Value Of Delta Cp Research Articles

Thermal properties of products based on ABS / PC

Polymer blends are capable of providing materials with extended useful properties beyond the range that can be obtained from single polymers. Acrylonitrile-butadiene-styrene and polycarbonate blends are important industrial materials, especially, in the machine building industry, as ABS improves the processing properties of parts and their toughness, and they also feature positive thermodynamic interaction between PC and styrene-acrylonitrile (SAN) copolymer as a part of ABS. DSC method was used to study the thermal properties of ABS and ABS/PC blends with various ratios. The result from this work shows that abnormal endothermic effect in the ABS glass transition region implies an inadequate manufacturing process for automotive components. The paper studied the influence of ABS/PC blend ratios on the value of glass transition temperature and delta Cp, and it was found that the Tg value for SAN/PC increased with decreasing ABS for ABS/PC blends with mass ratio of 75/25, 60/40, 50/50.

Probing neutrino oscillation parameters using high power superbeam from ESS

A high-power neutrino superbeam experiment at the ESS facility has been proposed such that the source-detector distance falls at the second oscillation maximum, giving very good sensitivity towards establishing CP violation. In this work, we explore the comparative physics reach of the experiment in terms of leptonic CP-violation, precision on atmospheric parameters, non-maximal theta23, and its octant for a variety of choices for the baselines. We also vary the neutrino vs. the anti-neutrino running time for the beam, and study its impact on the physics goals of the experiment. We find that for the determination of CP violation, 540 km baseline with 7 years of neutrino and 3 years of anti-neutrino (7nu+3nubar) run-plan performs the best and one expects a 5sigma sensitivity to CP violation for 48% of true values of deltaCP. The projected reach for the 200 km baseline with 7nu+3nubar run-plan is somewhat worse with 5sigma sensitivity for 34% of true values of deltaCP. On the other hand, for the discovery of a non-maximal theta23 and its octant, the 200 km baseline option with 7nu+3nubar run-plan performs significantly better than the other baselines. A 5sigma determination of a non-maximal theta23 can be made if the true value of sin^2theta23 lesssim 0.45 or sin^2theta23 gtrsim 0.57. The octant of theta23 could be resolved at 5sigma if the true value of sin^2theta23 lesssim 0.43 or gtrsim 0.59, irrespective of deltaCP.

Exploring the three flavor effects with future superbeams using liquid argon detectors

Recent measurement of a moderately large value of theta13 signifies an important breakthrough in establishing the standard three flavor oscillation picture of neutrinos. It has provided an opportunity to explore the sub-dominant three flavor effects in present and future long-baseline experiments. In this paper, we perform a comparative study of the physics reach of two future superbeam facilities, LBNE and LBNO in their first phases of run, to resolve the issues of neutrino mass hierarchy, octant of theta23, and leptonic CP violation. We also find that the sensitivity of these future facilities can be improved significantly by adding the projected data from T2K and NOvA. Stand-alone LBNO setup with a 10 kt detector has a mass hierarchy discovery reach of more than 7 sigma, for the lowest allowed value of sin^2theta23(true) = 0.34. This result is valid for any choice of true deltaCP and hierarchy. LBNE10, in combination with T2K and NOvA, can achieve 3 sigma hierarchy discrimination for any choice of deltaCP, sin^2theta23, and hierarchy. The same combination can provide a 3 sigma octant resolution for sin^2theta23(true) leq 0.44 or for sin^2theta23(true) geq 0.58 for all values of deltaCP(true). LBNO can give similar results with 10 kt detector mass. In their first phases, both LBNE10 and LBNO with 20 kt detector can establish leptonic CP violation for around 50% values of true deltaCP at 2 sigma confidence level. In case of LBNE10, CP coverage at 3 sigma can be enhanced from 3% to 43% by combining T2K and NOvA data, assuming sin^2theta23(true) = 0.5. For LBNO setup, CP violation discovery at 3 sigma is possible for 46% values of true deltaCP if we add the data from T2K and NOvA.

