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Circular Dichroism of the Laser–Induced Blue State Of CIRCULAR DICHROISM OF THE LASER–INDUCED BLUE STATE OF BACTERIORHODOPSIN, SPECTRAL ANALYSIS AND NEW INSIGHTS INTO THE PURPLE→BLUE COLOR CHANGE Thesis Submitted to The College of Arts and Sciences of the UNIVERSITY OF DAYTON In Partial Fulfillment of the Requirements for The Degree of Master of Science in Chemistry By Anusha Rudraraju Dayton, Ohio August, 2015 CIRCULAR DICHROISM OF THE LASER–INDUCED BLUE STATE OF BACTERIORHODOPSIN, SPECTRAL ANALYSIS AND NEW INSIGHTS INTO THE PURPLE→BLUE COLOR CHANGE Name: Rudraraju, Anusha APPROVED BY: _______________________ _______________________ Mark B. Masthay, Ph.D. Angela Mammana, Ph.D. Associate Professor Assistant Professor Committee Chairman Committee Member _______________________ _______________________ Matt Lopper, Ph.D. Shawn Swavey, Ph.D. Associate Professor Professor Committee Member Committee Member ii ABSTRACT CIRCULAR DICHROISM OF THE LASER–INDUCED BLUE STATE OF BACTERIORHODOPSIN, SPECTRAL ANALYSIS AND NEW INSIGHTS INTO THE PURPLE→BLUE COLOR CHANGE Name: Rudraraju, Anusha University of Dayton Advisor: Dr. Mark B. Masthay The purple membrane (PM) of the salt–loving bacterium H. Salinarium owes both its color and physiological function to the protein bacteriorhodopsin (BR). The PM is comprised of BR trimers arranged in a crystalline hexagonal lattice. PM converts to an ultraviolet–induced colorless membrane (UVCM) upon exposure to diffuse ultraviolet (UV) light through a 1–monomer 1–photon process and to a laser–induced blue membrane (LIBM) upon exposure to intense green laser pulses through a 1–monomer 2– photon process. The color changes which BR molecules undergo depend on the (1) BR aggregation state (e.g. crystalline trimeric PM and monomers) (2) wavelength of light (3) intensity of the light and (4) divalent cations, as earlier results indicate that calcium ions (Ca2+) are removed from the PM surface during the PM LIBM photoconversion. The origin of the unconventional biphasic band in CD spectrum has caused much debate. iii The principle purpose of the research is to further elucidate the mechanisms responsible for the well–known PMLIBM and PMUVCM photoconversions. Both of these processes have significant implications regarding potential photocooperative processes and exciton coupling between BR molecules within the PM. They also have important implications for the long–standing debate about the unusual “bisignate” circular dichroism (CD) spectrum of PM in the visible region of the spectrum, which has been attributed to both exciton coupling between the BR molecules and BR protein heterogeneity within the trimer. To accomplish this objective, I characterized the changes in the absorption and CD spectra of five separate BR species upon irradiation with intense 532 nm laser pulse and diffuse 254nm–UV–light: Native PM, Delipidated PM, Monomeric BR, cation–free blue membrane and calcium saturated PM. Conclusions were drawn about the relative roles of inter–trimer and intra–trimer photocooperativity, exciton coupling and BR protein heterogeneity in the photochemistry and color changes of BR in PM. iv ACKNOWLEDGEMENTS I would like to express my sincere gratitude towards Dr. Mark B. Masthay, for giving me the opportunity to work in his lab and for guiding me through my entire graduate course. Without his constant help and concern this thesis would not have been possible. Thank you for being so patient and kind to me. Further I would like to thank Dr. Angela Mammana who has helped me patiently though all the experiments. Thank you for your great concern and compassion. Thanks to both of them for making my stay at UD very memorable. I would like to express my appreciation to my committee members Dr. Matt Lopper and Dr. Shawn Swavey. I would also like to thank everybody in the chemistry department for being so nice and kind, which made my two years of study at the University of Dayton unforgettable. Last but not least, I would like to thank my family and friends, for all their love and support. v TABLE OF CONTENTS ABSTRACT……………………………………………………………………..……….iii ACKNOWLEDGEMENTS……………………………………………………………….v LIST OF FIGURES……………………………………………………………………….x LIST OF SCHEMES…………………………………………………………………….xv LIST OF GRAPHS……………………………………………………...........................xvi LIST OF TABLES…………………………………………………………………….xviii LIST OF ABBREVIATIONS…………………………………………………………....xx INTRODUCTION……………………………………………………………………..….1 MATERIALS AND METHODS………………………………………………………...11 A. BR Purchase……………………………………......………………………………11 B. Laser Studies and Spectroscopy………………………..…………………………..11 C. Laser and Hg–Lamp Description…………………………………………………...11 D. Sample Preparation…………………………………………………………………13 1. PM in Phosphate Buffer……..…………………...………………………....…13 (a) Laser–Induced Blue Membrane (LIBM).