1 / 68

MMG /BIOC 352

MMG /BIOC 352. Protein-DNA Interactions: Kinetics and Thermodynamics Example: the Bacteriophage l System. Spring 2006. Scott W. Morrical with special thanks to Margaret A. Daugherty. Contact Information. Scott W. Morrical Given B407 656-8260 Scott.Morrical@uvm.edu.

miriam
Download Presentation

MMG /BIOC 352

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. MMG /BIOC 352 Protein-DNA Interactions: Kinetics and Thermodynamics Example: the Bacteriophage l System Spring 2006 Scott W. Morrical with special thanks to Margaret A. Daugherty

  2. Contact Information Scott W. Morrical Given B407 656-8260 Scott.Morrical@uvm.edu

  3. Lecture outline: cI repressor protein Structure Dimerization Data Cro protein Structure Dimerization & DNA Binding An example of induced fit Data Kinetic Aspects of cI and cro binding Facilitated diffusion cro-DNA interactions Structure Analysis? cro-DNA vs. cI-DNA interactions Introduction to the system Bacteriophage lambda Lysogeny vs. lysis The molecular switch PR, PRM, cI repressor, cro Specific vs. Non-specific Interactions What makes a good DNA binding protein? Thermodynamic “Primer” DG = DH - TDS: importance Intrinsic Free Energy Cooperativity Techniques Quantitative DNAse Footprinting

  4. Reference list for this topic: Ref 1: Ptashne, M. (1992) A Genetic Switch, 2nd ed., Cell Press & Blackwell Scientific Publications, Cambridge, MA. **This an excellent general review of bacteriophage l with simple descriptions of thermodynamics and regulation. Ref 2: Johnson, A.D., Poteete, A.R., Lauer, G., Sauer, R.T., Ackers, G.K. & Ptashne, M. (1981) l Repressor and cro - components of an efficient molecular switch. Nature 294: 217-223. Review article of bacteriophage l, outdated, but ok for understanding the system in general. Ref 3: Chattophadhyay, R. & Ghosh, K. (2003) A comparative three-dimensional model of the carboxy-terminal domain of the lambda repressor and its use to build intact repressor tetramer models bound to adjacent operator sites. J. Struct. Biol. 141: 103-114. Ref 4: Oda, M. & Nakamura, H. (2000) Thermodynamic and kinetic analyses for understanding sequence-specific DNA recognition. Genes to Cells 5: 319-326. Just one of many reviews on thermo & kinetic aspects of DNA binding. Ref 5: Brenowitz, M., Senear, D.F., Shea, M.A. & Ackers, G.K. (1986) “Footprint” titrations yield valid thermodynamic isotherms. P.N.A.S. USA 83: 8462-8466

  5. Reference list - continued Ref 6: Koblan, K.S. & Ackers, G.K. (1992) Site-Specific Regulation of DNA Transcription at Bacteriophage l OR, Biochemistry 31: 57-67. Ref 7: Darling, P.J., Holt, J.M. & Ackers, G.K. (2000) Coupled Energetics of l cro Repressor Self-assembly and Site-specific DNA Operator Binding II: Cooperative Interactions of cro Dimers. J. Mol. Biol. 302: 625-638. Ref 8: Albright, R.A. & Matthews, B.W. (1998) Crystal structure of l-cro bound to a Consensus Operator at 3.0 Å Resolution, J. Mol. Biol. 280: 137-151. Ref 9: Spolar, R.S. and Record, M.T. (1994) Coupling of Local Folding to Site-Specific Binding of Proteins to DNA. Science 263: 777 - 784 a classic “must know” paper! Ref 10:von Hippel (1994) Protein - DNA Recognition : New Perspectives and Underlying Themes. Science 263: 769-770. (a review of Spolar & Record) Ref 11: Frankel, A.D. & Kim, P.S. (1991) Modular Structure of Transcription Factors: Implications for Gene Regulation. Cell 65: 717-719 (quick reading - introduces notion of induced fit) Ref 12: Takeda, Y., Ross.P.D. & Mudd,C.P. (1992) Thermodynamics of Cro-protein DNA interactions. Proc. Natl. Acad. Sci. USA 89: 8180-8184.

  6. Reference list - continued Ref 13: von Hippel, P.H. & Berg, O.G. (1989) Facilitated Target Location in Biological Systems. J. Biol. Chem. 264: 675-678. Nice mini-review. Ref 14:Kim, J.G., Takeda, Y., Matthews, B.W. & Anderson, W.F. (1987) Kinetic Studies of Cro-Repressor Operator DNA Interaction, J. Mol. Biol. 196: 149-158 Ref 15: Albright, R.A. and Matthews, B.W. (1998) How Cro and l-repressor distinguish between operators: The structural basis underlying a genetic switch. Proc. Natl. Acad. Sci. USA 95: 3431-3436.