Resolving the octant of θ 23 with T2K and NOνA

Preliminary results of MINOS experiment indicate that theta23 is not maximal. Global fits to world neutrino data suggest two nearly degenerate solutions for theta23: one in the lower octant (LO: theta23 < 45 degree) and the other in the higher octant (HO: theta23 > 45 degree). numu to nue oscillations in superbeam experiments are sensitive to the octant and are capable of resolving this degeneracy. We study the prospects of this resolution by the current T2K and upcoming NOvA experiments. Because of the hierarchy-deltacp degeneracy and the octant-deltacp degeneracy, the impact of hierarchy on octant resolution has to be taken into account. As in the case of hierarchy determination, there exist favorable (unfavorable) values of deltacp for which octant resolution is easy (challenging). However, for octant resolution the unfavorable deltacp values of the neutrino data are favorable for the anti-neutrino data and vice-verse. This is in contrast to the case of hierarchy determination. In this paper, we compute the combined sensitivity of T2K and NOvA to resolve the octant ambiguity. If sin^2\theta23 =0.41, then NOvA can rule out all the values of theta23 in HO at 2 sigma C.L., irrespective of the hierarchy and deltacp. Addition of T2K data improves the octant sensitivity. If T2K were to have equal neutrino and anti-neutrino runs of 2.5 years each, a 2 sigma resolution of the octant becomes possible provided sin^2\theta23 \leq 0.43 or \geq 0.58 for any value of deltacp.

Potential of optimized NOνA for large θ 13 &amp; combined performance with a LArTPC &amp; T2K

NOvA experiment has reoptimized its event selection criteria in light of the recently measured moderately large value of theta(13). We study the improvement in the sensitivity to the neutrino mass hierarchy and to leptonic CP violation due to these new features. For favourable values of deltacp, NOvA sensitivity to mass hierarchy and leptonic CP violation is increased by 20%. Addition of 5 years of neutrino data from T2K to NOvA more than doubles the range of deltacp for which the leptonic CP violation can be discovered, compared to stand alone NOvA. But for unfavourable values of deltacp, the combination of NOvA and T2K are not enough to provide even a 90% C.L. hint of hierarchy discovery. Therefore, we further explore the improvement in the hierarchy and CP violation sensitivities due to the addition of a 10 kt liquid argon detector placed close to NOvA site. The capabilities of such a detector are equivalent to those of NOvA in all respects. We find that combined data from 10 kt liquid argon detector (3 years of nu + 3 years of nubar run), NOvA (6 years of nu + 6 years of nubar run) and T2K (5 years of nu run) can give a close to 2 sigma hint of hierarchy discovery for all values of deltacp. With this combined data, we can achieve CP violation discovery at 95% C.L. for roughly 60% values of deltacp.

Getting the best out of T2K andNOνA

We explore the combined physics potential of T2K and NOvA in light of the moderately large measured value of theta13. For sin^2 2*theta13 = 0.1, which is close to the best fit value, a 90% C.L. evidence for the hierarchy can be obtained only for the combinations (Normal hierarchy, -170 <= deltaCP <= 0) and (Inverted hierarchy, 0 <= deltaCP <= 170), with the currently planned runs of NOvA and T2K. However, the hierarchy can essentially be determined for any value of deltaCP, if the statistics of NOvA are increased by 50% and those of T2K are doubled. Such an increase will also give an allowed region of deltaCP around its true value, except for the CP conserving cases deltaCP = 0 or 180. We demonstrate that any measurement of deltaCP is not possible without first determining hierarchy. We find that comparable data from a shorter baseline (L ~ 130 km) experiment will not lead to any significant improvement.

Hydration and Heat Stability Effects on Protein Unfolding

In summary, the thermal denaturation of proteins has been elucidated in terms of the chain free energy and the hydration free energy as follows. (1) Method to calculate the unfolding free energy. The free energy of unfolding consists of two contributions: the hydration around the molecule, and the intramolecular interactions. A method to calculate the free energy of hydration from the accessible surface area (ASA) of the constituent atomic groups in a protein has been developed. This assumes a proportionality between the free energy and the ASA, where the proportional constants were determined by least-squares fitting to the experimentally derived thermodynamic data on small molecules. Similarly, the free energy of unfolding for the chain in vacuo can be also calculated from the ASA, using the unfolding thermodynamics derived from the experimental data of the ten proteins. (2) Thermodynamics of protein unfolding predicted from the three-dimensional structures and from the amino acid content in proteins. First, our method is applied to predict the thermodynamics of protein unfolding from the X-ray structure. The predicted values of four test proteins agree well with the experimentally derived values. It also accounts for the temperature dependence of the free energy and of the enthalpy upon unfolding for 14 proteins. Second, this method is applied to the helix-coil transition of short peptides of poly(L-Ala)20 and Ac-(AAAAK)3A-NH2. The calculated enthalpy change is close to the experimental values for poly-L-Lys and poly-L-Glu. Since delta Hcu at 25 degrees C significantly contributes to delta Gu, the helix formation is enthalpy-driven through interactions in the chain. Third, the method is applied to predict the unfolding thermodynamics of a globular protein from its amino acid content. It also accounts for the temperature dependence of the free energy of unfolding for the 14 proteins. The agreement between the experimental and the calculated values by this method for the 14 proteins is not so different from those obtained with the three-dimensional structures. Fourth, the values of delta Cpu for 14 proteins may be closely approximated to the predicted values of delta Cp,hu. The delta Cp,hu value in a protein consists of the major contribution from the hydrophobic and the aromatic residues, and the minor one from the hydrophilic residues. (3) Dominant free energies in protein folding.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of Specific Solvent Rearrangement Regions of an Enzyme: NMR and ITC Studies with Aminoglycoside Phosphotransferase(3‘)-IIIa

