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PubMed1997

Three-way curve resolution applied to the study of solvent effect on the thermodynamic and conformational transitions related to the protonation of polycytidylic acid.

Solvent effect on the acid-base behavior of polycytidylic acid [poly(C)] is studied by means of potentiometric and spectrometric (UV and CD) procedures. Low-polarity biological environments are mimicked by using a 30% (v/v) dioxane-water mixture. Experiments performed in this medium are compared with previously reported results in aqueous solution in order to determine changes in both thermodynamic and structural aspects associated with modifications of the biomolecular surroundings. Potentiometric and spectrometric studies reveal the presence of a nonlinear polyelectrolytic effect associated with the protonation of poly(C) in the hydroorganic mixture, different from the analogous effect in aqueous solution. The curve resolution method alternating least squares is applied to the poly(C) spectrometric data. Concentration profiles and spectra of both deprotonated poly(C) and half-protonated [poly(C).poly(CH+)] are thus obtained. The fully protonated species poly(CH+) precipitates in the hydroorganic medium at pH values lower than 4. Evidence for the ordered structure of both poly(C) and [poly(C).poly(CH+)] species is seen through the comparison of the macromolecule spectra with those of the cytidine-3',5'-cyclic monophosphate monomer. Polarity decreases around the macromolecule produce significant hypochromicity in the CD spectra and hyperchromicity in the UV spectra, both signs of a disordering effect in the macromolecular structure due to the weakening of base stacking interactions.

Acid-Base Equilibrium↗

PubMed2002

A thermodynamic characterization of the binding of thrombin inhibitors to human thrombin, combining biosensor technology, stopped-flow spectrophotometry, and microcalorimetry.

The binding of a series of low-molecular-mass, active-site-directed thrombin inhibitors (399-575 Da) to human alpha-thrombin was investigated by surface plasmon resonance technology (BIACORE), stopped-flow spectrophotometry, and isothermal titration microcalorimetry (ITC). The equilibrium constants K(D) (nM to microM range) at 25 degrees C obtained from the BIACORE analysis correlated well with the inhibition constants K(i) in a chromogenic inhibition assay. The interactions between thrombin and three potent inhibitors, melagatran, inogatran, and CH-248, were further investigated at temperatures between 278 and 310K. A one-to-one binding stoichiometry found with ITC was supported by BIACORE data. K(i) and K(D) values increased with the temperature, mainly due to higher values for dissociation rate constants. The changes in enthalpy, DeltaH, and entropy, DeltaS, determined from the linear van't Hoff plots (R coefficient > 0.99), were linearly correlated by chemical compensation. Both techniques indicated clear differences in DeltaS for the three inhibitors, with a strong correlation to the number of rotational bonds. Immobilization of thrombin increased the binding stability at higher temperature and reduced the DeltaH by 20 kJ mol(-1). DeltaH values obtained from the inhibition kinetics and BIACORE were thus not identical, but correlated well with ITC data obtained at 37 degrees C. The two thermodynamic techniques allowed further differentiation between compounds of similar affinity; furthermore, kinetic analysis, hence analysis of the transition state, is complementary to ITC. A direct BIACORE binding assay might be a useful alternative to more elaborate inhibition studies.

Binding Sites↗

PubMed1994

Effect of the order of antibody variable regions on the expression of the single-chain HyHEL10 Fv fragment in E. coli and the thermodynamic analysis of its antigen-binding properties.

In order to physically stabilize the Fv fragment of anti-lysozyme monoclonal antibody, HyHEL10, the variable domains were linked covalently with a flexible linker. A marked difference in the level of expression in E. coli was observed between VH-linker-VL (scFvHL) and VL-linker-VH (scFvLH). The highly expressed scFvLH was purified by a single step of affinity chromatography from the culture supernatant with a typical yield of 3-5 mg per liter of culture. This HyHEL10 scFvLH showed reduced binding activity toward its antigen, HEL, in comparison with Fv. Thermodynamic study showed that this reduced activity was due to entropic loss upon binding to its antigen, although this interaction between scFvLH and its antigen was enthalpically favorable.

