AN ABSTRACT OF THE THESIS OF David W. Brown for the degree of Doctor of Philosophy in Biochemistry and Biophysics presented on 15 September, 1992. Title: Conformational Transitions of Nucleosome Core Particles Monitored with Time- Resolved Fluorescence SpectroscopyRedacted for Privacy Abstract Approved: Enoch W. Small Time-resolved fluorescence spectroscopy was used to monitor the effects of varying ionic strength on nucleosome core particle structure. Two main methods were used in these studies.First, the fluorescence anisotropy decay of bound ethidium was measured and was shown to reflect the rotational tumbling of the core particle through solution, the longest recovered decay time being a measure of the rotational correlation time of the particle. A rotational correlation time of 165 ns was recovered for the native core particle at 10 mM ionic strength, in excellent agreement with that predicted by hydrodynamic calculations based on the particle's known size and shape. This technique was then used to measure the rotational correlation time of the core particle as a function of ionic strength. Below 1 mM salt the recovered rotational correlation times suggested little change in shape throughout the region of the reversible low salt transition. At very low ionic strengths (below 0.2 mM), where the low salt transition becomes irreversible, the rotational correlation time increased sharply to 330 ns, suggesting a major change in the core particle structure. Computer modeling was performed to show that this increase was most likely due to a substantial elongation in the core particle structure, to at least a 5:1 axial ratio. At elevated ionic strengths, the rotational correlation time was seen to increase from the initial value of 165 ns to 240 ns as the salt concentration was raised from 10 mM to 0.35 M, with further increases being observed only above 0.65 M; we term this initial increase the moderate salt transition. Trypsinization of the core particles to remove the N- terminal histone domains completely abolished the increase, demonstrating that the moderate salt transition as measured by this technique involves the release of these protein domains from the body of the core particle. The second method used involved the measurement of the fluorescence decay of the intrinsic tyrosine residues of the core particle. This decay proved to be very complex, and was best represented by a distribution of lifetimes, suggesting different environments for the tyrosines. This distribution changed as the ionic strength of the solution changed, suggesting the movement of tyrosine residues to differing environments as the particle undergoes the low and moderate salt transitions, as well as the high salt dissociation. Conformational Transitions of Nucleosome Core Particles Monitored with Time-Resolved Fluorescence Spectroscopy by David W. Brown A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed 15 September 1992 Commencement June 1993 APPROVED: Redacted for Privacy Associate Professor of Biochemistry and Biophysics in charge of major Redacted for Privacy Chairman of Department of Biochemistry and Biophysics Redacted for Privacy Dean of Graduate cool Date thesis is presented 15 September 1992 ACKNOWLEDGEMENTS I would like to acknowledge the guidance, help, and instruction provided by my advisor, friend, and mentor, Dr. Enoch W. Small, throughout my studies. Special thanks are due to Dr. Louis J. Libertini, an associate of Dr. Small. His expertise,both in the laboratory and with the instrumentation, as well as his willingness to share this expertise with me, are greatly appreciated. Both of these individuals helped my family and I substantially during my final year off campus, when we relocated to Spokane, WA. Dr. Jeanne Rudzki Small provided many ideas regarding the format for this document; she also contributed materially to its printing. Thanks are extended to Dr. Michael J. Smerdon of Washington State University for the use of his facilities in preparing several of the figures. I gratefully acknowledge financial support from NIH Predoctoral Training Grant GM07774, as well as from NIH Grant GM25663 throughout my studies. Special acknowledgement is also extended to the many family members who supported me during my studies.I would like to thank my parents, Robert and Dolores Brown, for their continual belief in me, and also for their material assistance. My final year off-campus in Spokane would not have been possible without their financial and emotional support. My in-laws, Dr. Lawrence E. and Ruth Wittsell, expended considerable financial resources in providing housing for my family throughout the period of my schooling; their moral support and ideas also helped me through several rough times when it seemed like nothing was going right.I would like to thank my children Elizabeth, Kenneth, and Jennifer (who joined us midway) for their sacrifices as we went through this trying time, hoping