This work describes differential effects of solvent in complexes of the aminoglycoside phosphotransferase(3')-IIIa (APH) with different aminoglycosides and the detection of change in solvent structure at specific sites away from substrates. Binding of kanamycins to APH occurs with a larger negative DeltaH in H2O relative to D2O (DeltaDeltaH(H2O-D2O) < 0), while the reverse is true for neomycins. Unusually large negative DeltaCp values were observed for binding of aminoglycosides to APH. DeltaCp for the APH-neomycin complex was -1.6 kcal x mol(-1) x deg(-1). A break at 30 degrees C was observed in the APH-kanamycin complex yielding DeltaCp values of -0.7 kcal x mol(-1) x deg(-1) and -3.8 kcal x mol(-1) x deg(-1) below and above 30 degrees C, respectively. Neither the change in accessible surface area (DeltaASA) nor contributions from heats of ionization were sufficient to explain the large negative DeltaCp values. Most significantly, 15N-1H HSQC experiments showed that temperature-dependent shifts of the backbone amide protons of Leu 88, Ser 91, Cys 98, and Leu143 revealed a break at 30 degrees C only in the APH-kanamycin complex in spectra collected between 21 degrees C and 38 degrees C. These amino acids represent solvent reorganization sites that experience a change in solvent structure in their immediate environment as structurally different ligands bind to the enzyme. These residues were away from the substrate binding site and distributed in three hydrophobic patches in APH. Overall, our results show that a large number of factors affect DeltaCp and binding of structurally different ligand groups cause different solvent structure in the active site as well as differentially affecting specific sites away from the ligand binding site.

Thermodynamics of Ni2+, Cu2+, and Zn2+ Binding to the Urease Metallochaperone UreE

The two Ni2+ ions in the urease active site are delivered by the metallochaperone UreE, whose metal binding properties are central to the assembly of this metallocenter. Isothermal titration calorimetry (ITC) has been used to quantify the stoichiometry, affinity, and thermodynamics of Ni2+, Cu2+, and Zn2+ binding to the well-studied C-terminal truncated H144*UreE from Klebsiella aerogenes, Ni2+ binding to the wild-type K. aerogenes UreE protein, and Ni2+ and Zn2+ binding to the wild-type UreE protein from Bacillus pasteurii. The stoichiometries and affinities obtained by ITC are in good agreement with previous equilibrium dialysis results, after differences in pH and buffer competition are considered, but the concentration of H144*UreE was found to have a significant effect on metal binding stoichiometry. While two metal ions bind to the H144*UreE dimer at concentrations <10 microM, three Ni2+ or Cu2+ ions bind to 25 microM dimeric protein with ITC data indicating sequential formation of Ni/Cu(H144*UreE)4 and then (Ni/Cu)2(H144*UreE)4, or Ni/Cu(H144*UreE)2, followed by the binding of four additional metal ions per tetramer, or two per dimer. The thermodynamics indicate that the latter two metal ions bind at sites corresponding to the two binding sites observed at lower protein concentrations. Ni2+ binding to UreE from K. aerogenes is an enthalpically favored process but an entropically driven process for the B. pasteurii protein, indicating chemically different Ni2+ coordination to the two proteins. A relatively small negative value of DeltaCp is associated with Ni2+ and Cu2+ binding to H144*UreE at low protein concentrations, consistent with binding to surface sites and small changes in the protein structure.

