biochemical and structural studies of escherichia coli leucine-responsive regulatory protein

UNIVERSITY OF CALIFORNIA Santa Barbara Biochemical and Structural Studies of Escherichia coli Leucine-responsive regulatory protein A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology By Stephanie Snyder de los Ríos Committee in charge: Professor John J. Perona, Chair Professor David Low Professor Kevin Plaxco Professor Herbert Waite March 2006 UMI Number: 3206417 3206417 2006 UMI Microform Copyright All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 by ProQuest Information and Learning Company. The dissertation of Stephanie Snyder de los Ríos is approved by: ___________________________________ Professor D. Low ___________________________________ Professor K. Plaxco ___________________________________ Professor H. Waite ___________________________________ Professor J. J. Perona, Chair March 2006 iii Acknowledgements Graduate school has been a long, hard road – one that I could not have traveled alone. I would like to thank my advisor John Perona and all the past and present members of the Perona lab for their camaraderie, helpful advice, and friendship over the years. Particularly, I’d like to thank Kate Newberry for her patience, sound advice, willingness to answer my (seemingly) constant questions, and great friendship. I’d also like to acknowledge Kent Rossman for his willingness to teach me all about biochemistry, for pushing me to be independent, for setting a good example of optimism in the face of hardship, and for many years of friendship. Above all, I’d like to thank my husband, Miguel, whose encouragement, enthusiasm, care, and concern makes everything better. iv Vita Stephanie Snyder de los Rios March 2006 Education University of North Carolina at Chapel Hill, Chapel Hill, NC B.S. in Chemistry, May 1998 University of California at Santa Barbara, Santa Barbara, CA Ph.D. in Biochemistry, March 2006 Dissertation Topic: “Biochemical and Structural Studies of Escherichia coli Leucine-responsive Regulatory Protein” Experience 1997-1998 Teaching Assistant Department of Chemistry University of North Carolina, Chapel Hill Spring 2002 Teaching Assistant Department of Chemistry and Biochemistry University of California, Santa Barbara Winter 2003 Teaching Assistant Department of Chemistry and Biochemistry University of California, Santa Barbara Winter 2005 Teaching Assistant Department of Chemistry and Biochemistry University of California, Santa Barbara 2000-2006 Research Assistant Department of Chemistry and Biochemistry University of California, Santa Barbara v Publications de los Rios, S. and Perona, J.J. Crystal structure of Escherichia coli Leucine- responsive Regulatory Protein. Manuscript in preparation. Youngblood, B., Shieh, F K., de los Rios, S., Perona, J.J., and Reich, N.O. Specificity Engineering of the DNA Methyltransferase HhaI. Manuscript submitted. Fields of study Major field: X-ray crystallography Professor John J. Perona Minor field: Protein biochemistry Professor John J. Perona vi Abstract Biochemical and structural studies of Escherichia coli Lrp Transcriptional regulation of gene expression is an essential component for the life of any organism. For uropathogenic Escherichia coli, expression of the pap genes, generating the structural and regulatory components of pap pili, has been seen to be correlated with the ability of these bacteria to colonize and infect the urinary tract. Transcription at the pap operon is regulated by a large number of proteins including the global regulator Lrp (leucine-responsive regulatory protein) and the pap-specific regulator PapI. Transcription activation is sensitive to the methylation states of the two GATC sequences contained within the pap promoter. We have been interested in studying specifically how these two regulators, Lrp and PapI, interact with the pap promoter DNA using x-ray crystallography. Biochemical studies of Lrp and PapI were initiated to learn more about these proteins and enhance our ability to crystallize them. A crystal structure of Lrp in complex with one of its binding sites from the pap promoter has been solved to 3.2Å. While there is no clear electron density in the structure for the DNA, comparison of the Lrp structure and the homologous LrpA structure, crystallized in the absence of DNA, provide insight into conformational changes that may be related to DNA binding. In addition, several Lrp mutants were made, with the goal of aiding crystal growth, that have shed light on the ability of Lrp to bind DNA and the role of cooperativity in this process. vii Table of Contents List of Figures and Tables………… ……………………………………………… ix 1. Introduction………….