Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher Vmax and Ks values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of L-dihydroorotate were determined at 25 degrees C for hamster DHOase (deltaG = -6.9 kcal/mol, deltaH = -11.5 kcal/mol, TdeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (deltaG = -5.6 kcal/mol, deltaH = -4.2 kcal/mol, TdeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the kcat/Ks value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(double dagger), making kcat/Ks almost temperature independent. The pseudo-first-order rate constant for water attack on L-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-11) s(-1) at 25 degrees C, with deltaH(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (delta deltaH(double dagger) = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.     

Two types of arrestins expressed in medaka rod photoreceptors

Globular protein thermostability is characterized the cold denaturation, maximal stability (Tms) and heat denaturation temperatures. For mesophilic globular proteins, Tms typically ranges from -25 degrees C to +35 degrees C. We show that the indirect estimate of Tms from calorimetry and the direct estimate from chemical denaturation performed in a range of temperatures are in close agreement. The heat capacity change of unfolding per mol residue (delta Cp) alone is shown to accurately predict Tms. Delta Cp and hence Tms can be predicted solely from the protein sequence. The average difference in free energy of unfolding at the observed and predicted values of Tms is 1.0 kcal mol(-1), which is small compared to typical values of the total free energy of unfolding.     

Human platelet factor 4 subunit association/dissociation thermodynamics and kinetics

Platelet factor 4 (PF4) monomers (7800 daltons) form dimers and tetramers in varying molar ratios under certain solution conditions [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469]. The presence of a simplified aromatic region (one Tyr and two His) and resolved monomer, dimer, and tetramer Y60 3,5 ring proton resonances makes study of PF4 aggregate association/dissociation thermodynamics and kinetics possible. PF4 protein subunit association/dissociation equilibrium thermodynamic parameters have been derived by 1H NMR (500MHz) resonance line-fitting analysis of steady-state Y60 3,5 ring proton resonance monomer-dimer-tetramer populations as a function of temperature from 10 to 40 degrees C. Below 10 degrees C and above 40 degrees C, resonance broadening and overlap severely impaired analysis. Enthalpic and entropic contributions to dimer association Gibb's free energy [-5.1 kcal/mol (30 degrees C)] are +2.5 +/- 1 kcal/mol and +26 +/- 7 eu, respectively, and for tetramer association Gibb's free energy [-5.7 kcal/mol (30 degrees C)], they are -7.5 +/- 1 kcal/mol and -7 +/- 3 eu, respectively. These thermodynamic parameters are consistent with low dielectric medium electrostatic/hydrophobic interactions governing dimer formation and hydrogen bonding governing tetramer formation. Association/dissociation kinetic parameters, i.e., steady-state jump rates, have been derived from exchange-induced line-width increases and from 1H NMR (500 MHz) saturation-transfer and spin-lattice (Tl) relaxation experiments. From dissociation jump rates and equilibrium constants, association rate constants were estimated. For dimer and tetramer equilibria at 30 degrees C, unimolecular dissociation rate constants are 35 +/- 10 s-1 for dimer dissociation and 6 +/- 2 s-1 for tetramer dissociation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Global analysis of the thermal and chemical denaturation of the N-terminal domain of the ribosomal protein L9 in H2O and D2O. Determination of the thermodynamic parameters, deltaH(o), deltaS(o), and deltaC(o)p and evaluation of solvent isotope effects.

