A molecular simulation study of thermal and pH effects on apo-lactoferrin stability: implications for potential encapsulation function of gram-positive bacteria
Creators: name: Nhan, Carol
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 300699
subject: Food sciences not elsewhere classified
classificationCode: 340402
subject: Biomolecular modelling and design

description: In this Masters thesis computational modelling techniques were employed to investigate iron-free apo-Lactoferrin (apo-Lf) structural conformation changes in the presence of variant temperature and pH. These conditions represent the environment most milk protein goes through in food processing and the production of food products.<br><br>Lactoferrin (Lf) is an iron-binding glycoprotein present in secretory fluids of nasal, pancreatic, amniotic, seminal plasma, saliva and tears, and in milk secretions such as those from human and bovine sources. It is reported to have multifunctional roles such as antibacterial, antivirus, antifungal, anti-inflammatory and anticancer activities.<br><br>In order to explore apo-Lf’s potential as an encapsulant for probiotics, sequence alignment was employed to identify a region on the C-lobe of Lf capable of binding to bacterial cell surfaces, followed by all-atom explicit solvent molecular dynamics (MD) simulations which were applied to study the conformational changes of apo-Lf after exposure to three processing temperatures: pasteurization (72 °C), spray drying (90 °C) and close to ultra-high temperature (UHT) (135 °C) in a pH 7.0 environment.<br><br>Below 90 °C, the simulations indicate relatively minor changes in overall protein structure, dimensions, per-residue fluctuations and inter-residue contacts and motional correlations, relative to a low temperature (27 °C) control simulation, consistent with experimentally-known conservation of apo-Lf structure and properties at low thermal<br>processing temperatures. At conditions similar to UHT (127 °C), however, marked disruptions to protein structure are predicted to occur at a number of levels. There was a substantial decrease in protein dimensions due to collapse in the inter-lobe region, causing a reduction in separation between the N- and C-terminal lobes. The α-helical content was reduced, although much of the β-pleated sheet structure was retained. There was a marked increase in residue fluctuations in several regions of known functional importance, including the antibacterial and iron-binding regions, as well as a C-terminal area predicted to play a role in bacterial membrane surface binding. It is proposed that this putative membrane binding region was stabilized by a triplet of hydrophobic residues comprised of Leu446, Trp448 and Leu451, and that their mutual interactions are severed at 400 K, resulting in changes to the structure, and potential membrane binding propensity, of this region. Network analysis of disruptions to inter-residue contacts also identified large clusters of residues in the N-terminal lobe which lose contacts with their neighbours. Taken together, UHT conditions are therefore predicted to cause disruptions to multiple functional properties of apo-Lf.<br><br>Furthermore, a unique method was proposed for identifying thermal-induced protein unfolding based on examining the topology of networks of inter-residue motional correlation gain for high-temperature simulation trajectories relative to a low-temperature control simulation.<br><br>To further explore apo-Lf’s potential as an encapsulant for Gram-positive bacteria, MD simulations along with examining topology networks were applied again to study the pH-induced protein unfolding of apo-Lf after the exposure of pH conditions potentially experienced by the protein in the course of its lifetime as a food component product, from processing to consumption. This was achieved by studying the effects of “extreme” acidic (nominally pH 1.0) and basic (nominally pH 14.0) conditions on apo- Lf relative to neutral pH 7.0. These simulations predicted that pH 1.0 conditions affected parts of the N-Lobe, the lobe where the antibacterial peptides are located, while the pH 14.0 conditions affected the C-Lobe, the lobe in which the identified Gram-positive bacteria binding peptide is found. Overall, the MD simulation studies of apo-Lf, showed protein structural deviations which might have implications for the temperature- and pH-dependent properties of the bacterial cell binding regionsidentified in apo-Lf.<br><br>By enabling the thermal and pH sensitivity of several regions of functional importance to be identified, the results of these simulations can be used to further assist in the prediction of conditions suitable for successful protection and encapsulation of lactic acid bacteria using bovine milk protein materials such as lactoferrin. The outcome of this thesis can benefit the functional food and pharmaceutical industry by offering an alternative encapsulation material.<br>