Energy coupling between DNA binding and subunit association is responsible for the specificity of DNA-Arc interaction.

The effects of several DNA molecules on the free energy of subunit association of Arc repressor were measured. The association studies under equilibrium conditions were performed by the dissociating perturbation of hydrostatic pressure. The magnitude of stabilization of the subunit interaction was determined by the specificity of the protein-DNA interaction. Operator DNA stabilized the free energy of association by about 2.2 kcal/mol of monomeric unit, whereas poly(dG-dC) stabilized the subunit interaction by only 0.26 kcal. Measurements of the stabilizing free energy at different DNA concentrations revealed a stoichiometry of two dimers per 21 bp for the operator DNA sequence and for the nonspecific DNA poly(dA-dT). However, the maximum stabilization was much larger for operator sequence (delta p = 1,750 bar) as compared for poly(dA-dT) (delta p = 750 bar). The importance of the free-energy linkage for the recognition process was corroborated by its absence in a mutant Arc protein (PL8) that binds to operator and nonspecific DNA sequences with equal, low affinity. We conclude that the coupling accounts for the high specificity of the Arc-operator DNA interaction. We hypothesize a mutual coupling between the protein subunits and the two DNA strands, in which the much higher persistency of the associated form when Arc is bound to operator would stabilize the interactions between the two DNA strands.     

Cis proline mutants of ribonuclease A. I. Thermal stability.

A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.     

Thermodynamics of unfolding for turkey ovomucoid third domain: thermal and chemical denaturation.

We have used thermal and chemical denaturation to characterize the thermodynamics of unfolding for turkey ovomucoid third domain (OMTKY3). Thermal denaturation was monitored spectroscopically at a number of wave-lengths and data were subjected to van't Hoff analysis; at pH 2.0, the midpoint of denaturation (Tm) occurs at 58.6 +/- 0.4 degrees C and the enthalpy of unfolding at this temperature (delta Hm) is 40.8 +/- 0.3 kcal/mol. When Tm was perturbed by varying pH and denaturant concentration, the resulting plots of delta Hm versus Tm yield a mean value of 590 +/- 120 cal/(mol.K) for the change in heat capacity upon unfolding (delta Cp). A global fit of the same data to an equation that includes the temperature dependence for the enthalpy of unfolding yielded a value of 640 +/- 110 cal/(mol.K). We also performed a variation of the linear extrapolation method described by Pace and Laurents, which is an independent method for determining delta Cp (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 2520-2525). First, OMTKY3 was thermally denatured in the presence of a variety of denaturant concentrations. Linear extrapolations were then made from isothermal slices through the transition region of the denaturation curves. When extrapolated free energies of unfolding (delta Gu) were plotted versus temperature, the resulting curve appeared linear; therefore, delta Cp could not be determined. However, the data for delta Gu versus denaturant concentration are linear over an extraordinarily wide range of concentrations. Moreover, extrapolated values of delta Gu in urea are identical to values measured directly.     

Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions.

The objective of this study was to address the question of whether or not urea and guanidine hydrochloride (GdnHCl) give the same estimates of the stability of a particular protein. We previously suspected that the estimates of protein stability from GdnHCl and urea denaturation data might differ depending on the electrostatic interactions stabilizing the proteins. Therefore, 4 coiled-coil analogs were designed, where the number of intrachain and interchain electrostatic attractions (A) were systematically changed to repulsions (R): 20A, 15A5R, 10A10R, and 20R. The GdnHCl denaturation data showed that the 4 coiled-coil analogs, which had electrostatic interactions ranging from 20 attractions to 20 repulsions, had very similar [GdnHCl]1/2 values (average of congruent to 3.5 M) and, as well, their delta delta Gu values were very close to 0 (0.2 kcal/mol). In contrast, urea denaturation showed that the [urea]1/2 values proportionately decreased with the stepwise change from 20 electrostatic attractions to 20 repulsions (20A, 7.4 M; 15A5R, 5.4 M; 10A10R, 3.2 M; and 20R, 1.4 M), and the delta delta Gu values correspondingly increased with the increasing differences in electrostatic interactions (20A-15A5R, 1.5 kcal/mol; 20A-10A10R, 3.7 kcal/mol; and 20A-20R, 5.8 kcal/mol). These results indicate that the ionic nature of GdnHCl masks electrostatic interactions in these model proteins, a phenomenon that was absent when the unchanged urea was used. Thus, GdnHCl and urea denaturations may give vastly different estimates of protein stability, depending on how important electrostatic interactions are to the protein.     