The stability of the N-terminal domain of the ribosomal protein L9, NTL9, from Bacillus stearothermophilus has been monitored by circular dichroism at various temperatures and chemical denaturant concentrations in H2O and D2O. The basic thermodynamic parameters for the unfolding reaction, deltaH(o), deltaS(o), and deltaC(o)p, were determined by global analysis of temperature and denaturant effects on stability. The data were well fit by a model that assumes stability varies linearly with denaturant concentration and that uses the Gibbs-Helmholtz equation to model changes in stability with temperature. The results obtained from the global analysis are consistent with information obtained from individual thermal and chemical denaturations. NTL9 has a maximum stability of 3.78 +/- 0.25 kcal mol(-1) at 14 degrees C. DeltaH(o)(25 degrees C) for protein unfolding equals 9.9 +/- 0.7 kcal mol(-1) and TdeltaS(o)++(25 degrees C) equals 6.2 +/- 0.6 kcal mol(-1). DeltaC(o)p equals 0.53 +/- 0.06 kcal mol(-1) deg(-1). There is a small increase in stability when D2O is substituted for H2O. Based on the results from global analysis, NTL9 is 1.06 +/- 0.60 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 5.8 +/- 3.6 degrees C in D2O. Based on the results from individual denaturation experiments, NTL9 is 0.68 +/- 0.68 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 3.5 +/- 2.1 degrees C in D2O. Within experimental error there are no changes in deltaH(o) (25 degrees C) when D2O is substituted for H2O.     

Kinetic study of the activation process of frog epidermis pro-tyrosinase by trypsin

1. The rate of tyrosinase formation has been calculated by coupling the activatory process of frog epidermis pro-tyrosinase by trypsin to the oxidation of L-DOPA to dopachrome. Under certain conditions ([trypsin]/[pro-tyrosinase] greater than or equal to 300), the lag period of the coupled reactions, tau, is independent of trypsin concentration. 2. The specific rate constant of tyrosinase formation at different temperatures has been calculated, ranging from 0.025 sec-1, at 5 degrees C to 0.248 sec-1, at 30 degrees C. 3. Thermodynamic parameters of the activatory process (delta G not equal to = + 18.5 kcal/mol; delta H not equal to = + 14.8 kcal/mol; delta S not equal to = -12.4 e.u.; Ea = + 15.3 kcal/mol), have been determined by the study of the system at different temperatures. These values are characteristic for a normal chemical reaction. 4. From these kinetic data, the order of products formation in the proteolytic step, can be determined, active tyrosinase being the last product released.     

Thermodynamic analysis of the structural stability of the shiga toxin B-subunit

The conformational stability of Shiga toxin B-subunit (STxB), a pentameric protein from Shigella dysenteriae has been characterized by high sensitivity differential scanning calorimetry and circular dichroism spectroscopy under different solvent conditions. It is shown that the thermal folding/unfolding of STxB is a reversible process involving a highly cooperative transition between folded pentamer and unfolded monomers. The conformational stability of STxB is pH dependent and because of its pentameric nature is also concentration dependent. STxB is maximally stable in the pH range from 5 to 9 (Delta G upon unfolding is close to 13 kcal per mol of monomer at 25 degrees C), and its stability decreases both at lower and at higher pH values. The pH dependence of the Gibbs energy of stabilization between pH 2.5 and 5 is consistent with the change in the ionizable state of an average of four groups per monomer upon unfolding. Structural thermodynamic calculations show that the stabilization of the STxB pentamer is primarily due to the interactions established between monomers rather than intramonomer interactions. The folding of an isolated monomer into the conformation existing in the pentamer is unfavorable and expected to be characterized by a free-energy change upon folding in the order of 2.5 kcal mol(-1) at 25 degrees C. On the average, intersubunit interaction induced upon oligomerization of folded monomers should contribute close to -13.4 kcal per mol of monomer to bring the overall Gibbs energy to the experimentally determined value at this temperature.     

Thermodynamics of carbon monoxide binding to monomeric cytochrome c'

The thermodynamic parameters for carbon binding to monomeric Rhodopseudomonas palustris cytochrome c' are determined. An enthalpy change for CO(aq) binding to the cytochrome is measured directly by titration calorimetry as -6.7 +/- 0.2 kcal/mol of heme, the CO binding equilibrium constant is measured at 35 degrees C as (1.96 +/- 0.05) X 10(5) M-1, and the binding equilibrium constant at 25 degrees C is calculated from the van't Hoff equation as (2.8 +/- 0.1) X 10(5) M-1. Comparison of the results to the known energetics of CO binding to dimeric cytochrome c', where the CO binding site is buried in the protein interior, indicates that the heme binding site on the monomer form is, in contrast, more exposed.     