Amino Acid Sequence↗

PubMed1995

Thermodynamic characterization of the interaction between cytochrome b5 and cytochrome c.

In recent years many studies have been directed toward the elucidation of the interaction mechanisms within protein-protein complexes. One of the best studies protein-protein complexes has been the cytochrome b5 and cytochrome c electron transfer pair. Thermodynamic information about the association process has been obtained through methods which indirectly measure the binding between the proteins. We report here the use of Isothermal Titration Calorimetry to characterize the association of Rat cytochrome b5 and Horse cytochrome c. The association is accompanied by an unfavorable enthalpy change (+1.0 +/-0.1 Kcal/mole) and a large stabilizing change in entropy (33.9 +/-0.6 eu).

Animals↗

PubMed1998

Kinetic and thermodynamic principles determining the structural design of ATP-producing systems.

It is theoretically analysed whether the structural design of ATP-producing pathways, in particular the design of glycolysis, may be explained by optimization principles. On the basis of kinetic and thermodynamic principles conclusions are derived concerning the stoichiometry of these pathways in states of high ATP production rates. One of the extensions to previous investigations is that the concentrations of the adenine nucleotides are taken into account as variable quantities. This necessitates the consideration of an interaction of the ATP-producing system I with an external ATP-consuming system II. A great variety of pathways is studied which differ in the number and location of ATP-consuming reactions, ATP-producing reactions and reactions involving inorganic phosphate. The corresponding number of possible pathways may be calculated in an explicit manner as a function of the number of those reactions which do not couple to ATP or inorganic phosphate. The kinetics of the individual reactions are described by linear or bilinear functions of reactant concentrations and all rate equations are expressed in terms of equilibrium constants and characteristic times. A thermo-dynamical analysis of the two coupled systems yields upper and lower limits for the concentration of ATP and an explicit expression for the maximal difference between the number of ATP-producing and ATP-consuming reactions of system I. The following results of the optimization are obtained. (i) The ATP production rate always increases if the ATP-producing reactions as well as those reactions characterized by an uptake of inorganic phosphate are shifted as far as possible towards the end of system I. (ii) Explicit conditions for the optimal location of the ATP-consuming reactions are presented. The results are discussed in the context of characteristic times as well as in terms of enzyme kinetic parameters. (iii) For two sets of characteristic times the resulting stoichiometries and their corresponding steady-state fluxes are investigated in detail. One of these stoichiometries shows a close correspondence to contemporary standard glycolysis. (iv) It is shown that most possible pathways result in a very low steady-state flux, that is, the optimal stoichiometry is characterized by a significant selective advantage. (v) The standard free energy profile of a pathway with an optimal stoichiometry is discussed. It differs significantly from the free energy profiles of nonoptimized pathways.

Adenosine Triphosphate↗

PubMed2002

Thermodynamic study of small hydrophobic ions at the water-lipid interface.

The thermodynamics of binding of two small hydrophobic ions such as norharman and tryptophan to neutral and negatively charged small unilamellar vesicles was investigated at pH 7.4 using fluorescence spectroscopy. Vesicles were formed at room temperature from dimyristoyl phosphatidylcholine (DMPC) or DMPC/dimyristoylphosphatidic acid and DMPC/dimyristoylphosphatidylglycerol. The changes in fluorescence properties were used to obtain association isotherms at variable membrane surface negative charge and at different ionic strengths. The binding of both ions was found to be quantitatively enhanced as the percentage of negative phospholipid increases in the membrane. Also, a decrease in ion binding was found to occur as the concentration of monovalent salt was increased (0.045-0.345 M). If electrostatic effects were ignored, the experimental data showed biphasic behavior in Scatchard plots. When electrostatic effects were taken into account by means of the Gouy-Chapman theory, the same data yielded linear Scatchard plots that were described by a simple partition equilibrium of the hydrophobic ion into the lipid-water interface. We demonstrate that the effective interfacial charge, nu, of the ion is a determinant factor to obtain a unique value of the intrinsic (hydrophobic) binding constant independently of the surface charge density of the lipid membrane.

