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⁺ 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⁺ and Na⁺) 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 sp²O, amino sp³N, ring sp²N, methyl sp³C, and ring sp²C). All alkylureas interact favorably with nucleobase sp²C and sp³C atoms; these interactions become more favorable with an increasing level of alkylation of urea. Interactions with nucleobase sp²O 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 sp³C with nucleobase sp²C, sp³C, sp²N, and sp³N atoms are favorable while amide sp³C–nucleobase sp²O interactions are unfavorable. Strongly favorable interactions of urea with nucleobase sp²O but weakly favorable interactions with nucleobase sp³N indicate that amide sp²N–nucleobase sp²O and nucleobase sp³N–amide sp²O hydrogen bonding (NH···OC) interactions are favorable while amide sp²N–nucleobase sp³N 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 dμ₂/d<i>m</i>₃ = μ₂₃, 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 μ₂₃ values for four less-soluble solutes. Values of μ₂₃ 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 μ₂₃ 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>_p) 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 H₂O

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 sp²O, amide sp²N, aliphatic sp³C, and amide and aromatic sp²C unified atoms in water. Interactions of amide sp²O with urea and naphthalene are favorable, while amide sp²O–alkylurea interactions are unfavorable, becoming more unfavorable with increasing alkylation. Hence, amide sp²O–amide sp²N interactions (proposed n−σ* hydrogen bond) and amide sp²O–aromatic sp²C (proposed n−π*) interactions are favorable in water, while amide sp²O–sp³C interactions are unfavorable. Interactions of all ureas with sp³C and amide sp²N are favorable and increase in strength with increasing alkylation, indicating favorable sp³C–amide sp²N and sp³C–sp³C interactions. Naphthalene results show that aromatic sp²C–amide sp²N interactions in water are unfavorable while sp²C–sp³C 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^– 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 K₂SO₄) 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>₃) dependence of the unfolding equilibrium constant <i>K</i>_obs [<i>r</i>-value = −d ln <i>K</i>_obs/d<i>m</i>₃ = (1/<i>RT</i>) dΔ<i>G</i>_obs^°/d<i>m</i>₃ = <i>m</i>-value/<i>RT</i>]. <i>r</i>-Values for both stabilizing K⁺ salts and destabilizing GuH⁺ 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^–1 and K₂SO₄, KF, KCl, GuHCl, and GuHSCN <i>r</i>-values of 5.38, 1.05, 0.64, −1.38, and −3.00 m^–1, 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_R promoter DNA to form the unstable open intermediate I₂, which rapidly converts to much more stable open complexes (I₃, RP_o). Conversion of I₂ to RP_o 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 I₃. Overall rate constants (<i>k</i>_a) for promoter binding and DNA opening by ΔJAW RNAP are much smaller than for WT RNAP. Values of <i>k</i>_a for WT RNAP with DT+12 and full-length λP_R are similar, though contributions of binding and isomerization steps differ. Hence, the jaw plays major roles both early and late in RP_o 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 (-CH₂OH) and interior (-CH₂OCH₂-) 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_R 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_R 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 Å. 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