Probing large-scale conformational changes and other coupled processes in RNA polymerase, lac...
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Transcript of Probing large-scale conformational changes and other coupled processes in RNA polymerase, lac...
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 KohEscherichia coli as an osmotic system; solute-biopolymer interactions in vivo and in vitro
Scott Cayley Jonathan Cannon Jeff BallinCharles Anderson Jiang Hong Elizabeth Courtenay (MIT) Mike Capp Irina Shkel Dan Felitsky (Scripps)
Supported by the NIH
Biophysical Studies of Protein-DNA InteractionsSolute and Salt Effects In Vitro and In Vivo
Record Laboratory UW-Madison Departments of Chemistry and Biochemistry
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, (NH4)2SO4)
Processes: (∆ASA< 0)
Folding, Helix FormationDimerization, AssemblyCrystallization, Precipitation
Solute Series (Hofmeister ions, uncharged solutes):
Anions: Sulfate, Phosphate, F, Glu, Ac, Cl, Br, I, SCNCations: NR4, K, Na, GuHUncharged: MPD, TMAO, GB, Pro, Glycerol, Formamide, Urea
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 Kp
Kp = m3loc/m3
bulk
If Kp > 1, solute is accumulated; if Kp < 1, solute is excluded
Preferential Accumulation and Exclusion
Preferential interactions in principle are measurable byequilibrium dialysis.Preferential interaction coefficient is same as dialysisor Donnan coefficient.
H2O H2O
Solute Solute
Biopolymer
3
1
3
m3
m2
T,
1,
3
m3
m2
at dialysis equilibrium
Preferential Interaction Coefficient:
(1)
(2)
(3)
Although the dialysis analogy is useful conceptually, we find thatvapor pressure osmometry (VPO)is more efficient and as accurate as dialysis as a method ofcharacterizing preferential interactions
Local/Bulk Model
LocalBulk
n3bulk
n1bulk
m3bulk
m1
(n3 / n1)local
(n3 / n1)bulk bulk
bulk
n3local
n1local
m3local
m3
=Local
n3local
n1local
KP = =
3 = (ASA)(KP – 1)b1m3 / m1
> 0 accumulation < 0 exclusion
where b1(ASA) = B1 = n1local/n2
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)
Quantifying Preferential Interactions of Solutes With Native Biopolymer Surface (Enriched in Charged and Polar Groups):
Measure excess or deficit osmolality ∆Osm(m2,m3):
From ∆Osm(m2,m3) determine effect of solute on biopolymer chemical potential (activity coefficient)
From µ23, determine preferential interaction coefficient µ3
which is approximately equal to equilibrium dialysis coefficient
At low solute concentration, intensive quantity (per unit of biopolymer surface)
where
where Kp is solute partition coefficient and b1o is hydration (H20/A2)
J. Cannon &M. Capp, submitted ‘04
∆Osm is proportionalto m3 at constant m2 and increases with increasing m2 at constant m3
J. Cannon & M. Capp
is proportionalto m3, not a function of m2, and much larger for BSA thanfor HEWL at agiven m3
(J. Cannon & M. Capp)
Urea is Weakly Accumulated Near Native BSA Surface;Betaine is Strongly Excluded (from anionic carboxylate oxygens)
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
Quantifying Preferential Interactions of Solutes WithBiopolymer Surface Exposed in Unfolding/Melting(Enriched in Uncharged and Nonpolar Groups):
Measure or Tm as a function of solute concentration m3
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 dlnKobs/dm3 = “m-value”/RT = (Kp - 1)b1o(ASA)/ 55.5
“m-value” is the slope of a plot of -∆Gobso =RTlnKobs for unfolding
or other biopolymer process vs. solute concentration
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.
Urea Induced Unfolding of lacI HTH
Temperature (C) Urea Molarity
Fra
ctio
n U
nfo
lde
d Fra
ction U
nfo
lded
0
2
3
4
5
6Urea (M)
Felitsky et al., Biochemistry, ‘03
Betaine Effects on lacI HTH Stability
Temperature (C) Betaine Molarity
Fra
ctio
n U
nfo
lde
d Fra
ction U
nfo
lded
Betaine0 4 M
( Felitsky et al., Biochemistry,submitted)
(Felitsky et al. 2003)
Betaine has Qualitatively Different Interactions with Different Surfaces
lacI HTH Unfolding -0.38 0.05
(Felitsky, Cannon et al., 04)
3/(m3ASA) x 103ASA or ASA
native lysozyme
native bovine serum albumin
Other Polar22%
Nonpolar39%
Charged 39% -1.82 0.12
(Hong, Cannon et al, 04)
native DNA
-0.47 0.17
-0.83 0.05
Glycine Betaine: Correlation of Exclusion with AnionicBiopolymer Surface (carboxylate, phosphate oxygens)
Felitsky, ‘04
Urea: Correlation of Accumulation with Polar Amide Surface
Deviations for highly anionic surfaces suggest modest exclusion of urea fromvicinity of carboxylate and phosphate oxygens. Hong et al. ‘04
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
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
•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
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
(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
Linkage of Growth Rate and Cytoplasmic Water
Cayley et al., ‘03
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 m3 and to ASA, and Kp 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 (Kp = 0) from anionic (carboxylate, phosphate) oxygen surface and that hydration of this anionic surface is 2 layers of water (0.23 H20/A2). GB therefore drives biopolymer processes in which anionic surface is dehydrated.
Urea accumulates at polar amide surface of proteins and nucleic acid bases (Kp = 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). Inabsence 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?