Biophysical Studies of Protein-DNA Interactions
Solute and Salt Effects In Vitro and In Vivo
Record Laboratory
UW-Madison
Departments of Chemistry and Biochemistry
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
Carrie Davis
Wayne Kontur
Laurel Pegram
Kirk vanderMeulen
Melissa Anderson
Mike Capp
Junseock Koh
Oleg Tsodikov (Harvard)
Jill Holbrook (U. Heidelburg)
Escherichia coli as an osmotic system; solute-biopolymer
interactions in vivo and in vitro
Scott Cayley
Charles Anderson
Mike Capp
Jonathan Cannon
Jiang Hong
Irina Shkel
Jeff Ballin
Elizabeth Courtenay (MIT)
Dan Felitsky (Scripps)
Supported by the NIH
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 Formation
Dimerization, Assembly
Crystallization, Precipitation
Solute Series (Hofmeister ions, uncharged solutes):
Anions: Sulfate, Phosphate, F, Glu, Ac, Cl, Br,
I, SCN
Cations:
NR4, K, Na,
GuH
Uncharged: 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/m3bulk
If Kp > 1, solute is accumulated; if Kp < 1, solute is excluded
Preferential Accumulation and Exclusion
Preferential interactions in
principle are measurable by
equilibrium dialysis.
Preferential interaction
coefficient is same as dialysis
or Donnan coefficient.
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
Preferential Interaction Coefficient:

H 2O
H 2O
(1)
3
  
1 3
T, ,
1
Biopolymer (2)

Solute
Solute
(3)


m3 
m2 
m 3
m2
3
at dialy sis equilibrium
Local/Bulk Model
Bulk
Local
local
bulk
n3
n3
local
n1
bulk
n1
bulk
=
m3
m1
Local
local
n3
local
local
KP =
(n3 / n1)
bulk
(n3 / n1)
local
=
m3
n1
bulk
m3
bulk / m
=
(ASA)(K
–
1)b
m
P
1
3
1
3
where b1(ASA) = B1 = n1local/n2
> 0 accumulation
< 0 exclusion
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 µ
3
which is approximately equal to equilibrium dialysis coefficient
where
At low solute concentration, intensive quantity (per unit of biopolymer surface)
where Kp is solute partition coefficient and b1o is hydration (H20/A2)
∆Osm is proportional
to m3 at constant m2
and increases with
increasing m2 at
constant m3
J. Cannon &
M. Capp,
submitted ‘04
 is proportional
to m3, not a
function of m2,
and much larger
for BSA than
for HEWL at a
given m3
J. Cannon & M. Capp
Urea is Weakly Accumulated Near Native BSA Surface;
Betaine is Strongly Excluded (from anionic carboxylate oxygens)
(J. Cannon & M. Capp)
Preferential Interactions with B-DNA
Urea is Neither Strongly Accumulated Nor Excluded from ds DNA;
Betaine is Strongly Excluded (largely from anionic phosphate oxygens)
J. Hong
Quantifying Preferential Interactions of Solutes With
Biopolymer 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
Urea (M)
Fraction Unfolded
Fraction Unfolded
6
5
4
3
2
0
Temperature (C)
Urea Molarity
Felitsky et al., Biochemistry, ‘03
Betaine
0
4M
Temperature (C)
Fraction Unfolded
Fraction Unfolded
Betaine Effects on lacI HTH Stability
Betaine Molarity
( Felitsky et al., Biochemistry,submitted)
(Felitsky et al. 2003)
Betaine has Qualitatively Different Interactions with Different Surfaces
ASA or ASA
3/(m3ASA) x 103
-0.38  0.05
lacI HTH Unfolding
(Felitsky, Cannon et al., 04)
native lysozyme
native bovine serum albumin
Nonpolar
39%
Charged 39%
Other Polar
22%
native DNA
-0.47  0.17
-0.83  0.05
-1.82  0.12
(Hong, Cannon et al, 04)
Glycine Betaine: Correlation of Exclusion with Anionic
Biopolymer Surface (carboxylate, phosphate oxygens)
Felitsky, ‘04
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
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
1.2
MBM+GB
1
1
Growth rate (hr
-1
)
1.2
0.8
0.8
LB
0.6
0.6
0.4
0.4
MBM
0.2
0.2
0
0
0
1
2
3
External Osmolality (Osm)
(S. Cayley et al, ‘03)
Glycine Betaine (GB) increases growth rate at high osmolality and
therefore is a very effective osmoprotectant in E. coli
Passive and Active Responses to Osmotic Stress
•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
Accumulation of GB Increases the Amount of Cytoplasmic Water
Without Increasing the Total Amount of Osmolytes
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.
Steady State Amount of Cytoplasmic Water Decreases
with Increasing Osmolality of Growth
Cytoplasmic water ( l/mg DW)
2.4
1.9
+GB
1.4
-GB
0.9
0.4
0
1
2
Growth osmolality
(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
Linkage of Growth Rate and Cytoplasmic Water
1.2
1
1
-1
Growth rate (h )
1.2
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
0
50
100
150
Amount of cytoplasmic water
(mol/mg dry weight)
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). 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?