the protein could be observed under specified experimental conditions. The apparent isoelectric point of the sericin protein was about 3.7. The DLS method was used to investigate the effects of pH and NaCl on the size distribution, where a large polydispersity of the system could be observed. Compared to pH 4 or high NaCl concentration, at pH 3, 8 or low NaCl concentration the sericin aggregation shows a relatively smaller size but larger polydispersity. TEM was used to investigate the microstructure of the aggregated sericin protein, where a loose and pine-like branched form could be observed at pH 3 or 8; however, a relatively compact structure was observed near pH 4 or at high salt concentration At pH 4 the spherical monomer size can be calculated at around (60 +/- 6) nm (n = 10) by TEM measurement. These phenomena could be explained by the effects of the electrostatic repulsion, hydrogen bonding and Van der Waals attractive force, which provide a basic theory for the application of sericin as biomaterial.

Structural requirements of Angiotensin I-converting enzyme inhibitory peptides: quantitative structure-activity relationship study of di- and tripeptides.

A database consisting of 168 dipeptides and 140 tripeptides was constructed from published literature to study the quantitative structure--activity relationships of angiotensin I-converting enzyme (ACE) inhibitory peptides. Two models were computed using partial least squares regression based on the three z-scores of 20 coded amino acids and further validated by cross-validation and permutation tests. The two-component model could explain 73.2% of the Y-variance (inhibitor concentration that reduced enzyme activity by 50%, IC50) with the predictive ability of 71.1% for dipeptides, while the single-component model could explain 47.1% of the Y-variance with the predictive ability of 43.3% for tripeptides. Amino acid residues with bulky side chains as well as hydrophobic side chains were preferred for dipeptides. For tripeptides, the most favorable residues for the carboxyl terminus were aromatic amino acids, while positively charged amino acids were preferred for the middle position, and hydrophobic amino acids were preferred for the amino terminus. According to the models, the IC50 values of seven new peptides with matchable primary sequences within pea protein, bovine milk protein, and soybean were predicted. The predicted peptides were synthesized, and their IC50 values were validated through laboratory determination of inhibition of ACE activity.

Pattern similarity study of functional sites in protein sequences: lysozymes and cystatins.

BACKGROUND: Although it is generally agreed that topography is more conserved than sequences, proteins sharing the same fold can have different functions, while there are protein families with low sequence similarity. An alternative method for profile analysis of characteristic conserved positions of the motifs within the 3D structures may be needed for functional annotation of protein sequences. Using the approach of quantitative structure-activity relationships (QSAR), we have proposed a new algorithm for postulating functional mechanisms on the basis of pattern similarity and average of property values of side-chains in segments within sequences. This approach was used to search for functional sites of proteins belonging to the lysozyme and cystatin families. RESULTS: Hydrophobicity and beta-turn propensity of reference segments with 3-7 residues were used for the homology similarity search (HSS) for active sites. Hydrogen bonding was used as the side-chain property for searching the binding sites of lysozymes. The profiles of similarity constants and average values of these parameters as functions of their positions in the sequences could identify both active and substrate binding sites of the lysozyme of Streptomyces coelicolor, which has been reported as a new fold enzyme (Cellosyl). The same approach was successfully applied to cystatins, especially for postulating the mechanisms of amyloidosis of human cystatin C as well as human lysozyme. CONCLUSION: Pattern similarity and average index values of structure-related properties of side chains in short segments of three residues or longer were, for the first time, successfully applied for predicting functional sites in sequences. This new approach may be applicable to studying functional sites in un-annotated proteins, for which complete 3D structures are not yet available.

Enhanced anti-rotavirus action of human cystatin C by site-specific glycosylation in yeast.