  7. Bacteriophage l: an obligate parasite 8,000x Ref 1: Ptashne (1992) A Genetic Switch, 2nd ed., Cell Press & Blackwell Scientific Publications, Cambridge, MA. 100,000x

  8. Lambda, lysogeny and lysis R R R R R R R R R R infect inject lysogeny lysis prophage Ref 1

  9. An overview of l growth: Patterns of gene expression PL PR N cro 2 10 cI repressor 3 int 12 10 pattern of gene expression l chromosome Ref 1

  10. The molecular switch: Lysogeny to Lysis Polymerase can bind to PRM or PR ORi = right operator sites where i = 1,2 3 PR = right promoter; polymerase transcribes cro protein PRM = promoter of repressor maintenance; polymerase bound here transcribes cI repressor protein. cI repressor protein = maintains bacteria in lysogenic state cro protein = “control of repressor and other genes”; causes switch to lytic lifestyle

  11. l repressor vs. cro Key points: same operator sites; “reverse” affinities; cooperativity; bind as dimers Ref 1

  12. cI repressor: keeps cro turned off! X

  13. cro: turns off production of cI! X

  14. The switch: completing the story UV irradiation activates RecA cleavage of cI monomers. P-DNA complex is reversible. cI dimerization is reversible. When cI dimers fall off, they attempt to reestablish equilibrium; monomers get cleaved. Decrease in [cI dimers], hence DNA opens up for cro binding! Ref 1

  15. Designing an efficient DNA binding protein Purpose: To understand the factors that influence how efficiently a repressor protein occupies its operator in the cell. Given: the fraction of time that an operator is bound by repressor is determined by two factors i). Affinity of repressor for operator ii). Concentration of free repressor Problem: Non-specific binding! Goal: Understand how we can increase efficiency leads us to idea of cooperativity

  16. Designing an efficient DNA binding protein Equations on board The rationale for the arguments are taken from Appendix One in reference 1

  17. Designing an efficient DNA binding protein How can we increase specificity? 1). Increase protein concentration 2). Improve specificity directly *play with the KD/KOP ratio hold KD constant; improve KOP *increase number of contacts by increasing repressor twice the contacts, twice the energy! KOP = 10-20 M; KD = 10-8 M good idea, but…. Affinities become a problem, which give rise to kinetic problems!

  18. Increased efficiency with cooperativity

  19. cI binding to PR 10% 90% No cooperativity Cooperativity >99% occupied Ref 1

  20. cI binding to PR: OR1-OR2 species predominates cI binds strongly to OR1 and OR2; weakly to OR3; cooperative interactions enhance interactions at OR2 Ref 1

  21. Biological advantage of cooperativity “fast switch” for gene expression Ref 1

  22. cI repressor structure: low resolution Dimerization & regulation Kd ~ 6 nM Ref 1

  23. cI repressor structure: low resolution N-terminus: major groove interactions Linker region: flexible C-terminus: protein-protein interactions that give rise to cooperativity Ref 1

  24. cI repressor structure: “high resolution” (pdb1j5g) J. Struct. Biol. 141, 103-114; 2003

  25. REVIEW OF TUESDAY’S LECTURE lysis lysogeny Designing an efficient DNA-binding protein non-specific interactions “mess up” specific binding! KD/KOP and [R]T/[D]T determine binding efficiencies Best way to improve binding - COOPERATIVITY! Cooperativity gives rise to “faster” biological responses

  26. Cooperativity and Free Energy OR3 OR2 OR1 DG2 OR2+ OR2- DG3 OR2 DG1 ? OR3 OR1 DG1+DG2+DG3 > DG1+DG2+DG3 How do we determine that there is cooperativity? OR+ DG3 DG2 DG1

  27. [P•DNA] Keq = [P] [DNA] Thermodynamic Primer: Gibbs Free Energy Keq P + DNA P•DNA DGo = -RTln Keq Remember: more negative, more favorable reaction!

  28. = - dlnKeq DH d(1/T) R van’t Hoff equation: temperature dependence of Keq DG = DH - TDS Measure K as a function of T DH = +533 kJ/mol A <--> B Keq = [B]/[A] 62C 48C Linear: no DCp change Curvature: DCp change

  29. Thermodynamics and biological reactions DG = DH - TDS DG = criteria for spontaneity negative - reaction is favorable DH = direction of energy flow negative - exothermic information about chemical interactions DS = tells us about system organization positive - increase disorder can reflect conformational entropy or H20 entropy DCp = proportional to a change in hydrophobic surface area -- see Spolar reference - very important reference! e.g., negative - organization of protein structure upon DNA binding

  30. Microscopic binding configurations } Intrinsic binding DG Constraint: cooperativity can only occur between adjacent operators

  31. Autoradiogram of a “footprint”: false color image standard OR3 OR2 OR1 standard Brenowitz et al., (1986) P.N.A.S. 83: 8462-8466

  32. Individual site binding isotherms Langmuir isotherm-- single site interactions: Y = K1[X] / (1 + K1[X]) K1 = 1/[X] at Y = 0.5 For 2-site cooperative interaction: Y1 = (K1[X] + K1K2K12[X]2) / B Y2 = (K2[X] + K1K2K12[X]2) / B where B = (1 + (K1 + K2)[X] + K1K2K12[X]2) K1 and K2 are intrinsic binding constants for sites 1 & 2, and K12 is the interaction (or cooperativity) constant. K12 defines the extra free energy of binding 2 sites simultaneously compared to sum of individual free energies, i.e. DG12 = DGtotal - (DG1 + DG2) where DG = RTlnK. Fractional saturation OR3

  33. Individual site binding isotherms for cI - OR interactions OR1 OR3 OR2 OR3 OR2 OR2 OR3 OR1 1 2 3 1 3 Koblan, K. and Ackers, G.K., (1992) Biochemistry 31: 57-65.

