Introduction of a proline residue into position 31 of the loop of the dimeric 4-alpha-helical protein ROP causes a drastic destabilization

The exchange of an alanine with a proline residue in position 31 of the loop region of the dimeric 4-alpha-helical-bundle protein ROP causes a reduction in the alpha-helix content of 7% and a reduction in stability of about 40% compared to the wild type parameters. The Gibbs energy of unfolding by denaturants extrapolated linearly to zero denaturant concentration, delta G0D (buffer, 25 degrees C), has been determined to be 43 kJ (mol dimer)-1. The corresponding ROPwt value is 72 kJ (mol dimer)-1 (Steif et al., 1993). The extrapolated delta G0D values obtained from urea and GdmHCI un- and refolding studies are identical within error limits. Deconvolution of the stability values into enthalpy and entropy terms resulted in the following parameters. At T1/2 = 43 degrees C (Cprotein = 0.05 mg.ml-1) the ROP A31P mutant is characterized by delta Hv.H.0 = 272 kJ (mol dimer)-1, delta Cp = 7.2 kJ (mol dimer)-1 K-1, delta S0 = 762 J (mol dimer)-1 K-1. These parameters are only approximately 50% as large as the corresponding values of ROPwt. We assume that the significant reduction in stability reflects the absence of at least one hydrogen bond as well as deformation of the protein structure. This interpretation is supported by the reduction in the change in heat capacity observed for the A31P mutant relative to ROPwt, by the increased aggregation tendency of the mutant and by the reduced specific CD absorption at 222 nm. All results support the view that in the case of ROP protein the loop region plays a significant role in the maintenance of native structure and conformational stability.     

A correlation between the loss of hydrophobic core packing interactions and protein stability

The hydrophobic core packing in four-alpha-helical bundles appears to be crucial for stabilizing the protein structure. To examine the structural basis of hydrophobic stabilization, the crystal structures of the Leu-->Val (L41V) and Leu-->Ala (L41A) substitutions of the core residue Leu41 of the ROP protein have been determined. Both substitutions are destabilizing and lead to formation of cavities. The main responses to mutations are the collapse of the central part of the alpha-helix containing the site of mutation, shifts of internal water molecules, and in L41A, the trapping of a water molecule in a cavity engineered by the mutation. For both mutants, these effects limit the increase in cavity size to less than 10 A3, while an increase of 37 A3 and 100 A3 is expected for L41V and L41A, respectively, in the absence of any cavity size reducing effects. The mobility of internal side-chains is increased and in L41A, it reaches values typical for exposed residues. A parameter (Deltanh) is introduced as a measure of the number of van der Waals contacts lost. For ROP, barnase and T4 lysozyme mutants, there is a good correlation between Deltanh and the free energy of unfolding DeltaDeltaG relative to wild-type protein. The Deltanh value turns out to be more suitable for analysing structural and energetic responses to mutation than other parameter, such as cavity volumes and packing densities. Possible evolutionary implications of the DeltaDeltaG versus Deltanh relationship are discussed.     

Dimer-to-tetramer transformation: loop excision dramatically alters structure and stability of the ROP four alpha-helix bundle protein

The ROP loop excision mutant RM6 shows dramatic changes in structure and stability in comparison to the wild-type protein. Removal of the five amino acids (Asp30, Ala31, Asp32, Glu33, Gln34) from the loop results in a complete reorganization of the protein as evidenced by single crystal X-ray analysis and thermodynamic unfolding studies. The homodimeric four-alpha-helix motif of the wild-type structure is given up. Instead a homotetrameric four-alpha-helix structure with extended, loop-free helical monomers is formed. This intriguing structural change is associated with the acquisition of hyperthermophilic stability. This is evident in the shift in transition temperature from 71 degreesC characteristic of the wild-type protein to 101 degreesC for RM6. Accordingly the Gibbs energy of unfolding is increased from 71.7 kJ (mol of dimer)-1 to 195.1 kJ (mol of tetramer)-1. The tetramer-to-monomer transition proceeds highly cooperatively involving an enthalpy change of DeltaH=1073+/-30 kJ (mol of tetramer)-1 and a heat capacity change at the transition temperature of DeltaDNCp=14.9(+/-)3% kJ (mol of tetramerxK)-1. The two-state nature of the unfolding reaction is reflected in coinciding calorimetric and van't Hoff enthalpy values.     

