Study of the photophysical properties of oxyluciferin derivatives and their applications to the characterization of interactions between biomolecules Avisek Ghose To cite this version: Avisek Ghose. Study of the photophysical properties of oxyluciferin derivatives and their applica- tions to the characterization of interactions between biomolecules. Biological Physics [physics.bio-ph]. Université de Strasbourg, 2015. English. NNT : 2015STRAJ005. tel-01357655 HAL Id: tel-01357655 https://tel.archives-ouvertes.fr/tel-01357655 Submitted on 30 Aug 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE (ED-414) UMR CNRS 7213, Faculté de Pharmacie, Illkirch THÈSE présentée par : Avisek GHOSE Soutenue le : 17 Février 2015 pour obtenir le grade de : !"#$%&'($')R%+,-$&.,# ' de Strasbourg Discipline/ Spécialité : Biophysique TITRE de la thèse Etude des propriétés photophysiques de dérivés ($')R!01)%",2 rine $#')$%&.'344),"3#,!+.'')R #%($' (R,+#$&3"#,!+.'$+#&$'6,!7!) "%)$. THÈSE dirigée par : M. ARNTZ Youri Maître de Conférences, Université de Strasbourg RAPPORTEURS : Mme. NAVIZET Isabelle Professeur des Universités, Université Paris-Est M. GUSTAVSSON Thomas Directeur de Recherche, CNRS AUTRES MEMBRES DU JURY : M. LEONARD Jérémie Chargé de Recherche, Université de Strasbourg M. DIDIER Pascal Maître de Conférences, Université de Strasbourg UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE (ED-414) UMR CNRS 7213, Faculty of Pharmacy, Illkirch THESIS presented by: Avisek GHOSE on 17 Février 2015 to obtain degree of Doctorate of University of Strasbourg Discipline/ Specialisation : Biophysics Title of the thesis : Study of the photophysical properties of Oxyluciferin derivatives and their applications to the characterization of interactions between biomolecules Thesis Director : Dr. Youri ARNTZ Maître de Conférences, Université de Strasbourg Reporters : Prof. Isabelle NAVIZET Professeur des Universités, Université Paris-Est Dr. Thomas GUSTAVSSON Directeur de Recherche, CNRS Other members of the jury : Dr. Jérémie LEONARD Chargé de Recherche, Université de Strasbourg Dr. Pascal DIDIER Maître de Conférences, Université de Strasbourg Student: Dr. Einstein, aren't these the same questions as last year's [physics] final exam? Dr. Einstein: Yes; but this year the Panswers are different !Q RDr. Albert Einstein @AF7 A8 ):3@= R s my immense pleasure to express thanks to everyone allied with my three and half I years R stay in the Laboratory of Biophotonics and Pharmacology. I am greatly thankful to our beloved director Prof. Yves MELY for providing me an opportunity to pursue my Ph.D under the guidance of my supervisor Dr. Pascal DIDIER. I am deeply thankful to Dr. Guy DUPORTAIL for taking care of me and all my problems, as parent, ahead of my "##$%"&'$(' ) #"*+,-#.'$(' /"(-"#0R112'3'*456$"&'(, 5',7' 8"(9*' ,':#2';,-#$' 3<=>?'7,#' walking in parallel with us on every step of the project. I would like to express my special gratitude to Dr. Pascal DIDIER for supervising my work for the entire tenure and to bear with all discussions (scientific or not) and to answer all questions (sometimes silly) of mine with smile on the corner of his lips, always. His kind nature, intelligent troubleshooting capability and forever availability with an open heart and smile were the key for the successful completion of the work. Thanks to the President of Université de Strasbourg, Director of Ecole Doctorale, Dean of Faculté de Phamacie and all others for their concern. Furthermore, I would like to thank my jury members Prof. Isabelle NAVIZET, Dr. Thomas GUSTAVSSON and Dr. Jérémie LEONARD for accepting my request to evaluate our work. There evaluation will be having an added value to our work. I am thankful to Dr. Julien GODET, Dr. Ludovic RICHERT, Dr. Eleonore REAL, Dr. Nicolas HUMBERT and Dr. Kamal K. SHARMA for their support to our work. Thanks to Dr. Andrey KLYMCHENKO, Dr. Frédéric PRZYBILLA, Dr. Nicolas ANTON (especially for evaluating our work during mid thesis), Dr. Halina ANTON, Dr. Christian BOUDIER, Dr. Hugues de ROCQUIGNY and other members of LBP for their loving care. I appreciate involvement of our collaborators Prof. Lukas HINTERMANN, Dr. @"(65' =3BCDEF' Dr. Michel SLIWA, Dr. Mateusz REBARTZ, Dr. Oleg V. MALTSEV, Dr. Jun-ichi HOTTA for their contribution to the pleasant journey of the project. I have my thanks to Marlyse WERNERT, Ingrid BARTHEL and Ludovic FOUR for their help. Also, I thank my colleagues