  34. Temperature dependence of DG values for cI-OR OR1 van’t Hoff plots for cI-OR single-site interactions OR2 OR3 Koblan, K. and Ackers, G.K., (1992) Biochemistry 31: 57-65.

  35. cI-OR Interactions are Enthalpically Driven Koblan, K. and Ackers, G.K., (1992) Biochemistry 31: 57-65.

  36. Cro repressor structure & induced fit Cro: helix-turn-helix (like cI and CAP) Dimer subunits rotate 53o wrt each other upon binding to consensus OR Creation of extensive H-bond network plus van der Waals contacts along protein-DNA interface. DNA is bent 40o through 19 bp. Recognition helices of Cro dimer make extensive contacts with bp edges in major groove. Albright & Matthews, J. Mol. Biol. (1998) 280, 137-151

  37. Induced Fit (ref 9 -12) Observed in many specific protein-DNA recognition processes, and much less frequently in non-specific binding. Key thermodynamic feature of specific interactions: large negative DCp. hydrophobic effect: occlusion of hydrophobic surface area from water -- protein folding Also can arise from cation release into H20 Non-specific interactions occur with little or no change in DCp. largely electrostatically stabilized:hydration properties of individual components retained, driven by displacement of condensed monovalent cations from DNA. DNA: linear B-DNA to: smooth bends, kinks that disrupt base-pairing Protein: Quaternary rearrangement of domains or subunits ordering of disordered loops or N-termini Formation of a-helices from unfolded loops

  38. Binding isotherms for Cro repressor OR+ 3 1 2 OR1, OR2 & OR3 templates

  39. Cro-DNA Interactions are Entropically Driven Non-specific Specific (OR3)

  40. Specific protein-DNA interactions by induced-fit: large negative DCp Negative entropic contribution from local or global protein folding must be driven by binding free energy (i.e. formation of more extensive complementary interface, burying more macromolecular surface, releasing more water and ions)

  41. Comparison of cro vs. cI energetics Kd = 324 nM Kd = 6.2 nM

  42. Kinetics of protein-DNA interactions For function, regulatory proteins must reach target DNA. Problem: regulatory proteins show affinity to non-specific DNA result: competition, potential slowing down of interactions Early results on lac repressor show rates were increased above simple diffusion rates: 100 - 1000x faster!

  43. What limits biological reaction rates? In principle, limited by the rates at which diffusion can bring two molecules together. A + B AB kencounter = 4p(DA+DB)(rA+rB)N0/1000 DA+DB = diffusion constants for A & B rA & rB = hydrodynamic radii for A & B Smoluchowski’s equation

  44. Kinetics of biological reactions are complex! surfaces of macromolecules not uniformly reactive electrostatic forces may be attractive or repulsive asymmetric molecules (rate of diffusion decreases) complex interaction distance kassoc = 4pkaf(DA+DB)N0/1000 • represents fraction of A & B that interact a is the interaction distance f reflects attractive/repulsive electrostatic forces Modified Smoluchowski equation lets us calculate kassoc for protein & DNA (lac-DNA) at 108 M-1 sec-1. In reality, kassoc= 5 x 1010 M-1 sec-1.

  45. Forward rate constants for macromolecular association Biological molecules are not small inert spheres! Small diffusion constants General stickiness -- van der Waals Biological molecules are slow to drift apart! A classically defined “collision” can consist of many mini-collisions. Rotational rearrangements overcome steric factors Biological molecules are charged! Macromolecules set up electrostatic fields that guide substrates to functional sites; sometimes “shaped”

  46. Special Features of Protein-DNA Interactions Two macromolecules - hence inelastic collisions! Rotational diffusions allows protein to “hop” (4-8 bp) Most proteins have positively charged active site Initial interactions are with non-specific DNA More non-specific sites than specific sites Let N = number of base pairs in the DNA; we could expect the protein to form ~ N transient non-specific complexes before reaching specific site! Logically, in the non-specific sites are thought of as competitive targets, then increasing N should decrease rate. Experimentally, it increases rate! How?!

  47. Initial interactions are with non-specific DNA diffusion facilitated k1 k2 R+D+O <-> RD+O <-> RO + D k-1 k-2

  48. Non-specific binding speeds target location Two methods: Sliding Intersegment transfer Similarity: both involve diffusion while bound to non-specific DNA Net result: decrease in volume of solution that needs to be searched!

  49. SLIDING: “one-dimensional diffusion” Question: How does non-specific binding differ from site-specific binding, such that we can keep the protein associated with the DNA, but still be able to slide? Specific binding: Discrete hydrogen bond donors and acceptors between protein & DNA; energetically favorable. Non-specific binding: requires more of a delocalized type of interaction; charge-charge interactions

More Related