Nucleotide sequence of aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase.

In Escherichia coli, the phosphorylation and dephosphorylation of isocitrate dehydrogenase (IDH) are catalyzed by a bifunctional protein kinase/phosphatase. We have determined the nucleotide sequence of aceK, the gene encoding IDH kinase/phosphatase. This gene consists of a single open reading frame of 1,734 base pairs preceded by a Shine-Dalgarno ribosome-binding site. Examination of the deduced amino acid sequence of IDH kinase/phosphatase revealed sequences which are similar to the consensus sequence for ATP-binding sites. This protein did not, however, exhibit the extensive sequence homologies which are typical of other protein kinases. Multiple copies of the REP family of repetitive extragenic elements were found within the intergenic region between aceA (encoding isocitrate lyase) and aceK. These elements have the potential for combining to form an exceptionally stable stem-loop structure (delta G = -54 kcal/mol [ca. -226 kJ/mol]) in the mRNA. This structure, which masks the ribosome-binding site and start codon for aceK, may contribute to the downshift in expression observed between aceA and aceK. Another potential stem-loop structure (delta G = -29 kcal/mol [ca. 121 kJ/mol]), unrelated to the REP sequences, was found within aceK.     

Stability mutants of staphylococcal nuclease: large compensating enthalpy-entropy changes for the reversible denaturation reaction

By use of intrinsic fluorescence to determine the apparent equilibrium constant Kapp as a function of temperature, the midpoint temperature Tm and apparent enthalpy change delta Happ on reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a Tm of 53.3 +/- 0.2 degrees C and a delta Happ of 86.8 +/- 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 degrees C and 96 +/- 2 kcal/mol. The heat capacity change on denaturation delta Cp was estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of delta Happ and delta Sapp for a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated delta Cp for the mutant proteins and the d(delta Gapp)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions.     

Contribution of the hydrophobic effect to globular protein stability.

The decrease in conformational stability, delta(delta G), has been measured for 72 aliphatic side-chain mutants from four proteins in which a larger side-chain is replaced by a smaller side-chain so that steric effects are minimal. When these delta(delta G) values are corrected to the same accessibility, namely 100% buried, then the following -delta(delta G) values per -CH2- group (in kcal/mol) are obtained: Ile----Val (1.26), Ala (1.26), Gly (1.26); Leu----Ala (1.16), Gly (1.21); Val----Ala (1.23), Gly (1.53). The average of these values is 1.27(+/- 0.07) kcal/mol. The 72 individual values range from 0 to 2.4 kcal/mol with an average value of 1.27(+/- 0.51) (standard deviation) kcal/mol. When the delta Gtr values from n-octanol to water are corrected for the difference in volume between the solutes and the solvents, the average value for the same substitutions is 1.25(+/- 0.05) kcal/mol. This suggests that proteins gain 1.3(+/- 0.5) kcal/mol in stability for each -CH2- group buried in folding, and, furthermore, that the volume corrected delta Gtr values for n-octanol for the amino acid side-chains provide good estimates of the contribution of the hydrophobic effect to globular protein stability.     

Generating compatible translation initiation regions for heterologous gene expression in Escherichia coli by exhaustive periShine-Dalgarno mutagenesis. Human glutathione reductase cDNA as a model.