Thermohemolysis of erythrocytes in the temperature range including physiologic (autohemolysis)]

The activation energy of thermohemolysis of erythrocytes changes from 36 +/- 5 kcal/mol (35-45 degrees C) to 97 +/- 5 kcal/mol (45-55 degrees C) at the temperature about 45 degrees C in isotonic buffer. The break on Arhenius' plot is preserved also when erythrocytes are placed into plasma. The character of Arhenius' plot is the same when erythrocyte hemoglobin is totally oxidated into methemoglobin by chemical way, though thermal stability of such erythrocytes is decreased. The scheme is presented in which thermohemolysis of erythrocytes occurs by two independent ways: thermodenaturation of hemoglobin (limiting stage of the process when t greater than 45 degrees C) and modification of membrane proteins by hemin, the last being a product of hemoglobin oxidation (limiting stage of the process when t less than 45 degrees C).     

Microwave dielectric measurements of erythrocyte suspensions.

Complex dielectric constants of human erythrocyte suspensions over a frequency range from 45 MHz to 26.5 GHz and a temperature range from 5 to 40 degrees C have been determined with the open-ended coaxial probe technique using an automated vector network analyzer (HP 8510). The spectra show two separate major dispersions (beta and gamma) and a much smaller dispersion between them. The two major dispersions are analyzed with a dispersion equation containing two Cole-Cole functions by means of a complex nonlinear least squares technique. The parameters of the equation at different temperatures have been determined. The low frequency behavior of the spectra suggests that the dielectric constant of the cell membrane increases when the temperature is above 35 degrees C. The real part of the dielectric constant at approximately 3.4 GHz remains almost constant when the temperature changes. The dispersion shifts with temperature in the manner of a thermally activated process, and the thermal activation enthalpies for the beta- and gamma-dispersions are 9.87 +/- 0.42 kcal/mol and 4.80 +/- 0.06 kcal/mol, respectively.     

Spectral evidence for the existence of a second cytochrome o in whole cells of Vitreoscilla

Cytochrome o, a protoheme IX pigment, has been proposed as the terminal oxidase of the filamentous bacterium, Vitreoscilla. Aerobic and anaerobic photolysis of CO-liganded whole cells demonstrated the presence of a second CO-reactive pigment, cytochrome o'. At temperatures lower than -100 degrees C, anaerobic photolysis dissociated only about 25% of the total CO-liganded components to reveal the unliganded cytochrome o'. At these temperatures, the photolysis of cytochrome o could not be demonstrated. At warmer temperatures, recombination of CO with the reduced cytochrome o' occurred with an apparent energy of activation of 5.8 kcal/mol. Aerobic photolysis of whole cells demonstrated two oxygen-bound intermediates. At temperatures lower than -95 degrees C, a spectrally distinct compound with absorption maxima at 428, 534, and 564 nm appeared (form I'); the apparent second order rate constant (k+1) for the formation of this intermediate was found to be 9.1 M-1 s-1, the reverse rate (k-1) was 9.9 X 10(-5) s-1, and the equilibrium constant (Kd) was 1.1 X 10(-5) M. This oxygen intermediate of cytochrome o' is spectrally and kinetically similar to the oxygen intermediate of cytochrome o seen in Escherichia coli. At temperatures warmer than -90 degrees C, photolysis of aerobic samples resulted in the immediate formation of a second oxygen-bound intermediate (form I) with absorption maxima at 422, 534, and 564 nm. This second intermediate results from the binding of oxygen to the cytochrome o (oxygenated cytochrome o). These data support the proposal that whole cells of Vitreoscilla contain two alternative pathways of electron transport, one terminating with cytochrome o and the other with cytochrome o'.     

