Masters thesis of science a molecular simulation study of thermal and ph effects on apo lactoferrin stability implications for potential encapsulation function of gram positive bacteria

A Molecular Simulation Study of Thermal and pH effects on apo-Lactoferrin Stability: Implications for Potential Encapsulation Function of Gram-positive Bacteria A thesis submitted in fulfilment of the requirements for the degree of Master of Science Carol Nhan B Sci (App Sci) (Hons) RMIT University School of Science College of Science, Engineering and Health RMIT University April 2018 Declaration I certify that except where due acknowledgement has been made, the work is that of the author alone; the work has not been submitted previously, in whole or in part, to qualify for any other academic award; the content of the thesis is the result of work which has been carried out since the official commencement date of the approved research program; any editorial work, paid or unpaid, carried out by a third party is acknowledged; and, ethics procedures and guidelines have been followed I acknowledge the support I have received for my research through the provision of an Australian Government Research Training Program Scholarship Carol Nhan 24 April 2018 Acknowledgments As I begin to finish up my Masters by Research journey I’d like to firstly show acknowledgement and appreciation to my two supervisors, Dr Bee May and Dr Andrew Hung for their invaluable and kind advice, support, guidance and encouragement throughout my research years as I completed my honours’ and now Master’s thesis, without them, these two theses would not be possible This project was completed at RMIT University, City Campus I would like to thank the HDR School of Science for their fast replies, availability and friendliness to assist me during my postgrad candidature, especially with the travel grant procedure to Japan to present my honours thesis in my first symposium on “Modelling of pH effects on milk proteins and implications for binding to bacterial surface peptidoglycan” in 2016 and later, travel funds associated to Sydney, to present a poster based on my first result chapter of this thesis.at the Australian Institute of Food Science and Technology Convention in 2017, it was a very smooth journey Furthermore, the computational facilities utilised in this thesis was undertaken with the assistance of resources and services from the National Computational Infrastructure (NCI) and the Pawsey Supercomputing Centre, which are supported by the Australian Government I would also like to thank Associate Professor Colin Rix for his professional advice for my first result chapter and Associate Professor Darryl Small for his kind offer to view my second result chapter Others include postgraduate friends for offering their help and suggestions, my best friends for sticking with me and friends that I made have given support and encouragement during my research candidature Lastly to end, to thank my parents, these past 3+ years have not been easy and was difficult for them to understand research study and what it entails, for their support and understandings Publications and Presentations Journal publications Nhan C, Rix C, May BK, Hung A Temperature-induced structural changes of apolactoferrin and their functional implications: a molecular dynamics simulation study (Manuscript under revision, Molecular Simulation, April 2018) Nhan C, Small DM, May BK, Hung A pH-induced structural changes of apolactoferrin and implications for its function: a molecular dynamics simulation study Molecular Simulation 2018 DOI: 10.1080/08927022.2018.1535178 Other conference presentations Nhan C, May BK, Hung, A High-Temperature Molecular Simulations of Lactoferrin: Implications for Bacterial Attachment Poster session presented at: RMIT Research Day; 2017 Jul 10; Melbourne, AU Nhan C, May BK, Hung, A High-Temperature Molecular Simulations of Lactoferrin: Implications for Bacterial Attachment Poster session presented at: Australian Institute of Food Science & Technology (AIFST) 50th Anniversary Convention; 2017 Jul 17-18; Sydney, AU Thesis with Publications Declaration Chapter Declaration for Thesis with Publication School of Science Chapter Declaration for Thesis with Publications Chapter is represented by the following paper: Temperature-Induced Structural Changes of apo-Lactoferrin and Their Functional Implications: A Molecular Dynamics Simulation Study, Carol Nhan, Colin J Rix, Bee K May and Andrew Hung, Molecular Simulation (under review, April 2018) Declaration by candidate I declare that I wrote the initial draft of this manuscript, and my overall contribution to this paper is detailed below: Nature of Contribution Extent of Contribution (%) Performed all simulations unless duly acknowledged, analysed all of the data and prepared the graphs and tables, interpreted the results, prepared the manuscript draft, revised the manuscript 70 The following co-authors contributed to the work The undersigned declare that the contributions of the candidate and co-authors are correctly attributed below Author Nature of Contribution Colin J Rix Edited the first and final draft, providing advice on written English and scientific advice Supervised the project, edited the first