Identification and Thermodynamic Characterization of Molten Globule States of Periplasmic Binding Proteins

Molten globule-like intermediates have been shown to occur during protein folding and are thought to be involved in protein translocation and membrane insertion. However, the determinants of molten globule stability and the extent of specific packing in molten globules is currently unclear. Using far- and near-UV CD and intrinsic and ANS fluorescence, we show that four periplasmic binding proteins (LBP, LIVBP, MBP, and RBP) form molten globules at acidic pH values ranging from 3.0 to 3.4. Only two of these (LBP and LIVBP) have similar sequences, but all four proteins adopt similar three-dimensional structures. We found that each of the four molten globules binds to its corresponding ligand without conversion to the native state. Ligand binding affinity measured by isothermal titration calorimetry for the molten globule state of LIVBP was found to be comparable to that of the corresponding native state, whereas for LBP, MBP, and RBP, the molten globules bound ligand with approximately 5-30-fold lower affinity than the corresponding native states. All four molten globule states exhibited cooperative thermal unfolding assayed by DSC. Estimated values of DeltaCp of unfolding show that these molten globule states contain 28-67% of buried surface area relative to the native states. The data suggest that molten globules of these periplasmic binding proteins retain a considerable degree of long range order. The ability of these sequentially unrelated proteins to form highly ordered molten globules may be related to their large size as well as an intrinsic property of periplasmic binding protein folds.

Unusually Strong Binding to the DNA Minor Groove by a Highly Twisted Benzimidazole Diphenylether: Induced Fit and Bound Water

RT29 is a dicationic diamidine derivative that does not obey the classical "rules" for shape and functional group placement that are expected to result in strong binding and specific recognition of the DNA minor groove. The compound contains a benzimidazole diphenyl ether core that is flanked by the amidine cations. The diphenyl ether is highly twisted and gives the entire compound too much curvature to fit well to the shape of the minor groove. DNase I footprinting, fluorescence intercalator displacement studies, and circular dichroism spectra, however, indicate that the compound is an AT specific minor groove binding agent. Even more surprisingly, quantitative biosensor-surface plasmon resonance and isothermal titration calorimetric results indicate that the compound binds with exceptional strength to certain AT sequences in DNA with a large negative enthalpy of binding. Crystallographic results for the DNA complex of RT29 compared to calculated results for the free compound show that the compound undergoes significant conformational changes to enhance its minor groove interactions. In addition, a water molecule is incorporated directly into the complex to complete the compound-DNA interface, and it forms an essential link between the compound and base pair edges at the floor of the minor groove. The calculated DeltaCp value for complex formation is substantially less than the experimentally observed value, which supports the idea of water being an intrinsic part of the complex with a major contribution to the DeltaCp value. Both the induced fit conformational changes of the compound and the bound water are essential for strong binding to DNA by RT29.

Cold denaturation of ubiquitin at high pressure

Cold-induced conformational transition of ubiquitin was studied at pH 4.5 under a constant pressure of 2 kbar using variable pressure one-dimensional 1H and two-dimensional 15N/1H NMR spectroscopy as well as IR spectroscopy. Although a tendency for preferential stabilization of a peculiar locally disordered and partially hydrated conformer I, identical with that previously found with variable-pressure NMR at 0 degrees C, is recognized, the transition of the folded conformer N to the unfolded conformer U occurs largely cooperatively with decreasing temperature, reaching near completion at - 21 degrees C. NMR spectral features as well as the analysis of NMR relaxation parameters indicate that the polypeptide chain is almost fully unfolded, fairly well-hydrated and floppy at - 21 degrees C, whereas the IR spectrum shows a substantial decrease of the beta-sheet. The Gibbs energy change from the folded state (a mixture of N and I) to the unfolded state at 2 kbar obtained from the 1H NMR data is fitted well with a single DeltaCp value of 2.43 +/- 0.13 (kJ/K mol) for the entire temperature range between - 21 and 90 degrees C, covering both the cold denaturation and heat denaturation, showing that the two denatured states actually belong to a single thermodynamic phase of the protein. The DeltaCp value determined at 2 kbar is substantially smaller than the DeltaCp determined at 1 bar (3.8-5.8 (kJ/Kmol), which is consistent with the fact that the denaturation takes place from a mixture of N and I at 2 kbar rather than from pure N at 1 bar.

Why Is Trehalose an Exceptional Protein Stabilizer?: AN ANALYSIS OF THE THERMAL STABILITY OF PROTEINS IN THE PRESENCE OF THE COMPATIBLE OSMOLYTE TREHALOSE

Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 m trehalose was observed to raise the transition temperature, Tm of RNase A by as much as 18 degrees C and Gibbs free energy by 4.8 kcal mol-1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (DeltaCp) in trehalose solutions for all the studied proteins. An increase in the DeltaG and a decrease in the DeltaCp values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (DeltaG versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 m concentration 4-7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols.