Hussain, Vanille, Nedal, Rémy, Waseem, Salah, Lesia, Mariana, Manu, Redouane, Rajhans and others for their kind support. I would like to express my thanks to my friends here, in INDIA and anywhere in the world for their love, care and moral support during the hard times. Special note of thanks for Jasleen K. Bindra (especially for her continuous encouragement and concern during the tenure), Noémie, Bhaskar Mohan, Mohit, Abhineet, Pankaj & Sweta (and their little daughter Anwita), Shashi Kumar, Amita & Deepankar (Yes! the list is endless) for their kindness, motivation, moral support and for sharing their own experiences. I would be failing on my part if I do not acknowledge the love, affection, encouragement and inspiration given by my parents, who have been behind me in all stages of my life and are the sole reason for my achievements. This work is dedicated to the sacrifices they made for me. Last but not the least; I am thankful to Human Frontier Science Program for financially supporting our work through the project OExcited-State Structure of the Emitter and Color-Tuning Mechanism of the Firefly Bioluminescence (RGY- 0081/2011) P, Université de Strasbourg, Faculté de Pharmacie and CNRS for providing facilities and associated infrastructure to carry on the project. Cordially, I wish to express my gratitude to all the people involved in the writing of this thesis but really sorry if I forget someone to enlist. Mercy beaucoup à tous pour votre présence. Avisek GHOSE Illkirch, February 17 th , 2015 In his own time, in his own place, in what he really is and in the stage he has reached good or bad every human being is a specific element within the whole of the manifest divine Being. So, why be afraid of difficulties, sufferings and problems? When troubles come, try to understand the relevance of your sufferings. Adversity always presents opportunities for introspection. Dr. A P J Abdul Kalam, Wings of Fire (p4-5) Table of content Page Content No. Chapter 1: Introduction: A Bibliographic Review 1-12 1.1 Historical review 1 1.2 The reaction chemistry 4 1.3 The enzymatic regeneration of Oxyluciferin into Luciferin 5 1.4 The photophysics of firefly bioluminescence 6 1.5 Color tuning mechanism of firefly bioluminescence system 8 1.6 Oxyluciferin as a bio-analytical probe 11 An overview of the work 13-16 Chapter 2: Materials and Methods 17-27 2.1 Synthesis and 1HNMR study of Oxyluciferin derivatives 17 2.2 Sample preparation 17 2.3 Steady-state spectroscopic measurements 17 2.4 Time resolved fluorescence spectroscopic measurements 18 2.5 Expression and purification of firefly Luciferase 21 2.6 HIV-1 Peptide Synthesis & labeling 21 2.7 Two-photon excitation microscopy 23 2.8 Fluorescence Lifetime Imaging Microscopy (FLIM) 24 2.9 Fluorescence anisotropy measurements 26 2.10 Cell culture 27 2.11 Oligonucleotide sequences 27 2.12 Software used 27 Results and Discussions Chapter 3: pH Dependent Spectra of Oxyluciferin in Aqueous Buffer 28-57 3.1 Selection of model compounds 30 3.2 Optical Properties of Oxyluciferin in organic solvent 31 3.3 pH dependent absorption spectra 33 3.4 pH dependent Em. spectra & their time-resolved fluorescence decay 39 3.4.1 Emission spectra of the non-ionizable model compounds 39 3.4.2 Emission spectra of the ionizable model compounds 40 3.4.3 Emission spectra of Oxyluciferin (OxyLH 2) 43 3.5 Equilibrium and photodynamics in the excited state 48 3.6 Photoluminescence pathways of Oxyluciferin in aqueous solution 52 Appendix-A A.1 Geminate Recombination (GR) Process 54 A.2 Time resolved fluorescence decay of Oxyluciferin and its two 54 derivatives in DMSO-H 2O system A.3 Relative stability and photo-stability of keto-Oxyluciferin 55 Article- 1I'JK$**$,('@#,45# $5*',7'DL0&-6$75#$(GGG Chapter 4: Luciferase and Oxyluciferin Luminescence Mechanism 58-79 4.1 pH dependent bioluminescence of Luciferase/d-Luciferin/ATP 58 complex 4.2 Structure of Luciola cruciata Luciferase 59 i Page Content No. 4.2.1 Amino acid sequence of L. cruciata 62 4.3 Activity test of Luciferase 63 4.4 Oxyluciferin-Luciferase Assay Protocol 64 4.5 Steady state anisotropy titration of Oxyluciferin with Luciferase 65 4.6 pH dependent emission mechanism of Oxyluciferin with Luciferase 66 4.6.1 Emission of nonionizable model compounds with Luciferase 68 4.6.2 Emission of ionizable model compounds with Luciferase 69 4.6.3 Fluorescence emission of OxyLH 2 in complex with Luciferase 71 4.7 Conclusion 74 Appendix-B B.1 Scatchard Function 78 B.2 Fluorescence time resolved parameters