Adaptation of eucaryotic cDNA to heterologous expression was studied by mutating the translation initiation (TI) region upstream (mTI) and downstream (MTI) of the start codon. In the mTI subregion the 8 bases flanking the invariant Shine-Dalgarno motif GG-AG were mutagenized exhaustively, while the MTI subregion was subjected to random silent mutations at the wobble positions. The quality of a given TI sequence was judged on the basis of expressed enzyme activity. Low-yield and high-yield mutants of both TI subregions were selected and recombined systematically. The analysis of these double cartridges gave the following results: 1. As a rule, an unfavourable MTI subregion can be compensated for by mutations in the mTI subregion and vice versa. 2. The compatibility between mTI and MTI subregion is explainable at least in part by a low interaction tendency; a delta G(o)'-value of -10.7 kcal/mol appears to be a physical threshold for heterologous cDNA expression. 3. On the basis of periShine-Dalgarno mutations, the expression yield for different cDNA sequences could be increased by 1 to 2 orders of magnitude. One of these sequences encoded delta(1-15)human glutathione reductase, a mutant lacking the flexible N-terminal extension of the protein. In conclusion, to study and overcome TI region-based expression problems it is worthwhile to start out with a versatile vector containing exhaustive mutations in the periShine-Dalgarno sequences; as a rule the coding MTI subregion can be kept unchanged.     

Structural insight into the kinetics and DeltaCp of interactions between TEM-1 beta-lactamase and beta-lactamase inhibitory protein (BLIP)

In a previous study, we examined thermodynamic parameters for 20 alanine mutants in beta-lactamase inhibitory protein (BLIP) for binding to TEM-1 beta-lactamase. Here we have determined the structures of two thermodynamically distinctive complexes of BLIP mutants with TEM-1 beta-lactamase. The complex BLIP Y51A-TEM-1 is a tight binding complex with the most negative binding heat capacity change (DeltaG = approximately -13 kcal mol(-1) and DeltaCp = approximately -0.8 kcal mol(-1) K(-1)) among all of the mutants, whereas BLIP W150A-TEM-1 is a weak complex with one of the least negative binding heat capacity changes (DeltaG = approximately -8.5 kcal mol(-1) and DeltaCp = approximately -0.27 kcal mol(-1) K(-1)). We previously determined that BLIP Tyr51 is a canonical and Trp150 an anti-canonical TEM-1-contact residue, where canonical refers to the alanine substitution resulting in a matched change in the hydrophobicity of binding free energy. Structure determination indicates a rearrangement of the interactions between Asp49 of the W150A BLIP mutant and the catalytic pocket of TEM-1. The Asp49 of W150A moves more than 4 angstroms to form two new hydrogen bonds while losing four original hydrogen bonds. This explains the anti-canonical nature of the Trp150 to alanine substitution, and also reveals a strong long distance coupling between Trp150 and Asp49 of BLIP, because these two residues are more than 25 angstroms apart. Kinetic measurements indicate that the mutations influence the dissociation rate but not the association rate. Further analysis of the structures indicates that an increased number of interface-trapped water molecules correlate with poor interface packing in a mutant. It appears that the increase of interface-trapped water molecules is inversely correlated with negative binding heat capacity changes.     

The contribution of polar group burial to protein stability is strongly context-dependent