Carbolines↗

PubMed2002

Biomembrane phospholipid-oxide surface interactions: crystal chemical and thermodynamic basis.

Quartz has the least favored surface among many oxides for bacterial attachment and for lipid bilayer or micelle interactions. Tetrahedrally coordinated crystalline silica polymorphs are membranolytic toward liposomes, lysosomes, erythrocytes, and macrophages. Amorphous silica, the octahedral silica polymorph, (stishovite), and oxides such as Al2O3, Fe2 O3, and TiO2 are less cytotoxic. Existing theories for membrane rupture that invoke interactions between oxide surfaces and cell membrane phospholipids (PLs) do not adequately explain these differences in membranolytic potential of the oxides. The author presents a crystal chemical, thermodynamic model for the initial interaction of oxide surfaces with the quaternary ammonium component of the PL's polar head group. The model includes solvation energy changes and electrostatic forces during adsorption, represented by the dielectric constant of the solid and the charge-to-radius ratio of the adsorbing solute. The nature of oxide-solute interactions compared with oxide-water, solute-water, and water-water interactions determines the membranolytic activity of the oxide, where the solute is TMA+, the quaternary ammonium moeity. Significant membrane rupture, as on quartz, requires unfavorable adsorption entropy (DeltaS(ads,TMA+)<0) to maximize disruption of normal membrane structure and requires favorable Gibbs free energy of exchange between TMA+ and the ambient Na+ ions (DeltaG(exc,TMA+/Na+) = DeltaG(ads,TMA+)-DeltaG(ads,Na+)<0) to maximize the extent of membrane affected. For amorphous silica, DeltaS(ads,TMA+) >0, so disruption of structure is limited, even though G(exc,TMA+/Na+) is <0. Stishovite and other oxides have DeltaS(ads,TMA+) <0, but now DeltaG(exc,TMA+/Na+) is>0 at the acidic to circumneutral pHs of cellular and subcellular organelle fluids. The model predicts the correct sequence of membranolytic ability: quartz > or = amorphous SiO2 >Al2O3 >Fe2O3 >TiO2. The model thus explains the relatively poor adhesion of bacterial cells to quartz and the lack of quartz as a biomineral. It is proposed that one function of extracellular polymeric substances exuded by bacteria is to render mineral surfaces more hydrophilic.

Cell Membrane↗

PubMed1993

Conformation and thermodynamics of DNA "necks". Models for three-arm branch formation in a duplex.

The properties of three-arm DNA junctions differ from those of four-arm junctions in several respects. Most apparently, bases flanking the branch are reactive to single strand specific agents in three-arm junctions but not four-arm junctions. To determine the basis for this, we have designed and synthesized a series of complexes in which a short duplex, a neck, progressively extends from a parent 16-mer DNA duplex. These structure allow us to investigate how a three-arm branch forms, and how its properties change as the neck extends. Comparison of the properties of a nicked duplex with those of the neck structures using native gel electrophoresis with reporter arms attached reveals progressively greater geometrical perturbation of the complexes as the number of base-pairs in the neck increases. Footprinting by single-strand specific reagents indicates that the reactivity to single-strand reagents near the branch occurs when only a single pair is possible. The branch in each neck interacts tightly with ethidium, as does a nick in the same duplex. The thermodynamics of neck formation have been evaluated by calorimetry and from the concentration dependence of absorbance temperature profiles. Each neck complex is destabilized with respect to duplex DNA or a nicked duplex, and has a lower enthalpy of formation despite the increased number of base-pairs present. A model is proposed to account for these properties in which the bases in the pair adjacent to the duplex interact directly with the duplex, via transient insertion.

Base Sequence↗

PubMed1993

Contribution of hydration to protein folding thermodynamics. I. The enthalpy of hydration.

