Investigation of Interactions between Homeodomain Proteins and DNA Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen vorgelegt von Darius Vainius aus Sakiai, Litauen Göttingen 2004 D7 Referent: Prof. Dr. E. Neher Koreferent: Prof. Dr. W. Lauterborn Tag der mündlichen Prüfung: 18.05.2004 To Ruta and to my parents. Abbreviations ADC analog-to-digital converter a.u. arbitrary units Antp Antennapedia Bcd Bicoid bp base pair CD circular dichroism DNA deoxyribonucleic acid DTT dithiothreitol DMSO dimethylsulfonic acid E. coli Escherichia coli En Engrailed eve even-skipped Eq. equation FCS fluorescence correlation spectroscopy Fig. figure FRET fluorescence resonance energy transfer ftz fushi tarazu GFC gel filtration chromatography HEPES N-(2-Hydroxyethyl-)piperazin-N’-2-Ethansulfonic acid HLH helix-loop-helix HTH helix-turn-helix IAEDANS 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid kDa kilodalton NMR nuclear magnetic resonance mBBr monobromobimane PAGE polyacrylamide gel electrophoresis + pH negative decimal logarithm of H3O ion concentration PMT photomultiplier tube prd paired RISC reduced instruction set computer SDS sodiumdodecylsulfate TBP TATA-binding protein Ubx Ultrabithorax v/v volume/volume Abbreviations of amino acids: A (Ala) Alanine C (Cys) Cysteine D (Asp) Aspartic acid E (Glu) Glutamic acid F (Phe) Phenylalanine G (Gly) Glycine H (His) Histidine I (Ile) Isoleucine K (Lys) Lysine L (Leu) Leucine M (Met) Methionine N (Asn) Asparagine P (Pro) Proline Q (Gln) Glutamine R (Arg) Arginine S (Ser) Serine T (Thr) Threonine V (Val) Valine W (Trp) Tryptophan Y (Tyr) Tyrosine Contents 1. INTRODUCTION .......................................................................................................................1 2. REVIEW OF THE LITERATURE...................................................................................................3 2.1 Specific protein-DNA interactions ..................................................................................3 2.1.1 Forces between proteins and nucleic acids ..............................................................3 2.1.2 Structural tools of binding – DNA-binding motifs....................................................5 2.1.3 Common principles ...................................................................................................8 2.1.4 Changes in structure and dynamics of protein and DNA induced by interaction ....9 2.1.5 Thermodynamics and kinetics of Protein-DNA interaction....................................15 2.1.6 Influence of environmental factors .........................................................................22 2.2 Aspects of homeodomain-DNA interaction...................................................................26 3. AIMS OF THE PRESENT STUDY ...............................................................................................30 4. MATERIALS AND METHODS ...................................................................................................31 4.1 Materials ........................................................................................................................31 4.2 Protein preparation.........................................................................................................31 4.3 DNA preparation............................................................................................................33 4.4 Steady state absorption spectroscopy.............................................................................34 4.5 Circular dichroism .........................................................................................................34 4.6 Steady state fluorescence spectroscopy .........................................................................35 4.7 Fluorescence titrations ...................................................................................................36 4.8 Measurements of fluorescence resonance energy transfer (FRET) ...............................37 4.9 Stopped-flow kinetics ....................................................................................................40 4.10 Fluorescence correlation spectroscopy (FCS) .............................................................42 4.11 Molecular modeling.....................................................................................................46 5. RESULTS ...............................................................................................................................47 5.1 Spectroscopic characterization of homeodomain-DNA complexes at equilibrium.......47 5.1.1 Changes in intrinsic fluorescence of homeodomains upon binding to DNA ..........47 5.1.2 Changes of circular dichroism spectra of homeodomains upon binding to DNA ..49 5.1.3 Use of fluorescent labels to study the homeodomain-DNA interaction..................51 5.2 Analysis of homeodomain-DNA binding at equilibrium conditions.............................54 5.3 Quantitative analysis of FRET between the fluorescein and Cy3 labels linked to the DNA.....................................................................................................................................58 5.4 Analysis of the translational diffusion properties of the double-labeled oligonucleotide bFC and the Bcd-oligonucleotide complex using fluorescence correlation spectroscopy (FCS)....................................................................................................................................61 5.5 Investigation of the homeodomain-DNA interaction kinetics.......................................62 6. DISCUSSION ..........................................................................................................................69 7. SUMMARY.............................................................................................................................78 APPENDIX .................................................................................................................................80 A1. Function of Ultrabithorax and Engrailed homeodomain proteins ................................80 A2. Calculation of the kinetic fluorescence intensity profiles.............................................80 A3. Numerical methods of kinetic data analysis .................................................................81 REFERENCES .............................................................................................................................84 Introduction 1 1. Introduction The information required by every living organism for obtaining and maintaining its structures and functions is encoded in its DNA. At various levels of information handling such as storage (DNA packing into chromatin), maintenance (e.g. repair of damaged DNA), copying (replication) and transfer (i.e. the expression of genes involving transcription and translation) protein-nucleic acid interactions play a central role. Particularly important for metabolism, replication and development of each organism is the expression of its genes as proteins at correct locations, in proper amounts and with correct timing, relative to cellular and developmental cycles. Gene expression is primarily controlled at the level of transcription by the proteins that bind to specific DNA regulatory regions (operators, enhancers etc.) acting as activators or repressors [Jacob and Monod 1961]. These proteins, called transcription factors, constitute one of the largest and most diverse classes of DNA- binding proteins. Therefore, the elucidation of molecular details of specific interactions between transcription factors and DNA is critical for understanding the mechanisms involved in the control of the expression of genetic information. Structural studies of transcription factors have identified a number of different DNA- binding domains (see chapter 2.1.2), the molecular platforms upon which the protein components of the complementary recognition are positioned in space. Some of these domains form independently folding protein substructures, the DNA-binding properties of which often resemble those of the full protein [Struhl et al. 1989; Wilson et al. 1996]. If other regions of the protein do not influence these properties, the object of investigation can be reduced to the DNA-binding domain and studied separately. The present study focuses on the analysis of the DNA-binding properties of one of such domain present in homeotic transcription factors, the homeodomain (chapter 2.2). Homeodomain-containing proteins play fundamental roles at early developmental stages of eukaryotes [Gehring et al. 1994]. These proteins control the pattern formation and determine the identity of body segments by governing the choice between alternative developmental pathways depending on spatial position in the embryo [Gehring et al. 1994]. Such positional information in the fruit fly (Drosophila melanogaster) embryo is provided by the exponential anterio-posterior gradient of the maternal morphogen protein Bicoid (Bcd), a homeodomain- containing transcription factor. This protein activates a number of target genes at certain threshold levels of concentration and thus initiates position-specific developmental programs, 2 Introduction which are controlled by the products of these target genes (mostly other homeodomain- containing transcription factors). The ability of this protein to bind to specific DNA regions and activate the appropriate genes at concentrations