and final draft and approved the manuscript for submission, edited and approved the revised manuscript Supervised the project, edited the first and final draft and approved the manuscript for submission, edited and approved the revised manuscript and submitted to the journal Bee K May Andrew Hung Extent of Contribution (%) 12 12 Candidate’s Signature 23 April 2018 Senior Supervisor’s Signature 23 April 2018 School of Science Chapter Declaration for Thesis with Publications Chapter is represented by the following paper: pH-Induced Structural Changes of apo-Lactoferrin and Implications for Its Function: A Molecular Dynamics Simulation Study, Carol Nhan, Darryl M Small, Bee K May and Andrew Hung, Molecular Simulation (under review, April 2018, now accepted October 2018) Declaration by candidate I declare that I wrote the initial draft of this manuscript, and my overall contribution to this paper is detailed below: Nature of Contribution Extent of Contribution (%) Performed all simulations unless duly acknowledged, analysed all of the data and prepared the graphs and tables, interpreted the results, prepared the manuscript draft, revised the manuscript 70 The following co-authors contributed to the work The undersigned declare that the contributions of the candidate and co-authors are correctly attributed below Author Nature of Contribution Extent of Contribution (%) Darryl M Small Edited the final draft, providing advice on written English Bee K May Supervised the project, edited the first and final draft and approved the manuscript for submission, edited and approved the revised manuscript Andrew Hung 14 Supervised the project, edited the first and final draft and approved the manuscript for submission, edited and approved the revised manuscript and submitted to the journal 14 Candidate’s Signature 23 April 2018 Senior Supervisor’s Signature 23 April 2018 Abstract 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 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 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 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 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 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 lowtemperature control simulation 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 apoLf 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 Grampositive bacteria binding peptide is found Overall, the MD simulation studies of apoLf, showed protein structural deviations which might have implications for the temperature- and pH-dependent properties of the bacterial cell binding regions identified in apo-Lf 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 10 structure were found as demonstrated by a substantial decrease in protein dimensions due to collapse in the inter-lobe region There was also a marked increase in residue fluctuations in several regions of known functional importance, including antibacterial, iron-binding, and putative membrane binding regions, the latter is stabilised by a triplet hydrophobic residues which comprised of Leu446, Trp448 and Leu451 at low temperature, but which are disrupted under UHT conditions A unique network analysis based on examining gains in inter-residue motional correlation at elevated temperature, relative to the baseline simulations at 300 K, provides further insights on the effects of heating on the dynamical properties of apo-Lf, and is characterised by the formation of large clusters of residues with increased dynamical correlation in the N-terminal lobe To further elucidate protein stability under extreme conditions that apo-Lf may experience in its product lifetime as a potential component of novel dairy products, from processing through to ingestion/digestion, the effects of extreme pH on this located potential binding region as well as the other key functional properties of lactoferrin were studied This involved simulations under pH 1.0, pH 7.0 and pH 14.0 Based on the same analysis procedures as the temperature analysis methods, the changes in the overall protein fold and several residue-specific measures of structural and dynamical properties with respect to changes in pH were examined and discussed in terms of the implications of these differences on several functionally relevant regions on the protein, including a possible bacterial-binding region which was identified through sequence alignment Examination of the latter region at extremely acidic or basic pH enables prediction of the possible effects of different solvent environments on the bacterial-binding capacity of the protein, and its potential to act as a bacterial encapsulator In particular, simulations at acidic pH potentially enable prediction of the structural viability of apo-lactoferrin to persist in its bacterial encapsulation role within the gastric environment of the stomach after human consumption [11, 17, 104] Overall, it was found that extreme pH conditions alter protein structure and dynamics compared to neutral pH, and that basic pH condition induce far greater structural disturbance overall Increase in residue fluctuations and disrupted contacts at the CLobe of apo-Lf were more prominent at basic pH, while acidic pH was found to have the greatest influence on the N-Lobe (where