8 research outputs found
Contributions of Coulombic and Hofmeister Effects to the Osmotic Activation of <i>Escherichia coli</i> Transporter ProP
Osmosensing transporters mediate
osmolyte accumulation to forestall
cellular dehydration as the extracellular osmolality increases. ProP
is a bacterial osmolyte-H<sup>+</sup> symporter, a major facilitator
superfamily member, and a paradigm for osmosensing. ProP activity
is a sigmoid function of the osmolality. It is determined by the osmolality,
not the magnitude or direction of the osmotic shift, in cells and
salt-loaded proteoliposomes. The activation threshold varies directly
with the proportion of anionic phospholipid in cells and proteoliposomes.
The osmosensory mechanism was probed by varying the salt composition
and concentration outside and inside proteoliposomes. Data analysis
was based on the hypothesis that the fraction of maximal transporter
activity at a particular luminal salt concentration reflects the proportion
of ProP molecules in an active conformation. ProP attained the same
activity at the same osmolality when diverse, membrane-impermeant
salts were added to the external medium. Contributions of Coulombic
and/or Hofmeister salt effects to ProP activation were examined by
varying the luminal salt cation (K<sup>+</sup> and Na<sup>+</sup>)
and anion (chloride, phosphate, and sulfate) composition and then
systematically increasing the luminal salt concentration by increasing
the external osmolality. ProP activity increased with the sixth power
of the univalent cation concentration, independent of the type of
anion. This indicates that salt activation of ProP is a Coulombic,
cation effect resulting from salt cation accumulation and not site-specific
cation binding. Possible origins of this Coulombic effect include
folding or assembly of anionic cytoplasmic ProP domains, an increase
in local membrane surface charge density, and/or the juxtaposition
of anionic protein and membrane surfaces during activation
Quantifying Interactions of Nucleobase Atoms with Model Compounds for the Peptide Backbone and Glutamine and Asparagine Side Chains in Water
Alkylureas
display hydrocarbon and amide groups, the primary functional
groups of proteins. To obtain the thermodynamic information that is
needed to analyze interactions of amides and proteins with nucleobases
and nucleic acids, we quantify preferential interactions of alkylureas
with nucleobases differing in the amount and composition of water-accessible
surface area (ASA) by solubility assays. Using an established additive
ASA-based analysis, we interpret these thermodynamic results to determine
interactions of each alkylurea with five types of nucleobase unified
atoms (carbonyl sp<sup>2</sup>O, amino sp<sup>3</sup>N, ring sp<sup>2</sup>N, methyl sp<sup>3</sup>C, and ring sp<sup>2</sup>C). All
alkylureas interact favorably with nucleobase sp<sup>2</sup>C and
sp<sup>3</sup>C atoms; these interactions become more favorable with
an increasing level of alkylation of urea. Interactions with nucleobase
sp<sup>2</sup>O are most favorable for urea, less favorable for methylurea
and ethylurea, and unfavorable for dialkylated ureas. Contributions
to overall alkylurea–nucleobase interactions
from interactions with each nucleobase atom type are proportional
to the ASA of that atom type with proportionality constant (interaction
strength) α, as observed previously for urea. Trends in α-values
for interactions of alkylureas with nucleobase atom types parallel
those for corresponding amide compound atom types, offset because
nucleobase α-values are more favorable. Comparisons between
ethylated and methylated ureas show interactions of amide compound
sp<sup>3</sup>C with nucleobase sp<sup>2</sup>C, sp<sup>3</sup>C,
sp<sup>2</sup>N, and sp<sup>3</sup>N atoms are favorable while amide