Effect of temperature on Brettanomyces bruxellensis: metabolic and kinetic aspects

The effect of temperatures ranging from 15 to 35 degrees C on a culture of Brettanomyces bruxellensis was investigated in regards to thermodynamics, metabolism, and kinetics. In this temperature range, we observed an increase in growth and production rates. The growth behavior was well represented using the Arrhenius model, and an apparent activation energy of 16.61 kcal/mol was estimated. A stuck fermentation was observed at 35 degrees C as represented by high cell death. The carbon balance established that temperature had no effect on repartition of the glucose consumption between biomass and products. Hence, the same biomass concentration was obtained for all temperatures, except at 35 degrees C. Moreover, using logistic and Luedeking-Piret models, we demonstrated that production rates of ethanol and acetic acid were partially growth associated. Parameters associated with growth (alpha eth and alpha aa) remained constant with changing temperature, whereas, parameters associated with the population (beta eth and beta aa) varied. Optimal values were obtained at 32 degrees C for ethanol and at 25 degrees C for acetic acid.     

Relationship between lipid fluidity and water permeability of bovine tracheal epithelial cell apical membranes

Apical membrane vesicles were prepared from bovine tracheal epithelial cells. These membranes were enriched in alkaline phosphatase specific activity 35-fold compared to cellular homogenates. Steady-state fluorescence polarization studies of these membranes, using three fluorophores, demonstrated that they possessed a relatively low fluidity. Studies using the probe 1,6-diphenyl-1,3,5-hexatriene detected thermotropic transitions at 25.7 +/- 0.4 and 26.8 +/- 0.6 degrees C in these membranes and their liposomes, respectively. Analysis of the composition of these membranes revealed a fatty acyl saturation index of 0.59 +/- 0.02, a protein/lipid ratio (w/w) of 0.60 +/- 0.06, a cholesterol/phospholipid ratio (mol/mol) of 0.83 +/- 0.11, and a sphingomyelin/lecithin ratio (mol/mol) of 0.64 +/- 0.10. Membrane vesicles were osmotically active when studied by a stopped-flow nephelometric technique. Arrhenius plots of rates of osmotic water efflux demonstrated break points at approximately 28 and 18 degrees C, with activation energies of 16.7 +/- 0.2 kcal mol-1 from 35 to 28 degrees C, 8.3 +/- 0.5 kcal mol-1 from 28 to 18 degrees C, and approximately 3.0 kcal mol-1 below 18 degrees C. Treatment of membrane vesicles with benzyl alcohol, a known fluidizer, decreased lipid order (increased fluidity) and increased the rate of osmotic water efflux. The present results suggest that water crosses tracheal epithelial cell apical membranes by solubility-diffusion across the lipid domain and that increases in fluidity correlate with increases in the water permeability of these membranes.     

A time-resolved photoacoustic calorimetry study of the dynamics of enthalpy and volume changes produced in the photodissociation of carbon monoxide from sperm whale carboxymyoglobin

The dynamics of the enthalpy and volume changes produced in the photodissociation of carbon monoxide from sperm whale myoglobin is investigated by time-resolved photoacoustic calorimetry. The enthalpy and volume changes for the formation of the geminate pair, which occurs within 50 ns of photolysis, are delta H = -2.2 +/- 2.8 kcal/mol and delta V = -10.0 +/- 1.0 mL/mol relative to carboxymyoglobin. The enthalpy and volume changes associated with formation of deoxymyoglobin and solvated carbon monoxide, formed with a half-life of 702 +/- 31 ns at 20 degrees C, are delta H = 14.6 +/- 3.4 kcal/mol and delta V = 5.8 +/- 1.0 mL/mol relative to carboxymyoglobin.     