We previously suggested that proteins gain more stability from the burial and hydrogen bonding of polar groups than from the burial of nonpolar groups (Pace, C. N. (2001) Biochemistry 40, 310-313). To study this further, we prepared eight Thr-to-Val mutants of RNase Sa, four in which the Thr side chain is hydrogen-bonded and four in which it is not. We measured the stability of these mutants by analyzing their thermal denaturation curves. The four hydrogen-bonded Thr side chains contribute 1.3 +/- 0.9 kcal/mol to the stability; those that are not still contribute 0.4 +/- 0.9 kcal/mol to the stability. For 40 Thr-to-Val mutants of 11 proteins, the average decrease in stability is 1.0 +/- 1.0 kcal/mol when the Thr side chain is hydrogen-bonded and 0.0 +/- 0.5 kcal/mol when it is not. This is clear evidence that hydrogen bonds contribute favorably to protein stability. In addition, we prepared four Val-to-Thr mutants of RNase Sa, measured their stability, and determined their crystal structures. In all cases, the mutants are less stable than the wild-type protein, with the decreases in stability ranging from 0.5 to 4.4 kcal/mol. For 41 Val-to-Thr mutants of 11 proteins, the average decrease in stability is 1.8 +/- 1.3 kcal/mol and is unfavorable for 40 of 41 mutants. This shows that placing an [bond]OH group at a site designed for a [bond]CH3 group is very unfavorable. So, [bond]OH groups can contribute favorably to protein stability, even if they are not hydrogen-bonded, if the site was selected for an [bond]OH group, but they will make an unfavorable contribution to stability, even if they are hydrogen-bonded, when they are placed at a site selected for a [bond]CH3 group. The contribution that polar groups make to protein stability depends strongly on their environment.     

Crevice-forming mutants of bovine pancreatic trypsin inhibitor: stability changes and new hydrophobic surface.

Four mutants of bovine pancreatic trypsin inhibitor (BPTI) with replacements in the rigid core result in the creation of deep crevices on the surface of the protein. Other than crevices at the site of the mutation, few other differences are observed in the crystal structures of wild-type BPTI and the mutants F22A, Y23A, N43G, and F45A. These mutants are highly destabilized relative to wild type (WT). The differences between WT and mutants in the free energy change associated with cooperative folding/unfolding, delta delta G0 (WT-->mut), have been measured by calorimetry, and they are in good agreement with delta delta G0(WT-->mut) values from hydrogen exchange rates. For F22A the change in free energy difference is about 1.7 kcal/mol at 25 degrees C; for the other three mutants it is in the range of 5-7 kcal/mol at 25 degrees C. The experimental delta delta G0(WT-->mut) values of F22A, Y23A, and F45A are reasonably well accounted for as the sum of two terms: the difference in transfer free energy change, and a contribution from exposure to solvent of new surface (Eriksson, A.E., et al., 1992, Science 255, 178-183), if the recently corrected transfer free energies and surface hydrophobicities (De Young, L. & Dill, K., 1990, J. Phys. Chem. 94, 801-809; Sharp, K.A., et al., 1991a, Science 252, 106-109) are used and only nonpolar surface is taken into account. In N43G, three protein-protein hydrogen bonds are replaced by protein-water hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein structural plasticity exemplified by insertion and deletion mutants in T4 lysozyme.

To further investigate the ways in which proteins respond to changes in the length of the polypeptide chain, a series of 32 insertions and five deletions were made within nine different alpha-helices of T4 lysozyme. In most cases, the inserted amino acid was a single alanine, although in some instances up to four residues, not necessarily alanine, were used. Different insertions destabilized the protein by different amounts, ranging from approximately 1 to 6 kcal/mol. In one case, no protein could be obtained. An "extension" mutant in which the carboxy terminus of the molecule was extended by four alanines increased stability by 0.3 kcal/mol. For the deletions, the loss in stability ranged from approximately 3 to 5 kcal/mol. The structures of six insertion mutants, as well as one deletion mutant and the extension mutant, were determined, three in crystal forms nonisomorphous with wild type. In all cases, including previously described insertion mutants within a single alpha-helix, there appears to be a strong tendency to preserve the helix by translocating residues so that the effects of the insertion are propagated into a bend or loop at one end or the other of the helix. In three mutants, even the hydrophobic core was disrupted so as to permit the preservation of the alpha-helix containing the insertion. Translocation (or "register shift") was also observed for the deletion mutant, in this case a loop at the end of the helix being shortened. In general, when translocation occurs, the reduction in stability is only moderate, averaging 2.5 kcal/mol. Only in the most extreme cases does "bulging" or "looping-out" occur within the body of an alpha-helix, in which case the destabilization is substantial, averaging 4.9 kcal/mol. Looping-out can occur for insertions close to the end of a helix, in which case the destabilization is less severe, averaging 2.6 kcal/mol. Mutant A73-[AAA] as well as mutants R119-[A] and V131-[A], include shifts in the backbone of 3-6 A, extending over 20 residues or more. As a result, residues 114-142, which form a "cap" on the carboxy-terminal domain, undergo substantial reorganizations such that the interface between this "cap" and the rest of the protein is altered substantially. In the case of mutant A73-[AAA], two nearby alpha-helices, which form a bend of approximately 105 degrees in the wild-type structure, reorganize in the mutant structure to form a single, essentially straight helix. These structural responses to mutation demonstrate the plasticity of protein structures and illustrate ways in which their three-dimensional structures might changes during evolution.     