……………………………………………………………1 1.1 Overview of transcription regulation………… ……………… …….1 1.2 The pap operon… ……………………………………………………4 1.3 Leucine-responsive regulatory protein (Lrp)……………………… 15 2. Purification and characterization of Lrp and PapI ………………… ……… 19 2.1 Purification and characterization of Lrp … …………… …….20 2.1.1 Materials and Methods ………………………………….20 Purification of Lrp……………………………………………20 Biochemical characterization of Lrp…………………….… 21 Determination of Lrp activity……………………………… 23 Purification and characterization of Selenomethionine Lrp…24 2.1.2 Results and Discussion 25 2.2 Purification and characterization of PapI …………………… …….43 2.2.1 Materials and Methods 43 Purification of PapI………………………………………… 43 Biochemical characterization of PapI……………………… 44 Determination of PapI activity……………………………….45 2.2.2 Results and Discussion 45 3. Crystal structure of Lrp …… ……………… 56 3.1 Materials and Methods ……………………… ……… 56 Protein expression and purification………………………………….56 DNA oligonucleotide purification………………………………… 57 Crystallization and diffraction data collection……………………….58 Structure determination and refinement…………………………… 59 Structure analysis……………………………………………… … 60 Molecular replacement solution of alternate crystal form………… 62 viii 3.2 Results and Discussion …………………… ………………………63 Structure of the E. coli Lrp dimer……………………………………63 Monomer Comparisons………………………………………………69 Dimer Comparisons……………………………………………… 73 Octamer Comparisons……………………………………………… 77 Lrp Quaternary Structure…………………………………………….85 Electrostatic Potentials and Putative DNA Binding Locations…… 87 DNA Binding Model……………………………………………… 87 Conformational flexibility as related to DNA binding potential…….94 4. Purification and characterization of Lrp mutants ……… …97 4.1 Purification and characterization of Lrp truncation mutants …… 97 4.1.1 Materials and Methods 97 Mutation of Lrp and purification of truncation mutants… …97 Characterization of oligomerization state for LrpMN and LrpMC……………………… ……………………… ……100 Activity assays for truncation mutants………………… 100 4.1.2 Results and Discussion 101 4.2 Purification and characterization of an apparent oligomer-breaking mutant of Lrp ……… 109 4.2.1 Materials and Methods 109 Mutation and purification of S110E and V148R Lrp…… 109 Characterization of V148R Lrp………………………… 109 Activity of V148R Lrp…………………………………… 112 4.2.2 Results and Discussion 112 5. References 123 ix List of Figures and Tables Figures Figure 1.1 Diagram of the pap operon……… … 5 Figure 1.2 Protein binding sites within the pap promoter………….………… …6 Figure 1.3 DNA sequence of the pap promoter region 8 Figure 2.1 SDS-PAGE gel of purified Lrp …………………………26 Figure 2.2 Mass spectrum of Lrp …………………………………………… 27 . Figure 2.3 CD Wavelength scan of Lrp …… ………… 28 Figure 2.4 Thermal melting and unmelting of Lrp ……………………… 30 Figure 2.5 Chemical melt of Lrp ………………………….31 Figure 2.6 Gel filtration elution profile of Lrp …………………….…33 Figure 2.7 Batch light scattering of Lrp ……………………… …34 Figure 2.8 On-line light scattering of Lrp.……………………………… …35 Figure 2.9 Sedimentation equilibium analysis of Lrp ……37 Figure 2.10 Gel shift of 456 pap operon DNA by Lrp ………………… 38 Figure 2.11 Lrp and 456 pap operon DNA binding curve ………………… ….39 Figure 2.12 Mass spectrum of selenomethione-substituted Lrp … …41 Figure 2.13 SeMet Lrp and 456 pap operon DNA binding curve …… … … 42 Figure 2.14 SDS-PAGE gel of purified PapI …………………….… 46 Figure 2.15 Mass spectrum of PapI ………………… ….48 Figure 2.16 CD wavelength scan of PapI …………………… ……50 [...]