Activation Parameters for the Spontaneous and Pressure-Induced Phases of the Dissociation of Single-Ring GroEL (SR1) Chaperonin

We investigated the dissociation of single-ring heptameric GroEL (SR1) by high hydrostatic pressure in the range 0.5-3.0 kbar. The kinetics were studied as a function of temperature in the range 15-35 degrees C. The dissociation processes at each pressure and temperature showed biphasic behavior. The slower rate (k1,obs) was confirmed to be the self-dissociation of SR1 at any specific temperature at atmospheric pressure. This dissociation was pressure independent and followed concentration-dependent first-order kinetics. The self-dissociation rates followed normal Eyring plots (In k1,obs/T vs. 1/T) from which the free energy of activation (deltaG++ = 22 +/- 0.3 kcal mol(-1)), enthalpy of activation (deltaH++ = 18 +/- 0.5 kcal mol(-1)), and entropy of activation (deltaS++ = -15 +/- 1 kcal mol(-1)) were evaluated. The effect of pressure on the dissociation rates resulted in nonlinear behavior (ln k2,obs vs. pressure) at all the temperatures studied indicating that the activation volumes were pressure dependent. Activation volumes at zero pressure (V++o) and compressibility factors (beta++) for the dissociation rates at the specific temperatures were calculated. This is the first systematic study where the self-dissociation of an oligomeric chaperonin as well as its activation parameters are reported.     

The growth of sickle hemoglobin polymers

The measurement of polymer growth is an essential element in characterization of assembly. We have developed a precise method of measuring the growth of sickle hemoglobin polymers by observing the time required for polymers to traverse a photolytically produced channel between a region in which polymers are created and a detection region. The presence of the polymer is functionally detected by observing its ability to create new polymers through the well-established process of heterogeneous nucleation. Using this method, we have determined the rate constants for monomer addition to and release from polymer ends, as well as their temperature dependences. At 25°C we find k₊ = 84 ± 2 mM⁻¹ s⁻¹ and k₋ = 790 ± 80 molecules/s from each end. These numbers are in accord with differential interference contrast measurements, and their ratio gives a solubility measured on individual fibers. The single-fiber solubility agrees with that measured in sedimentation experiments. The concentration dependence of the monomer addition rate is consistent with monomer addition, but not oligomer addition, to growing polymers. The concentration dependence suggests the presence of an activation enthalpy barrier, and the rate of monomer addition is not diffusion-limited. Analysis of the temperature dependence of the monomer addition rate reveals an apparent activation energy of 9.1 ± 0.6 kcal/mol.     

Temperature dependence of enthalpy changes for ethidium and propidium binding to DNA: effect of alkylamine chains

Calorimetric titrations have been performed on the binding of ethidium and propidium to calf thymus DNA at temperatures in the 15-60 degrees C range. Enthalpy changes (delta HB) derived from these experiments performed with the new Omega reaction calorimeter have a precision of +/- 0.10 kcal/mol or less at all temperatures. For ethidium (a monocation), delta HB varies little with temperature, and the heat capacity change (delta CP) for the binding reaction derived from these parameters is 10 cal/deg/mol. In contrast, delta HB changes from -6.5 to -8.1 kcal/mol for DNA binding of propidium (a dication due to a charged amine group at the end of an alkyl chain attached to the phenanthridine ring nitrogen), and delta CP is -57 cal/deg/mol. At 21 degrees C a plot of delta HB vs mole ratio is curved downward for propidium in the 0.08-0.25 range, whereas the same plot at 45 degrees C is a straight line from 0.05 to 0.15 and sharply downward thereafter. Similar plots for ethidium follow the latter pattern between 25 and 50 degrees C. These observations and our analyses of delta HB and delta SB are consistent with the hypothesis that the location in the DNA complex and the rotational motion of the alkylamine chain change substantially over the temperature range in this study. Only near 50 degrees C is delta HB equal for the binding of these two cations to DNA, and caution must be used in analyses of enthalpic effects when the temperature dependence for delta HB is not available.     

Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima

Domain II (residues 189-338, M(r) = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.COLI: using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme.The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(p) value of 1.4 +/- 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.MARITIMA: glutamate dehydrogenase is a thermostable protein with a DeltaG(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T. MARITIMA: domain II glutamate dehydrogenase.     