The thermodynamics of bovine and porcine insulin and proinsulin association determined by concentration difference spectroscopy

Difference spectroscopy was used to determine the equilibrium constants and thermodynamic parameters for the monomer-dimer association of bovine and porcine insulin and bovine proinsulin at pH 2.0 and 7.0. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -6.6 kcal/mol, -18 cal/mol-deg, and -12 kcal/mol, respectively. Porcine insulin behaved similarly to bovine insulin in its dimerization properties in that delta G degree 25, delta S degree, and delta H degree were found to be -6.8 kcal/mol, -14 cal/mol-deg, and -11 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -7.2 kcal/mol, -16 cal/mol/deg, and -12 kcal/mol, respectively. At pH 7.0 delta G degree 25, delta S degree, and delta H degree for dimerization of porcine insulin were -6.7 kcal/mol, -11.6 cal/mol-deg, and -10 kcal/mol, respectively. The similarity in the thermodynamic parameters of both insulin species at the different pH's suggests that there are minimal structural changes at the monomer-monomer contact site over this pH range. The dimerization of both insulin species is under enthalpic control. This may suggest that the formation of the insulin dimer is not driven by hydrophobic bonding but, rather, is driven by the formation between subunits of four hydrogen bonds in an apolar environment. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine proinsulin were found to be -5.3 kcal/mol, -26 cal/mol-deg, and -13 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of proinsulin were -5.9 kcal/mol, -4.2 cal/mol-deg, and -7.2 kcal/mol, respectively. Although the presence of the C-peptide on proinsulin does not drastically affect the overall free energy change of dimer formation (as compared to insulin), the other thermodynamic parameters are rather drastically altered. This may be because of electrostatic interactions of groups on the C-peptide with groups on the B-chain which are near the subunit contact site in the insulin dimer.     

Intercalation binding of 6-substituted naphthothiopheneamides to DNA: enthalpy and entropy components.

N-(3-dimethylaminopropyl)naphtho[2,1-b]thiophene-4-carboxamide and the 6-substituted methoxy, methyl, fluoro, chloro, bromo, trifluoromethyl, and cyano derivatives have been shown to bind to DNA via intercalation with binding constants in the 35-900 X 10(3) range at 25 degrees C, pH 7, and [Na+] = 0.019M. Both electron-donating and -withdrawing substituents enhance intercalation binding, but the binding affinity is most enhanced by the cyano substituent. Calorimetric titrations for calf thymus DNA differ dramatically from those reported for ethidium [Hopkins et al. (1990) Biopolymers Vol. 29, pp. 449-459]. Apparent enthalpy parameters (delta HB) for intercalation are constant only at low coverage of sites and become much more positive as saturation is approached. In the plateau region, delta HB values for the parent and the cyano-, fluoro-, chloro-, and bromo-substituted compounds are nearly the same (approximately -5.9 kcal/mol). For the methyl- (-6.8 kcal/mol) and methoxy- (-7.5 kcal/mol) substituted compounds, the delta HB values are more exothermic than that for the unsubstituted compound, whereas delta HB for the trifluoromethyl compound is approximately 1 kcal/mol less exothermic. The corresponding delta SB values, corrected for mixing effects, are in the 7-15-cal/deg/mol range and are approximately linearly related to delta HB if the cyano derivative is excluded.     