... of PapI, further characterizations were performed on this protein to understand more about PapI and how to better work with it 19 2 Purification and Characterization of Lrp and PapI 2.1 Purification and characterization of Lrp 2.1.1 Materials and Methods Purification of E coli Leucine-resposive regulatory protein E coli Lrp was expressed in E coli strain JM105 containing the plasmid pJWD1 (Ernsting... understand its role in this particular operon and extend this information to better understand its role on a global scale in E coli The role of Lrp in the pap operon is very intriguing, considering the similarity of its different binding sites, the role of DNA methylation, and the presence of other regulators such as PapI, CAP and PapB 18 2 Purification and characterization of Lrp and PapI The leucine-responsive. .. system is the Escherichia coli (E coli) pap (pyelonephritis-associated pili) operon This operon contains structural and regulatory genes for the expression of pili on the surface of the bacteria (Figure 1.1) Pili are appendages of the bacteria that are attached to the cell wall, and that contain an adhesion protein on their tip These pili have been shown to be involved in the ability of E coli to cause... mechanism of phase variation (reviewed in (van der Woude et al., 1996 and Hernday et al., 2002)) The transcription of pap genes cycles between a phase OFF state and a phase ON state This variation is regulated by a number of proteins Upstream of the pBA promoter, there are six binding sites for the leucine-responsive regulatory 4 1 Introduction Figure 1.1 Figure 1.1 Diagram of the pap operon The majority of. .. unnecessary proteins, and production of unneeded proteins reduces production of other necessary proteins For these and other reasons, transcription is a highly regulated process 1.1 Overview of Transcription Regulation Regulation of transcription is an essential process for the life of any organism The regulation is complex and involves many different processes Many factors influence the ability of a gene... monomers of Lrp………….……72 Figure 3.4 Representation of the C-terminal dimer interface of Lrp .… 74 Figure 3.5 Superposition of the Lrp and LrpA dimers …………….76 Figure 3.6 Comparison of the Lrp and LrpA octamers .78 Figure 3.7 Representation of the Lrp and LrpA octamer interfaces … 81 Figure 3.8 Superposition of Lrp and LrpA tetramers .…………….83 Figure 3.9 Electrostatic surface potential diagram of. .. Phase OFF When Lrp binds to sites 1, 2, and 3 of the pap operon, site 5 is methylated, and transcription at the pBA promoter is turned off When Lrp binds to sites 4, 5, and 6 in conjunction with PapI, site 2 is methylated, and transcription at the pBA promoter is turned on Lrp is depicted in green and PapI is depicted in red 10 1.Introduction cofactor (Geier and Modrich, 1979) Dam’s methylation of GATC... make-up of an organism Through the process of transcription, an exact copy of the information encoded in the DNA is made in the form of RNA The ribosomes then read the genetic code embedded in the RNA and synthesize the proteins specified by the DNA sequence It is important for all life that proteins are made at the proper time Untimely production of proteins can be harmful to cells because some proteins... binding of CpxR-P appears to be methylation-independent since incubation of CpxR-P with methylated pap DNA sequences showed no effect on its binding affinity RimJ was implicated in the regulation of the pap operon by a study of the thermoregulatory control of transcription of the operon Transcription of pap pili has been seen to not occur at low temperatures of 23°C (Blyn et al., 1989) The RimJ protein. .. encoded within the DNA which serve as protein binding sites, (2) specific chemical modifications of the DNA which can alter protein recognition of these sequences, (3) 1 1 Introduction environmental factors which can influence protein binding to DNA sites, and (4) the overall structure (topology) of the DNA itself Regulatory regions of DNA sequences, which are bound by proteins during transcription initiation, . UNIVERSITY OF CALIFORNIA Santa Barbara Biochemical and Structural Studies of Escherichia coli Leucine-responsive regulatory protein A Dissertation submitted in partial satisfaction of the. Topic: Biochemical and Structural Studies of Escherichia coli Leucine-responsive Regulatory Protein Experience 1997-1998 Teaching Assistant Department of Chemistry University of North. Abstract Biochemical and structural studies of Escherichia coli Lrp Transcriptional regulation of gene expression is an essential component for the life of any organism. For uropathogenic Escherichia