sp<sup>3</sup>C–nucleobase sp<sup>2</sup>O interactions are
unfavorable. Strongly favorable interactions of urea with nucleobase
sp<sup>2</sup>O but weakly favorable interactions with nucleobase
sp<sup>3</sup>N indicate that amide
sp<sup>2</sup>N–nucleobase sp<sup>2</sup>O and nucleobase sp<sup>3</sup>N–amide sp<sup>2</sup>O hydrogen bonding (NH···OC)
interactions are favorable while amide sp<sup>2</sup>N–nucleobase
sp<sup>3</sup>N interactions are unfavorable. These favorable amide–nucleobase
hydrogen bonding interactions are prevalent in specific protein–nucleotide
complexes
Quantifying Additive Interactions of the Osmolyte Proline with Individual Functional Groups of Proteins: Comparisons with Urea and Glycine Betaine, Interpretation of <i>m</i>‑Values
To quantify interactions of the osmolyte l-proline with
protein functional groups and predict their effects on protein processes,
we use vapor pressure osmometry to determine chemical potential derivatives
dμ<sub>2</sub>/d<i>m</i><sub>3</sub> = μ<sub>23</sub>, quantifying the preferential interactions of proline (component
3) with 21 solutes (component 2) selected to display different combinations
of aliphatic or aromatic C, amide, carboxylate, phosphate or hydroxyl
O, and amide or cationic N surface. Solubility data yield μ<sub>23</sub> values for four less-soluble solutes. Values of μ<sub>23</sub> are dissected using an ASA-based analysis to test the hypothesis
of additivity and obtain α-values (proline interaction potentials)
for these eight surface types and three inorganic ions. Values of
μ<sub>23</sub> predicted from these α-values agree with
the experiment, demonstrating additivity. Molecular interpretation
of α-values using the solute partitioning model yields partition
coefficients (<i>K</i><sub>p</sub>) quantifying the local
accumulation or exclusion of proline in the hydration water of each
functional group. Interactions of proline with native protein surfaces
and effects of proline on protein unfolding are predicted from α-values
and ASA information and compared with experimental data, with results
for glycine betaine and urea, and with predictions from transfer free
energy analysis. We conclude that proline stabilizes proteins because
of its unfavorable interactions with (exclusion from) amide oxygens
and aliphatic hydrocarbon surfaces exposed in unfolding and that proline
is an effective in vivo osmolyte because of the osmolality increase
resulting from its unfavorable interactions with anionic (carboxylate
and phosphate) and amide oxygens and aliphatic hydrocarbon groups
on the surface of cytoplasmic proteins and nucleic acids
Experimental Atom-by-Atom Dissection of Amide–Amide and Amide–Hydrocarbon Interactions in H<sub>2</sub>O
Quantitative information about amide
interactions in water is needed
to understand their contributions to protein folding and amide effects
on aqueous processes and to compare with computer simulations. Here
we quantify interactions of urea, alkylated ureas, and other amides
by osmometry and amide–aromatic hydrocarbon interactions by
solubility. Analysis of these data yields strengths of interaction
of ureas and naphthalene with amide sp<sup>2</sup>O, amide sp<sup>2</sup>N, aliphatic sp<sup>3</sup>C, and amide and aromatic sp<sup>2</sup>C unified atoms in water. Interactions of amide sp<sup>2</sup>O with urea and naphthalene are favorable, while amide sp<sup>2</sup>O–alkylurea interactions are unfavorable, becoming more unfavorable
with increasing alkylation. Hence, amide sp<sup>2</sup>O–amide
sp<sup>2</sup>N interactions (proposed n−σ* hydrogen
bond) and amide sp<sup>2</sup>O–aromatic sp<sup>2</sup>C (proposed
n−π*) interactions are favorable in water, while amide
sp<sup>2</sup>O–sp<sup>3</sup>C interactions are unfavorable.