the antibacterial peptides are located) with 148 increased backbone fluctuations and disruptions of inter-residue contacts as well as greater solvent exposure of part of the iron-binding site 6.2 Major Conclusions Based on the studies conducted in this research project, the following final conclusions were drawn: Lactoferrin is a heavily researched multifunctional protein from bovine milk Much of the current literature describe its properties at an experimental, phenomenological level The research reported in this thesis is the first to further understanding characteristics of iron binding, antibacterial, protein denaturation and bacterial binding peptide stability of apo-Lf under thermal and pH conditions using all-atom computational simulations Below 90 °C, the simulations indicate relatively minor changes in overall protein structure relative to a low temperature (27 °C) control simulation At UHT conditions (127 °C) disruptions to protein structure are predicted to occur at a number of levels such as: reduction in separation between the N- and Cterminal lobes, α-helical content was reduced, disturbance to the antibacterial and iron-binding regions, as well as a C-terminal area predicted to play a role in bacterial membrane surface binding The triplets of hydrophobic residues comprised of Leu446, Trp448 and Leu451 is proposed to stabilize the bacterial membrane surface binding region At neutral pH 7.0 the N-terminal lobe surface is predominantly positive, while a mixture of positive and negative charges exists on the C-terminal lobe surface The extreme pH conditions predicted lobe-specific disturbance; with acidic pH affecting the N-Lobe more than the high pH, while at high pH, the C-lobe was more affected Neutral pH 7.0 resulted in a relatively rigid structure Much more overall structure disturbance occurred at basic pH with relative twist and 149 detachment of the N- and C-lobes, compared to acidic pH Monitoring the triplet hydrophobic residues (Leu446, Trp448 and Leu451), identified them to play a role in stabilizing the proposed bacterial membrane surface binding region, which showed extreme pH conditions disrupts the interactions between this triplet of hydrophobic residues, whereas an acidic environment caused greater effect to the identified bacterial binding region compared to alkaline conditions 6.3 Future Work This study has focused on apo-Lf and its implications to encapsulate Gram-positive bacteria It is recommended that both holo-Lf and native-Lf, are also studied for comparison measures, as they have a greater compact structure compared to apo-Lf In addition, it is recommended that mutations of the identified bacterial binding regions in apo-Lf may provide greater information if the peptides were simulated individually using the same parameters of temperature and pH conditions used in this study, with a special focus on the hydrophobic triads (Leu446, Trp448 and Leu451) Another area is to extend the study with the advancement in computational approach using other software programs to assist in further understanding of how the protein refolds back to its native conformation if it cools to room temperature which is the eventual temperature that the protein will be exposed to as a potential component of food products One further major area of possible improvement in simulation methodology relates to the study of “milder”, but arguably more realistic, pH conditions The current simulations were performed under the assumption that extreme acidic environments cause all titratable side chains to be protonated, while extreme basic environments cause all titratable side chains to be deprotonated While these assumptions may bear some validity for extreme pH conditions, simulations intended to model milder pH environments will require more advanced simulation methods, such as constant pH simulation methods, for which accurate methodologies are still actively being developed and refined Furthermore, the breakdown of covalent bonding in apoLf under extreme pH conditions (which cannot be modelled exclusively using the 150 classical molecular mechanics approach taken in the present work) will require more detailed computational and simulation studies, perhaps combining quantum mechanics and classical mechanical approaches It is the hope of the author that this work described in this thesis provides a strong basis for ongoing research in this important area Furthermore, the present studies may contribute to the use of apo-Lf or lactoferrin for potential encapsulation material of Gram-positive bacteria, better food processing applications and leading to enhancement of their nutritional value 151 References 10 11 12 13 14 15 16 17 18 Lukjancenko O, Ussery DW, Wassenaar TM Comparative Genomics of Bifidobacterium, Lactobacillus and Related Probiotic 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Structural Changes of apo- Lactoferrin and Their Functional Implications: A Molecular Dynamics Simulation Study, Carol Nhan, Colin J Rix, Bee K May and Andrew Hung, Molecular Simulation (under... revision, Molecular Simulation, April 2018) Nhan C, Small DM, May BK, Hung A pH- induced structural changes of apolactoferrin and implications for its function: a molecular dynamics simulation study