Isothermal titration microcalorimetric studies for the binding of octenoyl-CoA to medium chain acyl-CoA dehydrogenase

We investigated the binding of octenoyl-CoA to pig kidney medium chain acyl-CoA dehydrogenase (MCAD) by isothermal titration microcalorimetry under a variety of experimental conditions. At 25 degrees C in 50 mM phosphate buffer at pH 7.6 (ionic strength of 175 mM), the binding is characterized by the stoichiometry (n) of 0.89 mole of octenoyl-CoA/(mole of MCAD subunit), delta G = -8.75 kcal/mol, delta H = -10.3 kcal/mol, and delta S = -5.3 cal mol(-1) K(-1), suggesting that formation of MCAD-octenoyl-CoA is enthalpically driven. By employing buffers with various ionization enthalpies, we discerned that formation of the MCAD-octenoyl-CoA complex, at pH 7.6, accompanies abstraction (consumption) of 0.52 +/- 0.15 proton/(MCAD subunit) from the buffer media. We studied the effects of pH, ionic strength, and temperature on the thermodynamics of MCAD-octenoyl-CoA interaction. Whereas the ionic strength does not significantly influence the above interaction, the pH of the buffer media exhibits a pronounced effect. The pH dependence of the association constant of MCAD +octenoyl-CoA <==> MCAD-octenoyl-CoA yields a pKa for the free enzyme of 6.2. Among thermodynamic parameters, whereas delta G remains invariant as a function of temperature, delta H and deltaS(standard) both decrease with an increase in temperature. At temperatures of < 25 degrees C, delta G is dominated by favorable entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attain a value of zero at 23.8 degrees C, and then becomes unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy-entropy compensation. The temperature dependence of delta H yields the heat capacity change (delta Cp(0)) of -0.37 +/- 0.05 kcal mol(-1) K(-1), attesting to the fact that the binding of octenoyl-CoA to MCAD is primarily dominated by the hydrophobic forces. The thermodynamic data presented herein are rationalized in light of structural-functional relationships in MCAD catalysis.     

Differential inhibition of lysophosphatidate signaling by volatile anesthetics

cAMP receptor protein (CRP) is involved in regulation of expression of several genes in Escherichia coli. The protein is a homodimer and each monomer is folded into two distinct structural domains. The mechanism of the biological activity of the protein may involve the interaction between the subunits and domains. In order to determine the interaction between the subunits or domains of CRP, we have studied the reversible denaturation of the protein by guanidine hydrochloride. The unfolding and refolding kinetics of CRP was monitored using stopped-flow fluorescence spectroscopy at 20 degrees C and pH 7.9. The results of CRP denaturation indicate that the transition can be described by a three-state model: (CRP native)2<=> 2 (CRP native)<=>2 (CRP denatured). The faster process, characterized by the relaxation time tau 2 = 80 +/- 3 ms, corresponds to the dissociation of CRP dimer into monomers. The slower process has the relaxation time tau t = 1.9 +/- 0.1 s and corresponds to the cooperative unfolding of CRP monomer. The free energy change in the absence of denaturant upon CRP dissociation is delta G dis degrees = 46.9 +/- 2.5 kJ/mol and for monomer unfolding delta G unf degrees = 30.9 +/- 1.3 kJ/mol. The thermal unfolding of CRP was studied by circular dichroism and fluorescence spectroscopy at various guanidine hydrochloride concentrations. It has been found that the native protein is maximally stable at about 21 +/- 0.3 degrees C and is denatured upon heating and cooling from this temperature. The apparent free energy change for CRP unfolding at 21 degrees C is equal to 30.5 +/- 0.4 kJ/mol and the apparent specific heat change is equal to delta Cp, app = 10.7 +/- 0.7 kJ mol-1 K-1. The predicted values of cold denaturation midpoint is equal to tau G = -18.8 +/- 1.5 degrees C and for high-temperature transition tau G = 63.1 +/- 1.5 degrees C. The predicted midpoint of high-temperature unfolding transition is about the same as determined experimentally.     