Interactions of all ureas with sp<sup>3</sup>C and amide sp<sup>2</sup>N are favorable and increase in strength with increasing alkylation,
indicating favorable sp<sup>3</sup>C–amide sp<sup>2</sup>N
and sp<sup>3</sup>C–sp<sup>3</sup>C interactions. Naphthalene
results show that aromatic sp<sup>2</sup>C–amide sp<sup>2</sup>N interactions in water are unfavorable while sp<sup>2</sup>C–sp<sup>3</sup>C interactions are favorable. These results allow interactions
of amide and hydrocarbon moieties and effects of urea and alkylureas
on aqueous processes to be predicted or interpreted in terms of structural
information. We predict strengths of favorable urea–benzene
and <i>N</i>-methylacetamide interactions from experimental
information to compare with simulations and indicate how amounts of
hydrocarbon and amide surfaces buried in protein folding and other
biopolymer processes and transition states can be determined from
analysis of urea and diethylurea effects on equilibrium and rate constants
Positioning the Intracellular Salt Potassium Glutamate in the Hofmeister Series by Chemical Unfolding Studies of NTL9
<i>In vitro</i>, replacing
KCl with potassium glutamate
(KGlu), the <i>Escherichia coli</i> cytoplasmic salt and
osmolyte, stabilizes folded proteins and protein–nucleic acid
complexes. To understand the chemical basis for these effects and
rank Glu<sup>–</sup> in the Hofmeister anion series for protein
unfolding, we quantify and interpret the strong stabilizing effect
of KGlu on the ribosomal protein domain NTL9, relative to the effects
of other stabilizers (KCl, KF, and K<sub>2</sub>SO<sub>4</sub>) and
destabilizers (GuHCl and GuHSCN). GuHSCN titrations at 20 °C,
performed as a function of the concentration of KGlu or another salt
and monitored by NTL9 fluorescence, are analyzed to obtain <i>r</i>-values quantifying the Hofmeister salt concentration (<i>m</i><sub>3</sub>) dependence of the unfolding equilibrium constant <i>K</i><sub>obs</sub> [<i>r</i>-value = −d ln <i>K</i><sub>obs</sub>/d<i>m</i><sub>3</sub> = (1/<i>RT</i>) dΔ<i>G</i><sub>obs</sub><sup>°</sup>/d<i>m</i><sub>3</sub> = <i>m</i>-value/<i>RT</i>]. <i>r</i>-Values for both stabilizing K<sup>+</sup> salts and destabilizing
GuH<sup>+</sup> salts are compared with predictions from model compound
data. For two-salt mixtures, we find that contributions of stabilizing
and destabilizing salts to observed <i>r</i>-values are
additive and independent. At 20 °C, we determine a KGlu <i>r</i>-value of 3.22 m<sup>–1</sup> and K<sub>2</sub>SO<sub>4</sub>, KF, KCl, GuHCl, and GuHSCN <i>r</i>-values of
5.38, 1.05, 0.64, −1.38, and −3.00 m<sup>–1</sup>, respectively. The KGlu <i>r</i>-value represents a 25-fold
(1.9 kcal) stabilization per molal KGlu added. KGlu is much more stabilizing
than KF, and the stabilizing effect of KGlu is larger in magnitude
than the destabilizing effect of GuHSCN. Interpretation of the data
reveals good agreement between predicted and observed relative <i>r</i>-values and indicates the presence of significant residual
structure in GuHSCN-unfolded NTL9 at 20 °C
Key Roles of the Downstream Mobile Jaw of <i>Escherichia coli</i> RNA Polymerase in Transcription Initiation
Differences in kinetics of transcription initiation by
RNA polymerase
(RNAP) at different promoters tailor the pattern of gene expression
to cellular needs. After initial binding, large conformational changes
occur in promoter DNA and RNAP to form initiation-capable complexes.
To understand the mechanism and regulation of transcription initiation,
the nature and sequence of these conformational changes must be determined. <i>Escherichia coli</i> RNAP uses binding free energy to unwind
and separate 13 base pairs of λP<sub>R</sub> promoter DNA to
form the unstable open intermediate I<sub>2</sub>, which rapidly converts
to much more stable open complexes (I<sub>3</sub>, RP<sub>o</sub>).
Conversion of I<sub>2</sub> to RP<sub>o</sub> involves folding/assembly
of several mobile RNAP domains on downstream duplex DNA. Here, we
investigate effects of a 42-residue deletion in the mobile β′
jaw (ΔJAW) and truncation of promoter DNA beyond +12 (DT+12)
on the steps of initiation. We find that in stable ΔJAW open
complexes the downstream boundary of hydroxyl radical protection shortens
by 5–10 base pairs, as compared to wild-type (WT) complexes.