Cryopreservation of canine spermatozoa: theoretical prediction of optimal cooling rates in the presence and absence of cryoprotective agents

In the present study a shape independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of ejaculated canine sperm cells. Volumetric shrinkage during freezing of canine sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPAs (6 different combinations of freezing media were used, ranging from a media with no CPAs, and those with 0.5%, 3%, and 6% glycerol and with 0.5% and 3% Me(2)SO). Using previously published data, the canine sperm cell was modeled as a cylinder of length 105.7mum and a radius of 0.32mum with an osmotically inactive cell volume, V(b), of 0.6 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best fit" membrane permeability parameters at 5 and 10 degrees C/min for canine sperm cells in the absence of CPAs are: L(pg)=0.52x10(-15)m(3)/Ns (0.0029mum/min-atm) and E(Lp)=64.0kJ/mol (15.3kcal/mol) (R(2)=0.99); and the corresponding parameters in the presence of CPAs ranged from L(pg)[cpa]=0.46 to 0.53x10(-15) m(3)/Ns (0.0027-0.0031mum/min-atm) and E(Lp)[cpa]=46.4-56.0kJ/mol (11.1-13.4kcal/mol). These parameters are significantly different than previously published parameters for canine and other mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that optimal rates of freezing canine sperm cells ranges from 10 to 30 degrees C/min; these theoretical cooling rates are found to be in close conformity with previously published but empirically determined optimal cooling rates.     

Helix formation and the unfolded state of a 52-residue helical protein

A growing class of proteins in biological processes has been found to be unfolded on isolation under normal solution conditions. We have used NMR spectroscopy to characterize the structural and dynamic properties of the unfolded and partially folded states of a 52-residue alanine-rich protein (Ala-14) at temperatures from -5 degrees C to 40 degrees C. At 40 degrees C, alanine residues in Ala-14 adopt phi and psi angles, consistent with a significant ensemble population of polyproline II conformation. Analysis of relaxation rates in the protein reveals that a series of residues, Gln 35-Ala 36-Ala 37-Lys 38-Asp 39-Asp 40-Ala 41-Ala 42, displays slow motional dynamics at both -5 degrees C and 40 degrees C. Temperature-dependent chemical shift changes indicate that this region is the site of helix initiation. The remaining N-terminal residues become increasingly dynamic as they extend from the nucleation site. The C terminus remains dynamic and changes less with temperature, indicating it is relatively unstructured. Ala-14 provides a high-resolution portrait of the unfolded state and the process of helix nucleation and propagation in the absence of tertiary contacts, information that bears on early events in protein folding.     

A test of the model to predict unusually stable RNA hairpin loop stability

To investigate the accuracy of a model [Giese et al., 1998, Biochemistry37:1094-1100 and Mathews et al., 1999, JMol Biol 288:911-940] that predicts the stability of RNA hairpin loops, optical melting studies were conducted on sets of hairpins previously determined to have unusually stable thermodynamic parameters. Included were the tetraloops GNRA and UNCG (where N is any nucleotide and R is a purine), hexaloops with UU first mismatches, and the hairpin loop of the iron responsive element, CAGUGC. The experimental values for the GNRA loops are in excellent agreement (deltaG degrees 37 within 0.2 kcal/mol and melting temperature (TM) within 4 degrees C) with the values predicted by the model. When the UNCG hairpin loops are treated as tetraloops, and a bonus of 0.8 kcal/mol included in the prediction to account for the extra stable first mismatch (UG), the measured and predicted values are also in good agreement (deltaG degrees 37 within 0.7 kcal/mol and TM within 3 degrees C). Six hairpins with unusually stable UU first mismatches also gave good agreement with the predictions (deltaG degrees 37 within 0.5 kcal/mol and TM within 8 degrees C), except for hairpins closed by wobble base pairs. For these hairpins, exclusion of the additional stabilization term for UU first mismatches improved the prediction (AG degrees 37 within 0.1 kcal/mol and TM within 3 degrees C). Hairpins with the iron-responsive element loop were not predicted well by the model, as measured deltaG degrees 37 values were at least 1 kcal/mol greater than predicted.