The Ala/Val98 polymorphism of the hepatocyte nuclear factor-1alpha gene contributes to the interindividual variation in serum C-peptide response during an oral glucose tolerance test: evidence from studies of 231 glucose-tolerant first degree relatives of type 2 diabetic probands

The third form of maturity-onset diabetes of the young is caused by mutations in the hepatocyte nuclear factor-1alpha gene. Recently, we demonstrated an association between a prevalent polymorphism at codon 98, Ala/Val98, of this gene and a 20% decreased insulin release during an oral glucose tolerance test (OGTT) in middle-aged glucose-tolerant Danish Caucasian subjects. The major objective of the present study was to replicate this finding among glucose-tolerant first degree relatives of type 2 diabetic patients of the same ethnic origin. All participants, 231 glucose-tolerant offspring of 62 type 2 diabetic probands, underwent an OGTT with measurements of plasma glucose, serum insulin, and serum C peptide during the test. Thirty-three heterozygous carriers of the Ala/Val variant were identified, whereas no subjects had the variant in its homozygous form. Ala/Val carriers had a 20% reduction in serum C peptide at 30 min during the OGTT (1225+/-636 vs. 1507+/-624 pmol/L; P=0.02) compared to wild-type carriers. No significant differences in serum insulin levels during the OGTT were observed between carriers of the variant and Ala/Ala homozygotes. In conclusion, among Danish glucose-tolerant first degree relatives of type 2 diabetic patients the Ala/Val98 polymorphism of the hepatocyte nuclear factor-1alpha gene is associated with a decreased serum C-peptide secretion during an OGTT. This finding confirms our previously reported observation of the functional importance of the variant to insulin secretion during an OGTT among middle-aged healthy subjects.     

Thermodynamics of apocytochrome b5 unfolding

Apocytochrome b5 from rabbit liver was studied by scanning calorimetry, limited proteolysis, circular dichroism, second derivative spectroscopy, and size exclusion chromatography. The protein is able to undergo a reversible two-state thermal transition. However, transition temperature, denaturational enthalpy, and heat capacity change are reduced compared with the holoprotein. Apocytochrome b5 stability in terms of Gibbs energy change at protein unfolding (delta G) amounts to delta G = 7 +/- 1 kJ/mol at 25 degrees C (pH 7.4) compared with delta G = 25 kJ/mol for the holoprotein. Apocytochrome b5 is a compact, native-like protein. According to the spectral data, the cooperative structure is mainly based in the core region formed by residues 1-35 and 79-90. This finding is in full agreement with NMR data (Moore, C.D. & Lecomte, J.T.J., 1993, Biochemistry 32, 199-207).     

Volume and enthalpy changes after photoexcitation of bovine rhodopsin: laser-induced optoacoustic studies

Laser-induced optoacoustic measurements were performed with bovine rhodopsin in the temperature range 5-32 degrees C in its natural environment (i.e., in washed membranes) as well as solubilized in dodecyl-beta-D-maltoside. A signal deconvolution procedure using a simple sequential kinetic scheme for the photobaric time evolution revealed, in the case of the washed membranes, the presence of an intermediate with a 14-ns lifetime at 25 degrees C, of the same order as that reported for the BSI intermediate in solubilized rhodopsin (Hug, S. J., W. J. Lewis, C. M. Einterz, T. E. Thorgeirsson, and D. S. Kliger. 1990. Nanosecond photolysis of rhodopsin: evidence for a new, blue-shifted intermediate. Biochemistry. 29:1475-1485), with an energy content of (85 +/- 20) kJ/mol, and accompanied by an expansion of 26 +/- 3 ml/mol. The difference in energy content between BSI and the next transient lumi was estimated in only -1 +/- 5 kJ/mol, concomitant with an expansion of 9 +/- 3 ml/mol. Thus, this transition, which according to literature involves an equilibrium, should be controlled by an entropic change, rather than by an enthalpic difference. This is supported by the fact that both activation parameters for the decay of batho and BSI decrease upon solubilization. For detergent-solubilized rhodopsin, two time constants were enough to fit the sample signal. A short lifetime ascribable to BSI was not detected in this case. For the first intermediate (probably batho in equilibrium with BSI), an energy content of 50 +/- 20 kJ/mol and an expansion of 20 +/- 1 ml/mol, and for lumi an energy content of 11 +/- 20 kJ/mol and a further expansion of 11 +/- 2 ml/mol were determined. Thus, the intermediates of the membrane-embedded form of rhodopsin (in contrast to solubilized samples) are kept in a higher energy level, although the total expansion from rhodopsin to lumi is similar for both conditions (35 +/- 6 and 31 +/- 3 ml/mol). The expansions are interpreted as protein reorganization processes as a consequence of the photoisomerization of the chromophore. As a result, weak interactions are probably perturbed and the protein gains conformational flexibility.     