The cDNA encoding human cystatin C (HCC) was subjected to site-specific substitution of alanine for serine at the position 37, to obtain the Asn(35)-Lys(36)-Ser(37) sequence that is a signal for asparagine-linked (N-linked) glycosylation of protein in eukaryotes, and was transformed into Pichia pastoris X33. As a result, 1.2 mg/L oligomannosyl HCC with a carbohydrate chain of Man(10)GlcNAc(2) was produced by the Pichia transformant. The oligomannosyl HCC was more stable at the low ionic strength condition of 50 mM potassium phosphate buffer, pH 7.0, than the wild-type. In addition, the oligomannosylation substantially improved the molecular stability of cystatin against an aspartic proteinase, cathepsin D, in which the susceptibility decreased to less than 50% of nonglycosylated one. The anti-rotavirus activity of HCC was substantially enhanced by the site-directed glycosylation using the yeast expression system. A MA-104 cell line was used as a host cell for human rotavirus type-2 Wa strain in this study, to which both the wild-type and oligomannosyl HCCs did not show cytotoxicity at a concentration of 100 mug/mL. More than 80% viability of the host cell infected with 1.0 x 10(5) PFU/mL of rotavirus was conserved under the condition coexisting with 75 mug/mL of the oligomannosyl HCC, which was 15.2% higher than that of wild-type HCC. Thus, the in vitro anti-rotavirus assay indicated that the supplement of a proper amount of the oligomannosyl HCC could be used as an anti-rotavirus agent.

FT-Raman spectroscopy, fluorescent probe, and solvent accessibility study of egg and milk proteins.

Due to possible contribution of both electrostatic and hydrophobic interactions, use of anionic fluorescent probes such as 1-anilinonaphthalene-8-sulfonic acid (ANS) and cis-parinaric acid (CPA) for the measurement of protein surface hydrophobicity (S0) has been controversial. A neutral probe, 6-propionyl-2-(dimethylamino)-naphthalene (PRODAN), may circumvent this problem. To select the best indicator of S0, in this study, the data for nine model proteins in phosphate buffer, pH 7.5, measured using the above-mentioned probes, was compared to their FT-Raman spectra and calculated solvent accessibility values. Log S0 measured using CPA had the highest correlation (r = 0.874) with the intensities of Raman spectral signals at 760 cm(-1) and 2800-3100 cm(-1), which were combined using a mixture design based on the random-centroid optimization. The order of correlation of Raman spectral parameters with S0 values were CPA > PRODAN > ANS. FT-Raman spectroscopy, therefore, identified CPA, followed by PRODAN, as the fluorescent probe of choice for describing surface hydrophobicity. However, the amino acid surface accessibility calculated using the PredictProtein software was not useful in identifying the best fluorescent probe for the measurement of S0.

Pattern similarity analysis of amino acid sequences for peptide emulsification.

A new computer program for homology similarity search (HSS) was introduced. Application of the HSS to peptide sequences of short peptides with fewer 32 amino acid residues has explained the underlying mechanism of their emulsifying ability. It was found that certain regularity in the frequency of alternate polar/apolar cycle with high hydrophobic similarity density was required to obtain good emulsion. To supplement this required regularity, charge distribution, molecular flexibility, and a structural torsion caused by a proline residue might also play roles.

Homology similarity analysis of sequences of lactoferricin and its derivatives.

A new method, homology similarity analysis (HSA), was developed to investigate homology pattern similarities of selected segments within sequences of peptides. This new approach facilitated elucidation of the structure-function relationships of lactoferricin derivatives. Helix propensity of positions 4-9 in the lactoferricin sequence was the most important in determining the antimicrobial activity of lactoferricin against Escherichia coli, followed by cationic charge pattern at positions 4-9 and 1-3. The pattern similarity of segments within sequences could be a useful tool for representing the distribution attributes of amino acid residue properties to the structure-function relationships of proteins and peptides, especially when used in conjunction with principal component similarity analysis followed by the regression version of artificial neural networks.

In vitro anti-cancer activities in Caco-2 and HCT-116 cells of recombinant cystatin C prepared by a Pichia expression system.

Caco-2 and HCT-116 cells were used to access growth-inhibition and anti-invasion activity of recombinant cystatin C expressed in Pichia pastoris X33, G12W/H86V. The mutant G12W/H86V prepared by a pilot plant production system showed more than 10% growth inhibition of Caco-2 cells at 0.56-56 nM concentrations. Growth-inhibited cells had lower cathepsin L activity than the control cells that were not treated with the inhibitor. Conversely, the cathepsin B activity was not changed by treatment with G12W/H86V. The in vitro anti-invasion test using HCT-116 cells showed that G12W/H86V suppressed the cell invasion by 15%, while its wild-type cystatin, aspartic protease inhibitor pepstatin A, and matrix metalloproteinase (MMP) inhibitor MMP-2/MMP-9 inhibitor III did not suppress cell invasion. These results indicate that the recombinant cystatin C with higher protease inhibitory activity effectively retards the growth and invasiveness of human colon carcinoma cells.