The enthalpy of hydration of polar and non-polar groups upon protein unfolding has been estimated for four globular proteins in the temperature range 5 to 125 degrees C, using structural information on the groups in these proteins exposed to water in the native and unfolded states and volume-corrected calorimetric information on the enthalpy and heat capacity of transfer into water of various model compounds. It has been shown that the enthalpy of hydration of polar groups greatly exceeds the enthalpy of hydration of non-polar groups. At low temperatures both these enthalpies are negative and change in opposite direction with increasing temperature. Subtracting the total enthalpy of hydration of polar and non-polar groups from the calorimetrically determined enthalpy of protein unfolding, the total enthalpy of internal interactions maintaining the native protein structure has been determined. Using thermodynamic information on the sublimation of organic crystals, the total enthalpy was divided into two components: one associated with the interactions between the non-polar groups (van der Waals interaction) and the rest associated with the interactions between polar groups (hydrogen bonding). This made it possible to estimate the overall enthalpies of disruption of contacts between the polar groups with their exposure to water and between the non-polar groups with their exposure to water. It appears that these enthalpies have opposite signs in the temperature range considered and change in opposite directions with increasing temperature. The enthalpy of transfer of non-polar groups from the protein interior into water is negative below 25 degrees C and positive above. The enthalpy of transfer of polar groups from the protein interior into water is positive at low temperatures and becomes negative at higher temperatures. Over the considered temperature range, however, the enthalpy of transfer of non-polar groups dominates. This results in a positive enthalpy of protein unfolding at elevated temperatures. The opposite sign and temperature dependence of the specific values of these two enthalpies for the considered proteins explains the experimentally observed convergence of the specific enthalpies of globular protein unfolding at about 130 degrees C.

Amino Acids↗

PubMed1995

Kinetics and thermodynamics of the RNase P RNA cleavage reaction: analysis of tRNA 3'-end variants.

We have studied the interaction of 3'-end variants of a (pre-)tRNAGly with ribonuclease P (RNase P) RNAs from Escherichia coli and Thermus thermophilus. To dissect the thermodynamics of tRNA binding from the overall catalytic reaction, specific binding of mature tRNAGly variants to RNase P RNAs was studied by gel retardation. A newly developed assay, based on the reduction of Pb(2+)-hydrolysis at the CCA end due to complex formation of tRNA and RNase P RNA, was utilized to confirm the dissociation constants. The binding data were supplemented by single and multiple turnover kinetic analyses of the corresponding pre-tRNAGly variants. For E. coli RNase P RNA the following results were obtained. Extensions of CCA by pCp or three nucleotides (AUA) stabilized gel-resolved tRNAGly binding by 1 to 1.5 kcal/mol. Changing the first C in CCA to A, G or U resulted in a more than 100-fold reduction in binding affinity, which corresponds to a loss of 3.5 to 4.5 kcal/mol of binding energy. However, single turnover rate constants were only slightly affected, indicating that a disruption or loss of the tRNA 3'-end-mediated interaction with RNase P RNA does not preferentially destabilize the transition state. Our data suggest another kinetic step following initial substrate binding to E. coli RNase P RNA (possibly a conformational rearrangement). For T. thermophilus RNase P RNA, product release of wild-type tRNAGly CCAAUA was not rate-limiting in the multiple turnover reaction. However, the effects of CCA mutations were similar to those attained with E. coli RNase P RNA. This supports the notion that a high-affinity binding site for the tRNA 3'-end is a ubiquitous feature of eubacterial P RNAs. Finally, the results obtained here provide further evidence that the gel retardation assay is suitable for binding interference studies to identify the structural elements of RNase P RNAs and tRNAs that are crucial for the formation of a specific RNase P RNA-tRNA complex.

Base Sequence↗

PubMed1994

Thermodynamics of folding a pseudoknotted mRNA fragment.

