INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600 BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF THE ARCHAEAL HISTONE HMF FROM THE HYPERTHERMOPHILIC METHANOGEN METHANOTHERMUS FERVIDUS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Rowan Andrew Grayling, B.S., M.S. ale* Ik** The Ohio State University 1995 Dissertation Committee: Approved by C. J. Daniels J. A. Krzycki J. N. Reeve \J Adviser F. R. Tabita Department of Microbiology UMI Number: 9612186 UMI Microform 9612186 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 "The role of the infinitely small in nature is infinitely large" Louis Pasteur This work is dedicated to Life on Earth, which many admire, but few truly appreciate. ACKNOWLEDGEMENTS I thank especially my adviser, Dr. John Reeve, for his guidance, support and friendship over the past five years. I am also indebted to Dr. Kathleen Sandman for her expert advice, friendship and support, several successful collaborations, and a seemingly endless supply of recombinant Escherichia coli clones and recombinant proteins. Encouragement and advice from Dr. Wayne Becktel and my committee members, Dr. Joseph Krzycki, Dr. Charles Daniels, and Dr. F. Robert Tabita has been greatly appreciated. I thank Don Ordaz in the OSU Fermentation Facility, Dr. John Lowbridge in the OSU Biochemical Instrumentation Center, and Bill Swoager in the Department of Microbiology for their advice and technical assistance. Many thanks also to the members of the Reeve lab - past and present, international and domestic - for their friendship and support. Finally, my sincere thanks to my close friend Holly Hillyer, for her support, encouragement, and friendship over the last few years, and also to my parents, for their lifelong support and encouragement, without which none of this would have been possible. VITA November 25,1966 ......................................Bom — Auckland, New Zealand 1988 ...............................................................B.S. in Cellular and Molecular Biology, University of Auckland, Auckland, New Zealand 1990 ...............................................................M.S. in Cellular and Molecular Biology, University of Auckland, Auckland, New Zealand PUBLICATIONS Grayling, R. A., W. J. Becktel, and J. N. Reeve. 1995. Structure and stability of histone HMf from the hyperthermophilic archaeon Methanothermusfervidus. Biochemistry 34: 8441-8448. Sandm an, K., R. A. Grayling, and J. N. Reeve. 1995. Improved N-terminal processing of recombinant proteins synthesized in Escherichia coli. Bio/Technology 13: 504-506. Sandman, K., R. A. Grayling, and J. N. Reeve. 1995. Purification of the Histone HMf from Methanothermusfervidus, p. 303 (Methanogens). In F. Robb (ed.), Archaea: a laboratory manual. Cold Spring Harbor Laboratory Press, Plainview, NY. Grayling, R. A., K. Sandman, and J. N. Reeve. 1994. Archaeal DNA binding proteins and chromosome structure. System. Appl. Microbiol. 16: 582-590. iv Sandman, K., R. A. Grayling, B. Dobrinski, R. Lurz, and J. N. Reeve. 1994. Growth-phase-dependent synthesis of histones in the archaeon Methanothermus fervidus. Proc. Natl. Acad. Sci. USA 91: 12624-12628. Luthi, E., N. B. Jasmat, R. A. Grayling, D. R. Love and P. L. BergquisL 1991. Cloning, sequence analysis and expression in Escherichia coli of a gene coding for a P-mannase from the extremely thermophilic bacterium “ Caldocellum saccharolyticum". Appl. Environ. Microbiol. 57: 694-700. Saul, D. J., L. C. Williams, R. A. Grayling, L.W. Chamley, D. R. Love and P. L. Bergquist. 1990. celB, a gene coding for a bifunctional cellulase from the extreme thermophile “ Caldocellum saccharolyticum”. Appl. Environ. Microbiol. 56: 3117-3124. FIELDS OF STUDY Major field: Microbiology Studies in the Biochemical and Biophysical Characterization of Thermostable Proteins Dr. J. N. Reeve, Adviser v TABLE OF CONTENTS DEDICATION.....................................................................................................................ii ACKNOWLEDGEMENTS................................................................................................ iii VTTA.................................................................................................................................... iv TABLE OF CONTENTS..................................... vi LIST OF FIGURES.................................................................................................... xi LIST OF TABLES..............................................................................................................xv LIST OF ABBREVIATIONS............................................................................................. xvi CHAPTER I: GENERAL INTRODUCTION.............................................................. 1 DNA binding proteins and genome compaction ........................................ 1 Histones and nucleosome structure in eukaryotes ..................................... 2 Prokaryotic DNA binding proteins .............................................................8 Bacterial DNA binding proteins ........................................................ 8 Archaeal DNA binding proteins ........................................................ 10 Curved DNA ...............................................................................................26 Circular dichroism spectroscopy ........................................................30 Thermophiles, and protein and DNA thermal stability .............................. 32 Goals of this study ..................................................................................... 36 CHAPTER H: PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF NATIVE AND RECOMBINANT HMF PROTEINS....................................................................................37 Introduction .............................................................................................................37 Materials and methods ............................................................................................39 vi Chemicals and reagents .............................................................................. 39 Growth of Methanothermusfervidus.........................................................39 Growth of Escherichia coli .........................................................................40 HMf purification methods .......................................................................... 41 Protein quantitation ..................................................................................... 42 Reverse phase HPLC ................................................................... 43 Protein-protein crosslinking........................................................................43 Formation of rHMfA*rHMfB heterodimer-containing mixtures in vitro...................... 44 Electrophoresis techniques .........................................................................44 Acid hydrolysis (‘deformylation’) of rHMfA formylated methionine residues ....................................................................................46 Results.....................................................................................................................46 Development of fermentation and protein purification methods and conditions .................................................................................................46 Protein purification ...................................................................................50 Growth phase dependence of the HMfA/HMfB ratio ................................52 Protein-protein crosslinking studies ........................................................... 60 Development of non-denaturing gel systems to resolve (r)HMfA and (r)HMfB ...............................................................................................67 Analysis of native and recombinant HMf dimer populations