Enhancement of proteinase inhibitory activity of recombinant human cystatin C using random-centroid optimization.

We had previously written a random-centroid optimization computer program for genetics (RCG) to optimize protein engineering, which was successfully applied to modify single site of the 16 amino acid residues at the active site of B. stearothermophilys neutral protease for improving thermostability [J. Agric. Food Chem., 46 (1998) 1655]. The same program was applied in this study to double-site mutation of the entire sequence of human cystatin C (HCC) with 120 residues for improving its protease inhibitory activity. The RCG program selected two sites simultaneously and amino acid residues to replace the sites selected in the sequence in order to find the best papain-inhibitory activity and stability of the protease inhibitor. Twenty-three double mutants and twenty-two single mutants were expressed by Pichia pastoris. Of the total 45 mutants, G12W/H86V mutant showed a 5-fold increase in the bioactivity over the recombinant wild-type (WT) cystatin. Also, P13F mutant exhibited a half-life temperature (T1/2) 5.2 degrees C higher than 68.2 degrees C of WT in addition to a 56% greater papain inhibitory activity. Mutation for diminishing beta-sheet content reduced polymerization of cystatin C, thus improving papain-inhibitory activity. The approach using RCG was able to improve the functional properties of cystatin by least relying on the prior knowledge of its molecular structure.

Principal component similarity analysis of Raman spectra to study the effects of pH, heating, and kappa-carrageenan on whey protein structure.

Raman spectroscopy was used to elucidate structural changes of beta-lactoglobulin (BLG), whey protein isolate (WPI), and bovine serum albumin (BSA), at 15% concentration, as a function of pH (5.0, 7.0, and 9.0), heating (80 degrees C, 30 min), and presence of 0.24% kappa-carrageenan. Three data-processing techniques were used to assist in identifying significant changes in Raman spectral data. Analysis of variance showed that of 12 characteristics examined in the Raman spectra, only a few were significantly affected by pH, heating, kappa-carrageenan, and their interactions. These included amide I (1658 cm(-1)) for WPI and BLG, alpha-helix for BLG and BSA, beta-sheet for BSA, CH stretching (2880 cm(-1)) for BLG and BSA, and CH stretching (2930 cm(-1)) for BSA. Principal component analysis reduced dimensionality of the characteristics. Heating and its interaction with kappa-carrageenan were identified as the most influential in overall structure of the whey proteins, using principal component similarity analysis.

Stabilization of oil-in-water emulsions by beta-lactoglobulin-polyethylene glycol conjugates.

The disulfide bonds of beta-lactoglobulin (beta-lg) were modified by oxidative sulfitolysis to generate beta-lgSO(3). The native protein (beta-lg) and the modified protein (beta-lgSO(3)) were conjugated to activated polyethylene glycol (PEG) to generate beta-lgPEG and beta-lgSO(3)PEG, respectively. Oil-in-water (o/w) emulsions containing 1% beta-lg or beta-lg conjugates were prepared at pH 2.8, 5.0, and 7.0. Emulsion droplet diameters and zeta potentials were measured. For the same emulsifier, emulsion droplet diameters decreased when emulsion pH increased. Zeta potentials of emulsion droplets increased with pH for beta-lg and beta-lgSO(3). Zeta potentials of beta-lgPEG and beta-lgSO(3)PEG approached zero, suggesting that the protein molecule was covered by PEG chains. Accelerated and 7-day storage stabilities at 21 degrees C of the emulsions were monitored. The emulsifying activity index (EAI) of beta-lgPEG was not significantly different from the EAI of beta-lg. The EAI of beta-lg was enhanced following sulfitolysis of beta-lactoglobulin. The emulsifying activity increased more when the oxidatively modified protein was conjugated to polyethylene glycol. Emulsions made with beta-lgSO(3)PEG were more stable than emulsions made with beta-lg, beta-lgPEG, or beta-lgSO(3) under accelerated stability study and for 7 days at 21 degrees C. The stability of o/w emulsions stabilized with beta-lgSO(3)PEG increased because individual droplets were better protected, against protein bridging or coalescence, by the thick adsorbed protein-PEG layer.