………..………………………….13 (b) Ultraviolet–Induced Colorless Membrane (UVCM).……….……….....…...13 2. Delipidated Purple Membrane (DLPM)………...…………………………......13 (a) Laser–Induced Delipidated Blue Membrane (LI–DLBM)………….………14 vi (b) Ultraviolet–Induced Delipidated Purple Membrane (UV–DLPM)………....14 3. Monomer (MON)......……………………...……………………………………14 (a) Laser Induced Colorless Monomer (LI–CMON).……………..…..………..15 (b) Ultraviolet–Induced Colorless Monomer (UV–CMON).…………….…….16 4. Cation Free Blue Membrane (CFBM) ………………………………..……..…16 (a) Laser Induced Cation Free Blue Membrane (LI–CFBM)……….………….16 (b) Ultraviolet–Induced Cation Free Blue Membrane (UV–CFBM)…….…….16 5. Calcium Saturated Purple Membrane (CaPM)…………………………….……17 (a) Laser–Induced Calcium Saturated Purple Membrane (LI–CaPM)…………17 (b) Ultraviolet–Induced Calcium Saturated Purple Membrane (UV–CaPM)..…17 E. ICP Experiment……………………………………………………………..………17 RESULTS………………….…………………………………………………………….20 (1) Laser Species…………………………………………………………………………20 (a) LIBM–Absorption spectra…………………….……………………………..……20 (b) LIBM–CD spectra……………………………………..………………………….21 (c) LI–DLBM–Absorption spectra….………………………………………………..23 (d) LI–DLBM–CD spectra...………………………………………………………….24 (e) LI–CMON–Absorption spectra…………...………………………….……...……25 (f) LI–CMON–CD spectra………...…………………………………………….........26 (g) LI–CFBM–Absorption spectra…………………………………...…….……....…27 vii (h) LI–CFBM–CD spectra……………………………………………......…..………28 (i) LI–CaPM–Absorption spectra………………………………………………….....29 (j) LI–CaPM–CD spectra……………...……………………………………………...30 (2) Hg–Lamp Species………...……………………………………………………….…31 (a) UVCM–Absorption spectra………………………………...……………………..31 (b) UVCM–CD spectra……………………………………………...…...…………...32 (c) UV–DLPM–Absorption spectra…….………….………...……………………..…33 (d) UV–DLPM–CD spectra………………………….………………..……...……….34 (e) UV–CMON–Absorption spectra………………………………………..........……35 (f) UV–CMON–CD spectra…………………………...……………………………....36 (g) UV–CFBM–Absorption spectra……………………………...……..…………......37 (h) UV–CFBM–CD spectra………………………………..………………...………..38 (i) UV–CaPM–Absorption spectra…………………………………..………………...39 (j) UV–CaPM–CD spectra………………………………………………..……………40 (3) ICP Results……………………………………………….………………………..…41 (4) Spectral Analysis Tables………………………………………………..……………42 DISCUSSION……………………………………………………………………………47 (a) One Photon/ Two Photon Absorption–Spectral Analysis………………………...….47 (b) Proposed Mechanisms for Generation of LIBM and UVCM…………………..........50 (c) Photocooperativity…………………………………………….….…....………….….52 (d) Exciton Coupling/ Protein Heterogeneity…………………………………………....54 (e) Cation Binding……………………………………………………….………………58 viii CONCLUSIONS………..……………………………………….………………………60 FUTURE STUDIES………………………......…………………………………………61 REFERENCES………………………………….…………………….…………………63 APPENDIX…………………………………….………………………………………...72 ix LIST OF FIGURES Figure 1. (A) ATRPSB chromophore is located roughly at the center of the channel formed by the 7 α– helices……………….………………………………..3 Figure 1. (B) Chemical structure of ATR, ATRPSB chromophore and reduced ATRPSB……………………...…………………………………………….…….3 Figure 2.Structure of PM lattice which shows the Purple BR monomers arranged in a heptamer of trimers………………………………..………………………..3 Figure 3. Upper – CD spectrum of the PM depicting the bisignate band. Lower – Absorbance band of PM with absorbance maximum at 568 nm. The zero point of the bisignate band would coincide with the absorbance maximum of the absorption spectra…………………...…………………………………..6 Figure 4. Top – Schematic representations of the native PM; Middle – LIBM; Bottom – UVCM. The dark blue represents the BR570; lighter blue represents the P605 and white represent the P360……………...………………...…….8 Figure 5. Schematic of the sample irradiation with 532 nm pulses from a Spectra Physics Model INDI–40 Pulsed Nd:YAG laser……………………………..…..12 Figure 6. Absorption Spectra of the photo–conversion of PM to LIBM induced by 6ns, 15 mJ pulse–1 (3.46 MWcm–2), 532 nm pulses for 230 min (spectra taken after 0, 10, 30, 50, 70, 90, 110, 130, 150, 180, 230 minutes of irradiation)…...……………………………………………………………….……......…20 x Figure 7. CD Spectra of the photo–conversion of PM to LIBM induced by 6ns, 15 mJ pulse–1(3.6 MWcm–2), 532 nm pulses for 230 min (spectra taken after 0, 10, 30, 50, 70, 90, 110, 130, 150, 180, 230 minutes of irradiation)………………………………………………………………...……...………21 Figure 8. Absorption Spectra of the photo–conversion of DLPM to LI–DLBM induced by 6ns, 15 mJ pulse–1 (3.6 MWcm–2), 532 nm pulses for 120 min (spectra taken after 0, 3, 6, 9, 12, 18, 24, 30, 36, 48, 60, 84, 120 minutes)……….……………....23 Figure 9. CD Spectra of the photo–conversion of DLPM to LI–DLBM induced by 6ns, 15 mJ pulse–1 (3.6 MWcm–2), 532 nm pulses for 120 min (spectra taken after 0, 3, 6, 9, 12, 18, 24, 30, 36, 48, 60, 84, 120 minutes)……………….............…….24 Figure 10. Absorption
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