Thermodynamics of antigen-antibody binding using specific anti-lysozyme antibodies

Titration calorimetry measurements on the binding of hen lysozyme to the specific monoclonal IgG antibodies D1.3, D11.15, D44.1, F9.13.7, F10.6.6, their papain-cleaved antigen binding fragments (Fab) and their protein-engineered fragments consisting of non-covalently linked heavy variable chain and light variable chain domains (Fv) were performed between 6-50 degrees C in 0.15 M NaCl, 0.01 M sodium phosphate pH 7.1. The binding thermodynamic free energy change (delta G degrees b), enthalpy change (delta Hb), and entropy change (delta Sb) were the same for the whole IgG and its Fv and Fab fragments. With the exception of F9.13.7 at 13 degrees C, all the binding reactions were enthalpically driven with enthalpy changes ranging from -129 +/- 7 kJ mol-1 (D1.3 at 49.8 degrees C) to -26.2 +/- 0.6 kJ mol-1 (D44.1 at 8.0 degrees C). The heat capacity changes for the binding reaction (delta Cp) ranged from -2.72 +/- 0.16 kJ mol-1 K-1 (F9.13.7) to -0.95 +/- 0.06 kJ mol-1 K-1 (F10.6.6). The apolar surface areas buried at the binding sites estimated from the heat capacity changes indicate that the binding reactions are primarily hydrophobic, contrary to the mainly observed enthalpy-driven nature of the reactions. Conformational stabilization and the presence of water at the antigen-antibody interface may account for this discrepancy.     

In bacterial reaction centers rapid delivery of the second proton to QB can be achieved in the absence of L212Glu

In the reaction center (RC) of Rhodobacter capsulatus, residue L212Glu is a component of the pathway for proton transfer to the reduced secondary quinone, QB. We isolated phenotypic revertants of the photosynthetically incompetent (PS-) L212Glu-->Gln mutant; all of them retain the L212Glu-->Gln substitution and carry a second-site mutation: L227Leu-->Phe, L228Gly-->Asp, L231Arg-->Cys, or M231Arg-->Cys. We also characterized the L212Ala strain, which is a phenotypic revertant of the PS- L212Glu-L213Asp-->Ala-Ala mutant. The activities of the RCs of these strains--all of which lack L212Glu--were studied by flash-induced absorption spectroscopy. At pH 7.5, the rate of second electron transfer in the L212Q mutant is comparable to the wild-type rate. However, this mutant shows a marked decrease in the rate of cytochrome oxidation under strong continuous illumination and a very slow phase (0.66 s-1) of the proton transfer kinetics following the second flash, indicating that transfer of the second proton to QB is slowed more than 1000-fold. The levels of recovery of the functional capabilities in the revertant RCs vary widely; their rates of cytochrome oxidation were intermediate between those of the wild-type and the L212Q mutant. The kinetics of proton transfer following the second flash show a significant recovery in the L212Q + M231C and L212A RCs (330-540 s-1), but the L212Q + L227F RCs recover this function only partially. Compensation for the lack of L212Glu in revertant RCs is discussed in terms of (i) conformational changes that could allow water molecules to approach closer to QB and/or (ii) the increase in the negative electrostatic environment and the resultant rise in the free energy level of QB- that is induced by the mutations. The stoichiometries of H+/QB- proton uptake below pH 7.5 in the L212Q mutant, the L212Q + M231C revertant, and the wild-type strains are essentially equivalent, suggesting that L212Glu is protonated at neutral pH in wild-type RCs. This is also supported by the P+QB- charge recombination data. Comparison of H+/QB- proton uptake data with those obtained previously for the stoichiometries of H+/QA- proton uptake [Miksovska, J., Maróti, P., Tandori, J., Schiffer, M., Hanson, D. K., Sebban, P. (1996) Biochemistry 35, 15411-15417] suggests that L212Glu is the key to the electrostatic and perhaps structural interaction between the two quinone sites.     

Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complex

The high-affinity streptavidin-biotin complex is characterized by an extensive hydrogen-bonding network. A study of hydrogen-bonding energetics at the ureido oxygen of biotin has been conducted with site-directed mutations at Asn 23, Ser 27, and Tyr 43. A new competitive biotin binding assay was developed to provide direct equilibrium measurements of the alterations in Kd. S27A, Y43F, Y43A, N23A, and N23E mutants display DeltaDeltaG degrees at 37 degrees C relative to wild-type streptavidin of 2.9, 1.2, 2.6, 3.5, and 2.6 kcal/mol, respectively. The equilibrium-binding enthalpies for all of the mutants were measured by isothermal titration calorimetry, and the Y43A and N23A mutants display large decreases in the equilibrium binding enthalpy at 25 degrees C of 8.9 and 6.9 kcal/mol, respectively. The S27A and N23E mutants displayed small decreases in binding enthalpy of 1.6 and 0.9 kcal/mol relative to wild-type, while the Y43F mutant displayed a -2.6 kcal/mol increase in the binding enthalpy at 25 degrees C. At 37 degrees C, the Y43A and N23A mutants display decreases of 7.8 and 7.9 kcal/mol, respectively, while the S27A, N23E, and Y43F mutants displayed decreases of 4.9, 3.7, and 1.2 kcal/mol relative to wild-type. Kinetic analyses were also conducted to probe the contributions of the hydrogen bonds to the activation barrier. Wild-type streptavidin at 37 degrees C displays a koff of (4.1 +/- 0.3) x 10(-5) s-1, and the conservative Y43F, S27A, and N23A mutants displayed increases in koff to (20 +/- 1) x 10(-5) s-1, (660 +/- 40) x 10(-5) s-1, and (1030 +/- 220) x 10(-)5 s-1, respectively. The Y43A and N23E mutants displayed 93-fold and 188-fold increases in koff, respectively. Activation energies and enthalpies for each of the mutants were determined by transition-state analysis of the dissociation rate temperature dependence. All of the mutants except Y43F display large reductions in the activation enthalpy. The Y43F mutant has a more positive activation enthalpy, and thus a more favorable activation entropy that underlies the overall reduction in the activation barrier. For the most conservative mutant at each ureido oxygen hydrogen-bonding position, bound-state alterations account for most of the energetic changes in a single transition-state model, suggesting that the ureido oxygen hydrogen-bonding interactions are broken in the dissociation transition state.     

Recombinant human erythrocyte cytochrome b5

The gene encoding the human erythrocyte form of cytochrome b5 (97 residues in length) has been prepared by mutagenesis of an expression vector encoding lipase-solubilized bovine liver microsomal cytochrome b5 (93 residues in length) (Funk et al., 1990). Efficient expression of this gene in Escherichia coli has provided the first opportunity to obtain this protein in quantities sufficient for physical and functional characterization. Comparison of the erythrocytic cytochrome with the trypsin-solubilized bovine liver cytochrome b5 by potentiometric titration indicates that the principal electrostatic difference between the two proteins results from two additional His residues present in the human erythrocytic protein. The midpoint reduction potential of this protein determined by direct electrochemistry is -9 +/- 2 mV vs SHE at pH 7.0 (mu = 0.10 M, 25.0 degrees C), and this value varies with pH in a fashion that is consistent with the presence of a single ionizable group that changes pKa from 6.0 +/- 0.1 in the ferricytochrome to 6.3 +/- 0.1 in the ferrocytochrome with delta H degrees = -3.2 +/- 0.1 kcal/mol and delta S degrees = -11.5 +/- 0.3 eu (pH 7.0, mu = 0.10). The 1D 1H NMR spectrum of the erythrocytic ferricytochrome indicates that 90% of the protein binds heme in the "major" orientation and 10% of the protein binds heme in the "minor" orientation (pH 7.0, 25 degrees C) with delta H degrees = -2.9 +/- 0.3 kcal/mol and delta S degrees = -5.4 +/- 0.9 eu for this equilibrium.     