Structural studies of N- and C-terminally truncated human apolipoprotein A-I

Apolipoprotein A-I (apoA-I) plays an important structural and functional role in lipid transport and metabolism. This work is focused on the central region of apoA-I (residues 60-183) that is predicted to contain exclusively amphipathic alpha-helices. Six N- and/or C-terminally truncated mutants, delta(1-41), delta(1-59), delta(198-243), delta(209-243), delta(1-41,185-243), and delta(1-59,185-243), were analyzed in their lipid-free state in solution at pH 4.7-7.8 by far- and near-UV CD spectroscopy. At pH 7.8, all mutants show well-defined secondary structures consisting of 40-52% alpha-helix. Comparison of the alpha-helix content in the wild type and mutants suggests that deletion of either the N- or C-terminal region induces helical unfolding elsewhere in the structure, indicating that the terminal regions are important for the integrity of the solution conformation of apoA-I. Near-UV CD spectra indicate significant tertiary and/or quaternary structural changes resulting from deletion of the N-terminal 41 residues. Reduction in pH from 7.8 to 4.7 leads to an increase in the mutant helical content by 5-20% and to a large increase in thermal unfolding cooperativity. Van't Hoff analysis of the mutants at pH 4.7 indicates melting temperatures T(m) ranging from 51 to 59 degrees C and effective enthalpies deltaH(v)(T(m)) = 35 +/- 5 kcal/mol, similar to the values for plasma apoA-I at pH 7.8 (T(m) = 57 degrees C, deltaH(v) = 32 kcal/mol). Our results provide the first report of the pH effects on the secondary, tertiary, and/or quaternary structure of apoA-I variants and indicate the importance of the electrostatic interactions for the solution conformation of apoA-I.     

Filling small, empty protein cavities: structural and energetic consequences

Most proteins contain small cavities that can be filled by replacing cavity-lining residues by larger ones. Since shortening mutations in hydrophobic cores tend to destabilize proteins, it is expected that cavity-filling mutations may conversely increase protein stability. We have filled three small cavities in apoflavodoxin and determined by NMR and equilibrium unfolding analysis their impact in protein structure and stability. The smallest cavity (14 A3) has been filled, at two different positions, with a variety of residues and, in all cases, the mutant proteins are locally unfolded, their structure and energetics resembling those of an equilibrium intermediate of the thermal unfolding of the wild-type protein. In contrast, two slightly larger cavities of 20 A3 and 21 A3 have been filled with Val to Ile or Val to Leu mutations and the mutants preserve both the native fold and the equilibrium unfolding mechanism. From the known relationship, observed in shortening mutations, between stability changes and the differential hydrophobicity of the exchanged residues and the volume of the cavities, the filling of these apoflavodoxin cavities is expected to stabilize the protein by approximately 1.5 kcal mol(-1). However, both urea and thermal denaturation analysis reveal much more modest stabilizations, ranging from 0.0 kcal mol(-1) to 0.6 kcal mol(-1), which reflects that the accommodation of single extra methyl groups in small cavities requires some rearrangement, necessarily destabilizing, that lowers the expected theoretical stabilization. As the size of these cavities is representative of that of the typical small, empty cavities found in most proteins, it seems unlikely that filling this type of cavities will give rise to large stabilizations.     

