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Probing large-scale conformational changes and other coupled processes in RNA polymerase, lac repressor, and IHF - DNA interactions (DNA wrapping and/or.

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Presentation on theme: "Probing large-scale conformational changes and other coupled processes in RNA polymerase, lac repressor, and IHF - DNA interactions (DNA wrapping and/or."— Presentation transcript:

1 Probing large-scale conformational changes and other coupled processes in RNA polymerase, lac repressor, and IHF - DNA interactions (DNA wrapping and/or opening, protein folding) Ruth Saecker Kirk vanderMeulen Oleg Tsodikov (Harvard) Carrie Davis Melissa Anderson Jill Holbrook (U. Heidelburg) Wayne Kontur Mike Capp Laurel Pegram Junseock Koh Escherichia coli as an osmotic system; solute-biopolymer interactions in vivo and in vitro Scott CayleyJonathan CannonJeff Ballin Charles Anderson Jiang HongElizabeth Courtenay (MIT) Mike Capp Irina Shkel Dan Felitsky (Scripps) Supported by the NIH Biophysical Studies of Protein-DNA Interactions Solute and Salt Effects In Vitro and In Vivo Record Laboratory UW-Madison Departments of Chemistry and Biochemistry

2 ASA-Based Prediction or Interpretation of Solute Effects On Biopolymer Processes Solutes:Denaturants (e.g. urea, GuHCl) Osmolytes, Stabilizers (e.g.glycine betaine (GB)) Hofmeister Salts (e.g. KF, KGlu vs. KSCN, KI) Crystallization Agents (e.g. PEG, MPD, (NH 4 ) 2 SO 4 ) Processes: (∆ASA< 0) Folding, Helix Formation Dimerization, Assembly Crystallization, Precipitation Solute Series (Hofmeister ions, uncharged solutes): Anions: Sulfate, Phosphate, F, Glu, Ac, Cl, Br, I, SCN Cations: NR 4, K, Na, GuH Uncharged: MPD, TMAO, GB, Pro, Glycerol, Formamide, Urea

3 Solute effects arise from PREFERENTIAL INTERACTIONS (Timasheff): Solute and water compete for the biopolymer surface Preferential Accumulation of Solute: Solute-Biopolymer interactions more favorable than interactions of both species with water Local concentration of solute higher than bulk Preferential Exclusion of Solute (Preferential Hydration) Local concentration of solute lower than bulk To describe solute distribution: Schellman 1:1 solute: water competitive binding model Our solute partitioning model; partition coefficient K p K p = m 3 loc /m 3 bulk If K p > 1, solute is accumulated; if K p < 1, solute is excluded

4 Preferential Accumulation and Exclusion Preferential interactions in principle are measurable by equilibrium dialysis. Preferential interaction coefficient is same as dialysis or Donnan coefficient. H2OH2OH2OH2O Solute Biopolymer Preferential Interaction Coefficient: (1) (2) (3) Although the dialysis analogy is useful conceptually, we find that vapor pressure osmometry (VPO) is more efficient and as accurate as dialysis as a method of characterizing preferential interactions

5 Local/Bulk Model where b 1 (ASA) = B 1 = n 1 local /n 2

6 Systems investigated to date: Solutes: E. coli osmolytes (GB, Pro, trehalose, KGlu) Denaturants (urea, GuHCl, GuHSCN) Hofmeister salts (KF, KCl, KBr, KI) Biopolymer Surfaces (ranging from nonpolar and uncharged to highly charged): Surface exposed on unfolding: Globular proteins (lac I HTH; 73% nonpolar, Alpha-helix essentially uncharged) DNA double helix Native protein surface (20-30% charged) Native DNA surface (44% charged surface)

7 Quantifying Preferential Interactions of Solutes With Native Biopolymer Surface (Enriched in Charged and Polar Groups): Measure excess or deficit osmolality ∆Osm(m 2,m 3 ): From ∆Osm(m 2,m 3 ) determine effect of solute on biopolymer chemical potential (activity coefficient) From µ 23, determine preferential interaction coefficient  µ 3 which is approximately equal to equilibrium dialysis coefficient At low solute concentration, intensive quantity (per unit of biopolymer surface) where where K p is solute partition coefficient and b 1 o is hydration (H 2 0/A 2 )

8 J. Cannon & M. Capp, submitted ‘04 ∆Osm is proportional to m 3 at constant m 2 and increases with increasing m 2 at constant m 3

9 J. Cannon & M. Capp  is proportional to m 3, not a function of m 2, and much larger for BSA than for HEWL at a given m 3

10 (J. Cannon & M. Capp) Urea is Weakly Accumulated Near Native BSA Surface; Betaine is Strongly Excluded (from anionic carboxylate oxygens)