Dissociation kinetics of open complexes formed with ΔJAW RNAP
and/or DT+12 DNA resemble those deduced for the structurally uncharacterized
intermediate I<sub>3</sub>. Overall rate constants (<i>k</i><sub>a</sub>) for promoter binding and DNA opening by ΔJAW
RNAP are much smaller than for WT RNAP. Values of <i>k</i><sub>a</sub> for WT RNAP with DT+12 and full-length λP<sub>R</sub> are similar, though contributions of binding and isomerization
steps differ. Hence, the jaw plays major roles both early and late
in RP<sub>o</sub> formation, while downstream DNA functions primarily
as the assembly platform after DNA opening
Chemical Interactions of Polyethylene Glycols (PEGs) and Glycerol with Protein Functional Groups: Applications to Effects of PEG and Glycerol on Protein Processes
In this work, we obtain the data
needed to predict chemical interactions
of polyethylene glycols (PEGs) and glycerol with proteins and related
organic compounds and thereby interpret or predict chemical effects
of PEGs on protein processes. To accomplish this, we determine interactions
of glycerol and tetraEG with >30 model compounds displaying the
major
C, N, and O functional groups of proteins. Analysis of these data
yields coefficients (α values) that quantify interactions of
glycerol, tetraEG, and PEG end (-CH<sub>2</sub>OH) and interior (-CH<sub>2</sub>OCH<sub>2</sub>-) groups with these groups, relative to interactions
with water. TetraEG (strongly) and glycerol (weakly) interact favorably
with aromatic C, amide N, and cationic N, but unfavorably with amide
O, carboxylate O, and salt ions. Strongly unfavorable O and salt anion
interactions help make both small and large PEGs effective protein
precipitants. Interactions of tetraEG and PEG interior groups with
aliphatic C are quite favorable, while interactions of glycerol and
PEG end groups with aliphatic C are not. Hence, tetraEG and PEG300
favor unfolding of the DNA-binding domain of lac repressor (lacDBD),
while glycerol and di- and monoethylene glycol are stabilizers. Favorable
interactions with aromatic and aliphatic C explain why PEG400 greatly
increases the solubility of aromatic hydrocarbons and steroids. PEG400–steroid
interactions are unusually favorable, presumably because of simultaneous
interactions of multiple PEG interior groups with the fused ring system
of the steroid. Using α values reported here, chemical contributions
to PEG <i>m</i>-values can be predicted or interpreted in
terms of changes in water-accessible surface area (ΔASA) and
separated from excluded volume effects
Fluorescence Resonance Energy Transfer Characterization of DNA Wrapping in Closed and Open <i>Escherichia coli</i> RNA Polymerase−λP<sub>R</sub> Promoter Complexes
Initial
recognition of promoter DNA by RNA polymerase (RNAP) is
proposed to trigger a series of conformational changes beginning with
bending and wrapping of the 40–50 bp of DNA immediately upstream
of the −35 region. Kinetic studies demonstrated that the presence
of upstream DNA facilitates bending and entry of the downstream duplex
(to +20) into the active site cleft to form an advanced closed complex
(CC), prior to melting of ∼13 bp (−11 to +2), including
the transcription start site (+1). Atomic force microscopy and footprinting
revealed that the stable open complex (OC) is also highly wrapped
(−60 to +20). To test the proposed bent-wrapped model of duplex
DNA in an advanced RNAP−λP<sub>R</sub> CC and compare
wrapping in the CC and OC, we use fluorescence resonance energy transfer
(FRET) between cyanine dyes at far-upstream (−100) and downstream
(+14) positions of promoter DNA. Similarly large intrinsic FRET efficiencies
are observed for the CC (0.30 ± 0.07) and the OC (0.32 ±
0.11) for both probe orientations. Fluorescence enhancements at +14
are observed in the single-dye-labeled CC and OC. These results demonstrate
that upstream DNA is extensively wrapped and the start site region
is bent into the cleft in the advanced CC, reducing the distance between
positions −100 and +14 on promoter DNA from >300 to <100
Ã…. The proximity of upstream DNA to the downstream cleft in the
advanced CC is consistent with the proposed mechanism for facilitation
of OC formation by upstream DNA