Dissecting the contributions of a specific side-chain interaction to folding and catalysis of Bacillus stearothermophilus lactate dehydrogenase

X-ray crystallography predicts hydrogen-bonding interactions between the side chains of Thr198 and two other amino acid residues, Glu194 (adjacent to the catalytic His195) and Ser318 (on the alpha-H helix which rearranges on substrate binding). In order to investigate the contribution of this conserved amino acid residue, Thr198, two mutants of Bacillus stearothermophilus lactate dehydrogenase were created (Val198 and Ile198). The steady-state kinetic parameters for both mutant enzymes were very similar with increased substrate Km and reduced kcat when compared with the wild-type enzyme. The mutation Val198 allowed non-productive binding of pyruvate to the unprotonated form of His195. Steady-state kinetic parameters determined for the Val198 mutant enzyme in high solvent viscosity suggested both an altered rate-limiting step in catalysis and implicated Thr198 in allosteric activation by the effector fructose 1,6-bisphosphate (Fru1,6P2). A shift in the Fru1,6P2 activation constant for the Val198 mutant enzyme suggested that Thr198 stabilises the catalytically competent (Fru1,6P2-activated) form of the enzyme by 6.6 kJ/mol. However, Thr198 was not important for maintaining the thermal stability of the Fru1,6P2-activated form. Equilibrium unfolding in guanidinium chloride indicated that Thr198 contributes 17.2 kJ/mol subunits towards the tertiary structural stability. The results emphasise the importance of the side chain-hydroxyl group of Thr198 which is required for (a) productive substrate binding, (b) allosteric activation and (c) protein conformational stability. The characteristics of the B. stearothermophilus lactate dehydrogenase mutations reported here were significantly different from those of the same mutations made in the corresponding position of the analogous enzyme Thermus flavus malate dehydrogenase [Nishiyama, M., Shimada, K., Horinouchi, S., & Beppu, T. (1991) J. Biol. Chem. 266, 14294-14299].     

Effects of temperature on the kinetics of the gated electron-transfer reaction between zinc cytochrome c and plastocyanin. Analysis of configurational fluctuation of the diprotein complex

This is a study of the effects of temperature (in the range 273.3-307.7 K) and of ionic strength (in the range 2.5-100 mM) on the kinetics of photoinduced electron-transfer reaction 3Zncyt/pc(II)--> Zncyt+/pc(I) within the electrostatic complex of zinc cytochrome c and cupriplastocyanin at pH 7.0. In order to separate direct and indirect effects of temperature on the rate constants, viscosity of the solutions was fixed, at different values, by additions of sucrose. The activation parameters for the reaction within the preformed complex, at the low ionic strength, are delta H++ = 13 +/- 2 kJ/mol and delta S++ = -97 +/- 4 J/K mol. The activation parameters for the reaction within the encounter complex, at the higher ionic strength, are delta H++ = 13 +/- 1 kJ/mol and delta S++ = -96 +/- 3 J/K mol. Evidently, the two complexes are the same. The proteins associate similarly in the persistent and the transient complex, i.e., at different ionic strengths. In both complexes, however, electron transfer is gated by a rearrangement, as previous studies from this laboratory showed. Changes in the solution viscosity modulate this rearrangement by affecting delta H++, not delta S++. The activation parameters are analyzed by empirical methods. The thermodynamic parameters delta H and delta S for the formation of the complex Zncyt/pc(II) are determined and related to changes in hydrophilic and hydrophobic surfaces upon protein association in three configurations. A difference between the values of delta H for the configuration providing optimal electronic coupling between the redox sites and the configuration providing optimal docking equals the experimental value delta H++ = 13 kJ/mol for the rearrangement of the latter configuration into the former. Enthalpy of activation may reflect a change in the character of the exposed surface as the diprotein complex rearranges. Entropy of activation may reflect tightening of the contact between the associated proteins.     

Thermodynamic cycle between DNA and RNA constituents for conformation of the sugar ring from nuclear magnetic resonance study

The effect of a structural change of ribose to deoxyribose, by replacement of 2'-OH by 2'-H, on the conformational equilibrium of the sugar ring is described in terms of one thermodynamic cycle. The method is based on the observation that conformational correlations of the sugar ring--side chain ensemble in DNA and RNA components show one general pattern, reflecting an intrinsic physical property of this ensemble. The pattern determines a choice of model systems to study. The systems consist of pairs of DNA and RNA components, nucleosides and nucleotides in aqueous solution, where all conformational factors are fully controlled. This approach allowed us to describe the thermodynamic cycle and measure its fundamental parameters, equilibrium constants and free energy differences, delta delta G, from a nuclear magnetic resonance study. The delta delta G values as determined for pairs of ribo- and deoxyribo-nucleosides in classes of syn-constrained and anti-preferred models, are comparable and lie in a narrow range, delta delta G = 1.7 +/- 0.1 [kJ/mol]. For pairs of ribo- and deoxyribo-nucleotides, the delta delta G values also lie in narrow ranges, delta delta G = 1.7 +/- 0.1 [kJ/mol] for 5'-phosphate nucleotides and delta delta G = 1.9 +/- 0.1 [kJ/mol] for 3'-phosphate nucleotides, i.e. similar to those observed for nucleosides. The measured quantity, delta delta G, is generally observed in a relatively narrow range, delta delta G = 1.75 +/- 0.15 [kJ/mol], irrespective of the class of the model system. This quantity represents a "pure" constant contribution, pe one sugar moiety, as a "driving force" for the N-->S shift in the sugar ring conformational equilibrium, when one compares RNA and DNA. This important thermodynamic quantity, delta delta G, has not hitherto been determined for nucleic acids. Ultimately the delta delta G quantity is revealed in the tendency to adopt S(C2'endo) sugar puckering domain by the majority of DNA structures, whereas RNA generally adopt an N(C3'endo) puckering domain. A possible biological significance of the delta delta G quantity may include evolutionary aspects of nucleic acids.     