A sequence in the leader and first gene of the Escherichia coli alpha mRNA folds into a complex pseudoknot structure that is required for binding of a translational repressor. The thermal denaturation of a 112 nt RNA containing this structure has been followed by calorimetry and UV hyperchromicity. To determine the partially folded intermediates in unfolding, the denaturation of 13 mutants and of several fragments with successive deletions of helices were investigated as well. An unfolding pathway with seven states is proposed as the simplest mechanism that accounts for the data, and has several implications. (1) The lowest temperature transition appears only in the presence of moderate concentrations of Mg2+ or high concentrations of K+ (delta H approximately 45 kcal/mol), and is the unfolding of tertiary structures, rather than secondary structure. Under some conditions it is destabilized by increasing salt concentration. (2) Two of the intermediates unfolding at higher temperature must have non-canonical or tertiary interactions in addition to the known secondary structure. (3) Two alternative structures compete for formation of the complete pseudoknot, and form as the pseudoknot unfolds. Thus structures not present in the completely folded pseudoknot affect the overall thermodynamics, and probably the kinetics, of unfolding. (4) Approximately 16 kcal/mol of free energy is required to completely expose the coding region to ribosomes at 37 degrees C, though approximately 6.5 kcal/mol is regained by refolding of upstream regions after the pseudoknot is unfolded. The substantial energy needed to unfold the pseudoknot may affect the rate of translation from this ribosome binding site. A simple model of RNA folding in which an optimum secondary structure forms first, followed by tertiary interactions that further stabilize the secondary structure, does not hold in this RNA.

Base Sequence↗

PubMed1995

A thermodynamic study of mutant forms of Streptomyces subtilisin inhibitor. I. Hydrophobic replacements at the position of Met103.

The thermodynamic effects of replacing the Met residue at amino acid position 103 of Streptomyces subtilisin inhibitor with other non-polar aliphatic residues were studied by means of differential scanning calorimetry. All but the Leu mutant, which is as stable as the wild-type but has different cooperative units in the course of unfolding, showed destabilization in terms of free energy. Similar losses in free energy, however, were caused by different reasons, i.e. by increased entropy for the Ala mutant and by decreased enthalpy for the Ile mutant, with a tendency that increases in entropy are accompanied by increases in enthalpy. The gain in entropy that caused the largest loss in free energy for the Gly mutant was unexpectedly smaller than that for the Ala mutant. The changes in enthalpy and entropy induced by the mutations exhibited some correlations with hydrophobicity, while no clear correlation was found between the changes in free energy and hydrophobicity.

Bacterial Proteins↗

PubMed1995

A thermodynamic study of mutant forms of Streptomyces subtilisin inhibitor. III. Replacements of a hyper-exposed residue, Met73.

Amino acid replacements of Met73, which is hyper-exposed in the native structure, with Asp, Glu, Lys, Gly, Ala, Val, Leu or Ile changed the stability of Streptomyces subtilism inhibitor by +1.60 to -0.94 kcal/mol-1 in free energy at 82.21 degrees C at pH 7.0, with higher hydrophobicity of the replacing amino acid side-chain showing a correlation with lower stability. Thermodynamic parameters obtained from detailed calorimetric analyses gave a nearly proportional relation between the entropy change and the enthalpy change of denaturation, i.e. T delta delta S degrees = -0.03 (+/- 0.11) + 1.14 (+/- 0.03) delta delta H, with the linear correlation coefficient 0.996 for 18 data points. This proportionality observed uniquely for the substitutions at position 73 was caused primarily by the water/side-chain interaction, or hydration effect, which can account for the majority of the changes in enthalpy and entropy induced by the mutations.

Bacterial Proteins↗

PubMed1995

Thermodynamics of unfolding of ribonuclease A under high pressure. A study by proton NMR.

