Imed Hasni

Milk β-lactoglobulin complexes with tea polyphenols.

The effect of milk on the antioxidant capacity of tea polyphenols is not fully understood. The complexation of tea polyphenols with milk proteins can alter the antioxidant activity of tea compounds and the protein secondary structure. This study was designed to examine the interaction of β-lactogolobulin (β-LG) with tea polyphenols (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG) at molecular level, using FTIR, CD and fluorescence spectroscopic methods as well as molecular modelling. The polyphenol binding mode, the binding constant and the effects of polyphenol complexation on β-LG stability and secondary structure were determined. Structural analysis showed that polyphenols bind β-LG via both hydrophilic and hydrophobic interactions with overall binding constants of KC-β-LG=2.2 (±0.8)×10(3)M(-1), KEC-β-LG=3.2 (±1)×10(3)M(-1), KECG-β-LG=1.1 (±0.6)×10(4)M(-1) and KEGCG-β-LG=1.3 (±0.8)×10(4)M(-1). The number of polyphenols bound per protein molecule (n) was 1.1 (C), 0.9 (EC), 0.9 (ECG) and 1.3 (EGCG). Molecular modelling showed the participation of several amino acid residues in polyphenol-protein complexation with extended H-bonding network. The β-LG conformation was altered in the presence of polyphenols with an increase in β-sheet and α-helix suggesting protein structural stabilisation. These data can be used to explain the mechanism by which the antioxidant activity of tea compounds is affected by the addition of milk.

Folic acid complexes with human and bovine serum albumins

The interaction of folic acid with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various folic acid contents was investigated. FTIR, UV-visible and fluorescence spectroscopic methods as well as molecular modelling were used to analyse folic acid binding sites, the binding constant and the effect on HSA and BSA stability and conformations. Structural analysis showed that folic acid binds HSA and BSA via both hydrophilic and hydrophobic contacts with overall binding constants of Kfolic acid-HSA=8.1 (±0.5)×10(4)M(-1) and Kfolic acid-BSA=1.0 (±0.3)×10(5)M(-1). The number of bound acid molecules per protein was 1.7 (±0.4) for HSA and 1.5 (±0.3) for BSA complexes. Molecular modelling showed participation of several amino acids in folic acid-protein complexes stabilised by hydrogen bonding network. Folic acid complexation altered protein secondary structure by major reduction of α-helix from 59% (free HSA) to 35% (acid-complex) and 62% (free BSA) to 25% (acid-complex) with an increase in random coil, turn and β-sheet structures indicating protein unfolding. The results suggest that serum albumins might act as carrier proteins for folic acid in delivering it to target molecules.

Binding of biogenic and synthetic polyamines to β-lactoglobulin.

The bindings of biogenic polyamines spermine (spm), spermidine (spmd) and synthetic polyamines 3,7,11,15-tetrazaheptadecane·4HCl (BE-333) and 3,7,11,15,19-pentazahenicosane·5HCl (BE-3333) with β-lactoglobulin (β-LG) were determined in aqueous solution. FTIR, UV-vis, CD and fluorescence spectroscopic methods as well as molecular modeling were used to determine the polyamine binding sites and the effect of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind β-LG via both hydrophilic and hydrophobic contacts. Stronger polyamine-protein complexes formed with synthetic polyamines than biogenic polyamines, with overall binding constants of K(spm-β-LG)=3.2(±0.6)×10(4) M(-1), K(spmd-β-LG)=1.8(±0.5)×10(4) M(-1), K(BE-333-β-LG)=5.8(±0.3)×10(4) M(-1) and K(BE-3333-β-LG)=6.2(±0.05)×10(4) M(-1). Molecular modeling showed the participation of several amino acids in the polyamine complexes with the following order of polyamine-protein binding affinity: BE-3333>BE-333>spermine>spermidine, which correlates with their positively charged amino group content. Alteration of protein conformation was observed with a reduction of β-sheet from 57% (free protein) to 55-51%, and a major increase of turn structure from 13% (free protein) to ∼21% in the polyamine-β-LG complexes, indicating a partial protein unfolding.

Binding of cationic lipids to milk β-lactoglobulin.