Quantitative analysis of DNA secondary structure from solvent-accessible surfaces: the B- to Z-DNA transition as a model

Solvent structure and its interactions have been suggested to play a critical role in defining the conformation of polynucleotides and other macromolecules. In this work, we attempt to quantitate solvent effects on the well-studied conformational transition between right-handed B- and left-handed Z-DNA. The solvent-accessible surfaces of the hexamer sequences d(m5CG)3, d(CG)3, d(CA)3, and d(TA)3 were calculated in their B- and Z-DNA conformations. The difference in hydration free energies between the Z and the B conformations (delta delta GH(Z-B] was determined from these surfaces to be -0.494 kcal/mol for C-5 methylated d(CG), 0.228 kcal/mol for unmethylated d(CG), 0.756 kcal/mol for d(CA)-d(TG), and 0.896 kcal/mol for d(TA) dinucleotides. These delta delta GH(Z-B) values were compared to the experimental B- to Z-DNA transition energies of -0.56 kcal/mol that we measured for C-5 methylated d(CG), 0.69-1.30 kcal/mol reported for unmethylated d(CG), 1.32-1.48 kcal/mol reported for d(CA)-d(TG), and 2.3-2.4 kcal/mol for d(TA) dinucleotides. From this comparison, we found that the calculated delta delta GH(Z-B) of these dinucleotides could account for the previous observation that the dinucleotides were ordered as d(m5CG) greater than d(CG) greater than d(CA)-d(TG) greater than d(TA) in stability as Z-DNA. Furthermore, we predicted that one of the primary reasons for the inability of d(TA) sequences to form Z-DNA results from a decrease in exposed hydrophilic surfaces of adjacent base pairs due to the C-5 methyl group of thymine; thus, d(UA) dinucleotides should be more stable as Z-DNA than the analogous d(TA) dinucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flanking DNA-sequences contribute to the specific binding of cI-repressor and OR1.

The binding of cI-repressor to a series of mutant operators containing OR1 of the right operator of bacteriophage lambda was investigated. Sites OR2 and/or OR3 were inactivated by either point or deletion mutations. The free energy of binding repressor to OR1 in the wildtype operator, delta G1, is -13.7 +/- 0.3 kcal/mol. delta G1 determined for an OR2- operator created by a single point mutation in OR2 is -13.6 +/- 0.2 kcal/mol. In contrast, delta G1 for the binding of repressor to a cloned synthetic OR1 operator containing only 24 bp of lambda sequence is -12.2 +/- 0.1 kcal/mol. When sequence 5' to OR1 is present, the binding affinity increases to -13.0 +/- 0.1 kcal/mol. In addition, the proximity of OR1 to a fragment-end decreases delta G1 from -13.7 to -12.3 +/- 0.1 kcal/mol. These results suggest that the DNA sequence outside the 17 bp OR1 binding-site contributes to the specific binding of cI-repressor.     

The role of salicylic acid in the induction of cell death in Arabidopsis acd11

Salicylic acid (SA) is implicated in the induction of programmed cell death (PCD) associated with pathogen defense responses because SA levels increase in response to PCD-inducing infections, and PCD development can be inhibited by expression of salicylate hydroxylase encoded by the bacterial nahG gene. The acd11 mutant of Arabidopsis (Arabidopsis thaliana L. Heynh.) activates PCD and defense responses that are fully suppressed by nahG. To further study the role of SA in PCD induction, we compared phenotypes of acd11/nahG with those of acd11/eds5-1 and acd11/sid2-2 mutants deficient in a putative transporter and isochorismate synthase required for SA biosynthesis. We show that sid2-2 fully suppresses SA accumulation and cell death in acd11, although growth inhibition and premature leaf chlorosis still occur. In addition, application of exogenous SA to acd11/sid2-2 is insufficient to restore cell death. This indicates that isochorismate-derived compounds other than SA are required for induction of PCD in acd11 and that some acd11 phenotypes require NahG-degradable compounds not synthesized via isochorismate.