Thermodynamic stability of ribonuclease A (6.2 mM pH 1.0, 0.15 M KCl, in 2H2O) has been studied in the pressure range of 1 to 2000 atm and in the temperature range of 7.5 to 40 degrees C with a high pressure 1H NMR technique at 400 MHz. His epsilon proton resonances were used as reporter groups to measure fractions of folded and unfolded species. Gibbs energy differences between folded and unfolded species were obtained as functions of pressure for different temperatures and as functions of temperature for different pressures. The volume increase upon unfolding, delta V, was negative and temperature-dependent, decreasing from -10 ml/mol at 7.5 degrees C to -30 ml/mol at 37 degrees C. From the least squares-fitting of experimental Gibbs energy differences to a theoretical expression holding pressure and delta Cp constant, we determined best-fit values of delta G, delta H, delta S and delta Cp for different values of pressure in the temperature range 7.5 to 40 degrees C. We found that delta Cp is dependent on pressure, decreasing from 1.79 kcal/mol K at 1 atm to 1.08 kcal/mol K at 2000 atm. These findings appear to be consistent with a notion that the state of hydration of non-polar side-chains upon unfolding of the protein is a major factor that determines the pressure dependence of the conformational stability of ribonuclease A under the chosen experimental condition.

Animals↗

PubMed1995

The unfolding thermodynamics of c-type lysozymes: a calorimetric study of the heat denaturation of equine lysozyme.

The energetics of the temperature-induced unfolding of equine lysozyme was studied calorimetrically and compared with that of two structurally homologous proteins: hen egg white lysozyme and alpha-lactalbumin. The structure of each of these proteins is characterized by the presence of a deep cleft that divides the molecule into two regions called the alpha and beta domains. In equine lysozyme and alpha-lactalbumin the latter domain specifically binds Ca2+. It is shown that, in contrast to hen egg white lysozyme in which the alpha and beta domains unfold as a single cooperative unit, in equine lysozyme the two domains unfold in two separate cooperative stages even in the presence of excess Ca2+. The calcium binding beta-domain unfolds at a lower temperature and with more extensive heat absorption than the alpha-domain. Binding of Ca2+ increases the stability of the beta-domain, but even in the holo form it is less stable than the alpha-domain. The thermodynamic characteristics of Ca2+ binding have been determined, and indicate that it is an entropically driven process. The unfolding of equine lysozyme largely resembles the unfolding of alpha-lactalbumin, which also unfolds in two stages, but in the latter case the second stage is much less cooperative and proceeds with a smaller and diffuse heat absorption. As a result, the total enthalpy of unfolding of equine lysozyme is significantly larger than that of alpha-lactalbumin, being almost of the same magnitude as the enthalpy of egg white lysozyme unfolding, which proceeds as a single two-state transition. Analyses of the unfolding enthalpy function of various lysozymes, which bind or do not bind Ca2+, and unfold in one or two stages, have led us to the conclusion that the main reason for the loss of interdomain cooperativity in equine lysozyme is not the cluster of negative charges forming the calcium binding site, but the difference in atomic packing in the interior and at the interface between the alpha and beta domains.

Animals↗

PubMed1995

Calculation of relative binding free energies and configurational entropies: a structural and thermodynamic analysis of the nature of non-polar binding of thrombin inhibitors based on hirudin55-65.

Free energy calculations were carried out on a series of exosite-binding inhibitors of thrombin. These inhibitors are based on the C-terminal fragment of hirudin and have the sequence Phe-Glu-Glu-IleH59-Pro-Glu-Glu-Tyr- Leu, where the superscript over Ile indicates its relative position in the natural sequence of hirudin. In this study, the effect of replacing IleH59 with ten other non-polar amino acids was examined. Three preferred interaction sites for methyl/methylene groups for the various XaaH59 side-chains in the complex were identified from conformational search calculations. The corresponding thermodynamic changes were determined using a combination of systematic search and energy minimization in a manner that locates the local minima in the system and in the process simultaneously builds up the partition function. The free energy, internal energy and entropic contributions are readily calculated from the partition function. Very good agreement in the resulting relative binding free energies was obtained between theory and experiment. The calculations allowed us to dissect out the enthalpic, entropic and solvation contributions to delta delta G. The contribution from desolvation was found to be relatively weak. The binding of these non-polar side-chains to thrombin is found to be driven mainly by favorable protein-ligand interactions rather than by the desire for non-polar groups to be desolvated. We also find that the configurational entropy contributes about 0.48 kcal/mol (0.81 kappa T) in average for each torsional angle "frozen" in binding.