We determined the bindings of several lipids such as cholesterol (CHOL), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethyl-ammoniumbromide (DDAB), and dioleoylphosphatidylethanolamine (DOPE) to β-lactoglobulin (β-LG) at physiological conditions. FTIR, CD, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of lipid-protein complexes. Structural analysis showed that lipids bind β-LG via both hydrophilic and hydrophobic interactions with overall binding constants of K(CHOL-β-LG) = 6.0 (±0.6) × 10(3) M(-1), K(DOPE-β-LG) = 6.5 (±0.7) × 10(3) M(-1), K(DDAB-β-LG) = 1.6 (±0.3) × 10(4) M(-1), and K(DOTAP-β-LG) = 2.2 (±0.67) × 10(4) M(-1). The number of lipid bound per protein molecule (n) was 0.8 (CHOL), 0.7 (DOPE), 1.0 (DDAB), and 1.3 (DOTAP). Molecular modeling showed the participation of several amino acid residues in lipid-protein complexation with the order of binding DOTAP > DDAB > DOPE > CHOL. Alterations of the protein conformation were observed in the presence of lipids with a minor decrease in β-sheet and an increase in turn structure.

Destabilization of the Oxygen Evolving Complex of Photosystem II by Al3

The inhibitory effect of Al3+ on photosynthetic electron transport was investigated in isolated thylakoid membranes of spinach. A combination of oxygen evolution, chlorophyll fluorescence induction (FI) and decay and thermoluminescence measurements have been used to characterize photosystem II (PSII) electron transport in the presence of this toxic metal cation. Our results show that below 3 mm, Al3+ already caused a destabilization of the Mn4O5Ca cluster of the oxygen evolving complex (OEC). At these concentrations, an increase in the relative amplitude of the first phase (OJ) of FI curve and retardation of the fluorescence decay kinetics following excitation with a single turnover flash were also observed. A transmembrane structural modification of PSII polypeptides due to the interaction of Al3+ at the OEC is proposed to retard electron transfer between the quinones QA and QB. Above 3 mm, Al3+ strongly retarded fluorescence induction and significantly reduced Fv/Fm together with the maximal amplitude of chlorophyll fluorescence induced by a single turnover flash. This chlorophyll fluorescence quenching was attributed to the formation of P680+ due to inhibition of electron transfer between tyrosine 161 of D1 subunit and P680.

Effect of biogenic polyamine spermine on the structure and function of photosystem I.

We located the binding sites of spermine (Spm) to PSI sub-membrane proteins and the impact of this interaction on the photoprotection of PSI activity, using spectroscopic methods and molecular modeling. Our results showed that at high Spm content the polyamine binds PSI polypeptides through H-bonding and induces major protein conformational changes with the reduction of α-helix from 52% to 42% and an increase of the β-sheet from 26% to 29%. However, polyamine does not affect significantly the photooxidizable P700 in control sample and considerably protects it against strong illumination. On the contrary, protein conformational changes coincide with an important inhibition of O2 uptake rates by polyamine, which revealed that the protein of the PSI donor side plastocyanin is a main target for Spm inhibition. The photoprotection of PSI photochemical activity may be due to the stabilization of the PSI stromal polypeptides by Spm as shown by the docking results. Spm binds to different amino acids with hydrophilic and hydrophobic characters, while the presence of several H-bondings stabilizes Spm-PSI complexation.

Characterization of the structural changes and photochemical activity of photosystem I under Al3+ effect

The photochemical activity of photosystem I (PSI) as affected by Al(3+) was investigated in thylakoid membranes and PSI submembrane fractions isolated from spinach. Biophysical and biochemical techniques such as oxygen uptake, light induced absorbance changes at 820nm, chlorophyll fluorescence emission, SDS-polyacrylamide gel electrophoresis, and FTIR spectroscopy have been used to analyze the sites and action modes of this cation on the PSI complex. Our results showed that Al(3+) above 3mM induces changes in the redox state of P700 reflected by an increase of P700 photooxidation phase and a delay of the slower rate of P700 re-reduction which reveals that Al(3+) exerted an inhibitory action at the donor side of PSI especially at plastocyanin (PC). Furthermore, results of P700 photooxidation monitored in the presence of DCMU with or without MV suggested that the same range of Al(3+) concentrations impairs the photochemical reaction centers (RC) of PSI, as shown by the decline in the amount of active population of P700, and disrupts the charge separation between P700 and the primary electron acceptor A0 leading to the inhibition of electron transfer at the acceptor side of PSI. These inhibitory actions were also accompanied by an impairment of the energy transfer from light harvesting complex (LHCI) to RC of PSI, following the disconnection of LHCI antenna as illustrated by an enhancement of chlorophyll fluorescence emission spectra at low temperature (77K). The above results coincided with FTIR measurements that indicated a conformational change of the protein secondary structures in PSI complex where 25% of α-helix was converted into β-sheet, β-antiparallel and turn structures. These structural changes in PSI complex proteins are closely related with the alteration photochemical activity of PSI including the inhibition of the electron transport through both acceptor and donor sides of PSI.