but not quite believing that things would get better in the end. Finally, I would like to thank my beloved wife, Lorna, for her unfailing support through all of my time spent in this work. This work would not have been possible without her. She cheered me on when things went right, she cheered me up when things went wrong, and throughout all of it, she never lost her faith in me. This work is dedicated to her. TABLE OF CONTENTS Chapter 1 Introduction 1 Chapter 2Experimental 10 Chapter 3Rotational Diffusion Of Nucleosome Core Particles Indicates An Extended Structure At Low Ionic Strength 26 Introduction 26 Materials and Methods 28 Results 29 Discussion 50 Conclusion 61 Chapter 4 The Effects Of N-Terminal Histone Domains On Core Particle Structural Transitions 62 Introduction 62 Materials and Methods 63 Results 80 Discussion 86 Conclusions 97 Chapter 5Using the Decay of Intrinsic Tyrosine Fluorescence of Core Particles to Monitor Conformational Changes 99 Introduction 99 Materials and Methods 101 Results and Discussion 102 Chapter 6Conclusions 121 Bibliography 125 Appendix 134 LIST OF FIGURES Figure Page Figure 2.1 Characterization of the Core Particle Preparation: SDS-PAGE 18 Figure 2.2 Characterization of the Core Particle Preparation: Denatured DNA Gel 20 Figure 2.3 Characterization of the Core Particle Preparation: Non-Denaturing DNA Gel 22 Figure 2.4 Polarized Fluorescence Measurement 24 Figure 3.1 Fluorescence Intensity Decays 30 Figure 3.2 Polarized Fluorescence Decays 33 Figure 3.3 Examples of Fluorescence Anisotropy Decays of Ethidium Bound to Core Particles 35 Figure 3.4 Reciprocal of the Longest Recovered Anisotropy Decay Lifetime of Chromatin Core Particles (max) Plotted as a Function of T/1 38 Figure 3.5 Fluorescence Intensity and Omax vs Ionic Strength 43 Figure 3.6 Anisotropy Decays of Ethidium Bound to Core Particles and Core Particle DNA 46 Figure 3.7 Cartoon Interpretation of the Structure of the Nucleosome Core Particle at Various Steps in the Low-Salt Transition 57 Figure 4.1 Trypsinization of Core Particles 70 Figure 4.2 Cut-Away Ellipsoid 72 Figure 4.3 Omax vs. [NaCl], Native Core Particles 76 Figure 4.4 Omax vs. [NaC1], Trypsinized Core Particles 78 Figure 4.5 Trypsinized Core Particles at Low Salt Concentrations 83 Figure 4.6 Expected Values for the Rotational Correlation Times for Several Prolate Ellipsoids 90 Figure 4.7 Cartoon Representation of the Moderate Salt Transition 95 Figure 5.1 Examples of the Fluorescence Decay Data 103 Figure 5.2 DeviationPlots 105 Figure 5.3 Tyrosine Intensity and Average Lifetime 109 Figure 5.4 Low-Resolution Distribution Aanalysis 111 Figure 5.5 Effects of Low Ionic Strengths 115 Figure 5.6 Effects of High Ionic Strengths 117 LIST OF TABLES Table Page Table 3.1 Analysis Results on Total Fluorescence Intensity Decays of Ethidium Bound to Core Particles or Free DNA 32 Table 3.2 Results of Analyses on Anisotropy Decays for Ethidium Bound to Core Particles 37 Table 3.3 Irreversibility of the Low-Salt Transition 45 Table 3.4 Comparison of Our Rotational Diffusion Results with Values from the Literature 53 Table 4.1 Axial Dimensions For Prolate and Oblate Ellipsoids of Varying Axial Ratios Having the Volume of the Core Particle (-5.6 X 105 A3) 74 Table 4.2 Predicted Rotational Diffusion Coefficients (Di) and Correlation Times (0i) for Various Prolate and Oblate Ellipsoids of Revolution 75 Table 4.3 Irreversibility of the Low-Salt Transition for Trypsinized Core Particles 85 Table 4.4 Least-Squares Analysis Results for Simulated Oblate Ellipsoids of Revolution 89 LIST OF APPENDIX FIGURES Figure Page Figure A.1 The Instrument Optics 136 Figure A.2 The InstrumentElectronics 138 Figure A.3 Pulse Height Distributions 149 Figure A.4 Effects of Gating on the Excitation Profile 151 Conformational Transitions of Nucleosome Core Particles Monitored with Time-Resolved Fluorescence Spectroscopy Chapter 1 Introduction The eukaryotic genome is organized in vivo into a complex of DNA and proteins called chromatin. The nucleosome is a repeating subunit of chromatin and is composed of an invariant core particle and a variable length of linker DNA which connects the nucleosomes together to form the chromatin strand. The nucleosome core particle contains 146 by of DNA wrapped in 1 3/4 left-handed superhelical turns around an inner protein core containing two each of the inner histones H2A, H2B, H3, and H4. These histones are organized into a predominantly a-helical central globular region and an 'unstructured', highly basic N-terminal tail. Recent X-ray analysis of the protein core permits one to trace the path of the polypeptide chains of the globular regions (Arents et al., 1991); the location and role of the N-terminal domains has not been determined, although they may play a role in the maintenance of higher order chromatin structure [reviewed in van Holde (1988)]. The core histones are organized into two H2A-H2B dimers and a single (H3-H4)2 tetramer.