Conformational transition of insulin induced by n-alkyltrimethylammonium bromides in aqueous solution

A surfactant-induced conformational transition of bovine insulin has been detected by difference spectroscopy for a homologous series of n-alkytrimethylammonium bromides, chain length C10-C16 at pH 10.0, 25 degrees C. The transition was followed as a function of surfactant concentration by absorbance measurements at 275 nm and the data were analysed to obtain the Gibbs energy of the transition in water (delta Gw degree) and in a hydrophobic environment (delta Ghc degree) for saturated protein-surfactant complexes. A value of delta Gw degree of -11.8 +/- 1.8 kJ mol-1 was found independent of n-alkyl chain length, which is similar to the value found for the n-alkylsulfate-induced transition in a previous study (-14.6 +/- 3.0 kJ mol-1). The values of delta Ghc degree were in the range approximately -88 to -100 kJ mol-1 for chain lengths from C10 to C16. The values of delta Ghc degree vs. chain length for both the n-alkyltrimethylammonium bromides and the n-alkylsulfates lie on the same curve, demonstrating that delta Ghc degree is independent of the nature of the surfactant head group.     

Energy characteristics of an ATP-hydrolase reaction catalyzed by solubilized Ca2+,Mg2+-ATPase from smooth muscle cell membrane

The effects of temperature, dielectric permeability and ionic strength on the activity of purified Ca2+, Mg(2+)-ATPase solubilized from myometrial sarcolemma have been studied under saturation of the enzyme with Ca2+, Mg2+ and ATP. The values of activation energy calculated from Arrhenius plots for both ATP hydrolase reactions catalysed by solubilized and reconstituted into azolectin liposomes Ca2+, Mg(2+)-ATPase and Mg2+, ATP-dependent Ca2+ transport by the reconstituted enzyme were 56.4 +/- 1.5, 68.0 +/- 5.1 and 63.1 +/- 2.9 kJ/mol, respectively. Analysis of experimental data in terms of the Laidler-Scatchard and Bronsted-Bjerrum theories revealed that the separation of the reaction products--the chelate MgADP complex--from the active site of the enzyme bearing one unity positive charge is the limiting step of the Ca2+, Mg(2+)-dependent enzymatic ATP-hydrolysis under conditions of substrate saturation. The values of the electrostatic components of the free energy, enthalpy and entropy of activation of the ATP hydrolase reaction were 46.6 +/- 0.3 kJ/mol, -(20.5 +/- 0.4) kJ/mol and -(214.2 +/- 4.3) J/(mol.degrees K), respectively. The nonelectrostatic component of activation enthalpy was 76.9 kJ/mol. The results obtained suggest that changes in polarity of the incubation medium markedly affect the activity of transport Ca2+, Mg(2+)-ATPase solubilized from smooth muscle cell plasma membranes and that the electrostatic interactions between the enzyme active site and specific reagents (MgADP, in particular) significantly contribute to the energetics of the ATP hydrolase reaction.     