Algorithms↗

PubMed1996

Compensating effects of opposing changes in putrescine (2+) and K+ concentrations on lac repressor-lac operator binding: in vitro thermodynamic analysis and in vivo relevance.

Ion concentrations (K+, Glu-) in the cytoplasm of growing Escherichia coli cells increase strongly with increases in the osmolarity of a defined growth medium. While in vitro experiments demonstrate that the extent of protein-nucleic acid interactions (PNAI) depends critically on salt concentration, in vivo measurements indicate that cells maintain a relatively constant extent of PNAI independent of the osmolarity of growth. How do cells buffer PNAI against changes in the cytoplasmic environment? At high osmolarity, the increase in macromolecular crowding which accompanies the reduction in amount of cytoplasmic water in growing cells appears quantitatively sufficient to compensate for the increase in [K+]. At low osmolarity, however, changes in crowding appear to be insufficient to compensate for changes in [K+], and additional mechanisms must be involved. Here we report quantitative determinations of in vivo total concentrations of polyamines (putrescine(2+), spermidine(3+)) as a function of osmolarity (OsM) of growth, and in vitro binding data on the effects of putrescine concentration on a specific PNAI (lac repressor-lac operator) as a function of [K+]. The total concentration of putrescine in cytoplasmic water decreases at least eightfold from low osmolarity (approximately 64 mmol (l H2O)-1 at 0.03 OsM) to high osmolarity (approximately 8 mmol (l H2O)-1 at 1.02 OsM). Over this osmotic range the total [K+] increases from approximately 0.2 mol (l H2O)-1 to approximately 0.8 mol (lH2O)-1. We find that the effect of putrescine concentration on the repressor-operator interaction in vitro is purely competitive and is quantitatively described by a simple competition formalism in which lac repressor behaves a a specific-binding oligocation (ZR = 8+/-3). We demonstrate that this thermodynamic result is consistent with a structural analysis of the number of positively charged side-chains on two DNA binding domains of repressor which interact with the phosphodiester backbone of the operator site. Since this oligocation character of the binding surface of DNA-binding proteins appears to be general, we propose the competitive effects of putrescine and K+ concentrations on the strength of specific binding are general. At low osmolarity, compensating changes in putrescine and K+ concentration in response to changes in external osmolarity provide a general mechanism for E. coli to vary cytoplasmic osmolarity while maintaining a constant extent of PNAI.

Base Sequence↗

PubMed1996

Thermodynamic prediction of conserved secondary structure: application to the RRE element of HIV, the tRNA-like element of CMV and the mRNA of prion protein.

An algorithm for prediction of conserved secondary structure of single-stranded RNA is presented. For each RNA of a set of homologous RNAs optimal and suboptimal secondary structures are calculated and stored in a base-pair probability matrix. A multiple sequence alignment is performed for the set of RNAs. The resulting gaps are introduced into the individual probability matrices. These homologous probability matrices are summed to give a consensus probability matrix emphasizing the conserved secondary structure elements of the RNA set. Thus the algorithm combines the advantages of thermodynamic structure prediction by energy minimization with the information obtained from phylogenetic alignment of sequences. The algorithm is applied to three examples. The REV-responsive element of HIV, the structure of which is well known from the literature, was chosen to test the algorithm. The second example is the 3' terminal segment of genomic single-stranded RNAs of cucumber mosaic viruses; a structure similar to that of the related brome mosaic virus was expected and was confirmed. The third example is the prion-protein mRNA from different organisms; the structure of this mRNA is not known. By application of the algorithm highly conserved hairpins were found in the prion-protein mRNA.

Algorithms↗