11 Urea is Neither Strongly Accumulated Nor Excluded from ds DNA; Betaine is Strongly Excluded (largely from anionic phosphate oxygens) J. Hong Preferential Interactions with B-DNA

12 Quantifying Preferential Interactions of Solutes With Biopolymer Surface Exposed in Unfolding/Melting (Enriched in Uncharged and Nonpolar Groups): Measure or T m as a function of solute concentration m 3 For uncharged solutes (Wyman) For Electrolyte solutes () For uncharged solutes Interpret as for interaction of solute with biopolymer surface exposed in unfolding (u) At low solute concentration and dlnK obs /dm 3 = “m-value”/RT = (K p - 1)b 1 o (ASA)/ 55.5

13 “m-value” is the slope of a plot of -∆G obs o =RTlnK obs for unfolding or other biopolymer process vs. solute concentration

14 lacI HTH as a Model System for Folding Studies Small helix-turn-helix protein Two state reversible equilibrium unfolding Marginal stability; population not 100% in folded state even at temperature of maximum stability Broad thermal and solute-induced transitions permit experimental study over wide ranges of temperatures and solute concentrations.

15 Urea Induced Unfolding of lacI HTH Temperature (  C) Urea Molarity Fraction Unfolded 0 2 3 4 5 6 Urea (M) Felitsky et al., Biochemistry, ‘03

16 Betaine Effects on lacI HTH Stability Temperature (  C) Betaine Molarity Fraction Unfolded Betaine 0 4 M ( Felitsky et al., Biochemistry,submitted)

17 (Felitsky et al. 2003)

18 Betaine has Qualitatively Different Interactions with Different Surfaces lacI HTH Unfolding -0.38  0.05 (Felitsky, Cannon et al., 04)   3 /(m 3 ASA) x 10 3  ASA or ASA native lysozyme native bovine serum albumin Other Polar 22% Nonpolar 39% Charged 39% -1.82  0.12 (Hong, Cannon et al, 04) native DNA -0.47  0.17 -0.83  0.05

19 Glycine Betaine: Correlation of Exclusion with Anionic Biopolymer Surface (carboxylate, phosphate oxygens) Felitsky, ‘04

20 Urea: Correlation of Accumulation with Polar Amide Surface Deviations for highly anionic surfaces suggest modest exclusion of urea from vicinity of carboxylate and phosphate oxygens. Hong et al. ‘04

21 Applications Effect of Uptake of GB on Amount of Cytoplasmic Water and Growth Rate of Osmotically-Stressed E. coli Urea and GB as Probes of Coupled Folding or Unfolding and of Other Coupled Processes in the Steps of RNA Polymerase-Promoter Binding

22 Osmotic Stress Reduces Growth Rate of E. coli (S. Cayley et al, ‘03) Glycine Betaine (GB) increases growth rate at high osmolality and therefore is a very effective osmoprotectant in E. coli

23 Initial (passive) response to osmotic stress: loss of water and turgor pressure Subsequent (active) response: accumulation of osmolytes, resulting in uptake of water Cayley et al, ‘03 Passive and Active Responses to Osmotic Stress

24 Propose that GB is a more efficient osmolyte than Kglu or trehalose because it is so highly excluded from anionic surface of DNA, RNA, and proteins. Accumulation of GB Increases the Amount of Cytoplasmic Water Without Increasing the Total Amount of Osmolytes

25 (Cayley et al., ‘03) Accumulation of solutes does not prevent reduction in steady state amount of cytoplasmic water with increasing growth osmolality Accumulation of betaine increases amount of cytoplasmic water at a given Osm Steady State Amount of Cytoplasmic Water Decreases with Increasing Osmolality of Growth

26 Linkage of Growth Rate and Cytoplasmic Water Cayley et al., ‘03

27 Summary of Results For the homologous series of surfaces exposed in unfolding globular proteins (with similar surface compositions and a wide range of ASA, values of  for preferential interactions of urea and GuHCl are proportional to m 3 and to ASA, and K p is the same for all proteins in the series. Analysis of the exclusion of GB from different biopolymer surfaces indicates that GB is completely excluded (K p = 0) from anionic (carboxylate, phosphate) oxygen surface and that hydration of this anionic surface is 2 layers of water (0.23 H 2 0/A 2 ). GB therefore drives biopolymer processes in which anionic surface is dehydrated. Urea accumulates at polar amide surface of proteins and nucleic acid bases (K p = 1.8 if hydration is a monolayer); urea appears to be weakly excluded from anionic oxygen surface. Conclusion: Can quantitatively predict effects of urea, GB on biopolymer processes from structure (∆ASA; composition). In absence of structure, can interpret effects of urea, GB in terms of ∆ASA if assume a particular surface composition. CAN THIS BE EXTENDED TO OTHER SOLUTES AND PROCESSES?


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