Unfolding of the tetrameric loop deletion mutant of ROP protein is a second-order reaction

Comprehensive kinetic studies were carried out on the unfolding properties of RM6 as a function of GdnHCl concentration and temperature. This protein is a mutant resulting from the dimeric wild-type CoLE1-ROP protein by deletion of 5 amino acids (Asp 30, Ala 31, Asp 32, Glu 33, Gln 34) in the loop of each monomer. The deletion has dramatic consequences. The dimeric 4-alpha-helix structure characteristic of the wild-type protein is completely reorganized and the RM6 structure can be described as a tetrameric alpha helix of extended monomers without loops. These extraordinary structural changes are accompanied by an enormous increase in transition temperature from 71 to 101 degreesC. These features have been discussed in a separate publication (1). The remarkable change in thermal stability of RM6 should be reflected in significant changes in the folding rate constants. This was observed in the present unfolding studies. Decay of tetrameric RM6 was monitored by circular dichroism (CD) and fluorescence to probe for changes in both secondary and tertiary structure, respectively. The identity of the kinetic parameters obtained from the two techniques supports the view that secondary and tertiary structure break down simultaneously. However, the most intriguing result is the finding that unfolding of tetrameric RM6 can be described very well by a second-order reaction. The magnitude of the second-order rate constant k2 varies dramatically with both temperature and denaturant concentration. At 25 degreesC and 6.5 M GdnHCl concentration k2 is 4200 L.(mol of dimer)-1.s-1, whereas at 4.4 M GdnHCl a value of k2 = 0.9 L.(mol of dimer)-1.s-1 is observed. Correspondingly, apparent activation enthalpies show a strong increase from DeltaH# = 29.1 kJ.mol-1 at 6. 5 M GdnHCl to Delta H# = 79.7 kJ.mol-1 at 4.4 M GdnHCl. A mechanism involving a dimeric intermediate is suggested which permits a consistent interpretation of the findings.     

Analysis of the reaction coordinate of photosynthetic water oxidation by kinetic measurements of 355 nm absorption changes at different temperatures in photosystem II preparations suspended in either H2O or D2O

Flash-induced absorption changes at 355 nm were measured at different temperatures within the range of 2 degrees C S2) = 14 kJ/mol, EA(S2-->S3) = 35 kJ/mol, and EA(S3-->-->S0 + O2) = 21 kJ/mol for theta > 11 degrees C, 67 kJ/mol for theta < 11 degrees C in PS II core complexes dissolved in H2O; (b) replacement of exchangeable protons by deuterons causes only minor changes ( S2, S2 --> S3, and S3 -->--> S0 + O2, respectively. The corresponding values of PS II membrane fragments are 1.3, 1.3, and 1. 4. Based on these results and corresponding EA data reported in the literature for PS II membrane fragments from spinach [Renger, G., & Hanssum, B. (1992) FEBS Lett. 299, 28-32] and PS II particles from the thermophilic cyanobacterium Synechococcus vulcanus Copeland [Koike, H., Hanssum, B., Inoue, Y., & Renger, G. (1987) Biochim. Biophys. Acta 893, 524-533], the reaction coordinate of the redox sequence in the WOC is inferred to be almost invariant to the evolutionary development from cyanobacteria to higher plants. Furthermore, the rather high activation energy of the S2 --> S3 transition provides evidence for a significant structural change coupled with this reaction. Implications for the mechanism of photosynthetic water oxidation are discussed.     

Effect of replacing a conserved proline residue on the function and stability of bovine adrenodoxin

A proline residue in the C-terminal part of the polypeptide chain is highly conserved among many [2Fe-2S] ferredoxins. To investigate the requirement for proline at this position, we constructed steric (4-108W), charged (4-108K), polar (4-108S) and non-polar (4-108A) truncated mutants of adrenodoxin and studied them for biological function and stability. Although the variants were expressed in Escherichia coli with a significantly lower yield compared with wild-type adrenodoxin, successful incorporation of the iron-sulfur cluster suggested their proper folding. Similar absorption, CD and EPR spectra indicated that the cluster environment was not affected by the mutations. No evidence for an essential role of Pro108 in determining the redox potential of adrenodoxin or its interactions with the redox partners was found. However, replacement of this residue results in a dramatic decrease in the overall protein stability. The differences in the Gibbs energy of unfolding at 37 degrees C, delta[delta(d)G(37 degrees C)], are -5.0, -7.8, -10.1 and -10.7 kJ/mol for 4-108A, 4-108S, 4-108W and 4-108K mutants, respectively, compared with 4-108P as a control. We conclude that the principle function of Pro108 is to stabilize adrenodoxin threefold: (i) through limitation of the conformation of the polypeptide chain in this region, (ii) through a hydrogen bond to Arg14 and (iii) favorable hydrophobic contacts.