The sequence-specific association of the ETS domain of murine PU.1 with DNA exhibits unusual energetics.

PU.1 belongs to the ETS family of transcription factors whose DNA-binding domains recognize purine-rich sequences containing the consensus 5'-GGAA/T-3'. We have characterized the sequence-specific association of the ETS domain of murine PU.1 to the lambdaB site of the Ig(lambda)2-4 enhancer as a function of temperature and pH by electrophoretic mobility shift, filter binding, and CD spectroscopy. From 0 to 25 degrees C, the dissociation equilibrium constant KD is, within experimental uncertainty, insensitive to temperature, and is only a weak function of temperature from 25 to 52 degrees C. van't Hoff analysis yielded a small value of DeltaCp = -2.1 kJ x mol(-1) x K(-1) in phosphate buffer, pH 7.4, containing 250 mM Na+. KD also shows a weak dependence at 25 degrees C on pH from 6.7 to 9.0 in phosphate, cacodylate, and Tris buffers that have disparate heats of ionization. The CD spectrum of the protein-DNA complex could be accounted for by a simple linear combination of the spectra of the free components throughout the binding temperature range. Structural calculations indicate that dehydration of solvent-accessible contact surfaces on the protein and DNA accounts for up to DeltaCp approximately -1 kJ x mol(-1) x K(-1). Taken together, these observations suggest that the hydrophobic effect and, in particular, coupled folding do not contribute significantly to the energetics of sequence-specific association. This is unusual with respect to other sequence-specific protein-DNA interactions for which significant enthalpic contributions and large negative heat capacities are commonly observed.

Compulsory Order of Substrate Binding to Herpes Simplex Virus Type 1 Thymidine Kinase: A CALORIMETRIC STUDY

Isothermal titration calorimetry has been used to investigate the thermodynamic parameters of the binding of thymidine (dT) and ATP to herpes simplex virus type 1 thymidine kinase (HSV1 TK). Binding follows a sequential pathway in which dT binds first and ATP second. The free enzyme does not bind ATP, whose binding site becomes only accessible in the HSV1 TK.dT complex. At pH 7.5 and 25 degrees C, the binding constants are 1.9 x 10(5) m(-1) for dT and 3.9 x 10(6) m(-1) for ATP binding to the binary HSV1 TK.dT complex. Binding of both substrates is enthalpy-driven and opposed by a large negative entropy change. The heat capacity change (DeltaCp) obtained from DeltaH in the range of 10-25 degrees C is -360 cal K(-1) mol(-1) for dT binding and -140 cal K(-1) mol(-1) for ATP binding. These large DeltaCp values are incompatible with a rigid body binding model in which the dT and ATP binding sites pre-exist in the free enzyme. Values of DeltaCp and TDeltaS strongly indicate large scale conformational adaptation of the active site in sequential substrate binding. The conformational changes seem to be more pronounced in dT binding than in the subsequent ATP binding. Considering the crystal structure of the ternary HSV1 TK.dT.ATP complex, a large movement in the dT binding domain and a smaller but substantial movement in the LID domain are proposed to take place when the enzyme changes from the substrate-free, presumably more open and less ordered conformation to the closed and compact conformation of the ternary enzyme-substrate complex.

A more unified picture for the thermodynamics of nucleic acid duplex melting: a characterization by calorimetric and volumetric techniques.

We use a combination of calorimetric and volumetric techniques to detect and to characterize the thermodynamic changes that accompany helix-to-coil transitions for five polymeric nucleic acid duplexes. Our calorimetric measurements reveal that melting of the duplexes is accompanied by positive changes in heat capacity (DeltaCP) of similar magnitude, with an average DeltaCP value of 64.6 +/- 21.4 cal deg-1 mol-1. When this heat capacity value is used to compare significantly different transition enthalpies (DeltaHo) at a common reference temperature, Tref, we find DeltaHTref for duplex melting to be far less dependent on duplex type, base composition, or base sequence than previously believed on the basis of the conventional assumption of a near-zero value for DeltaCP. Similarly, our densimetric and acoustic measurements reveal that, at a given temperature, all the AT- and AU-containing duplexes studied here melt with nearly the same volume and compressibility changes. In the aggregate, our results, in conjunction with literature data, suggest a more unified picture for the thermodynamics of nucleic acid duplex melting. Specifically, when compared at a common temperature, the apparent large differences present in the literature for the transition enthalpies of different duplexes become much more compressed, and the melting of all-AT- and all-AU-containing duplexes exhibits similar volume and compressibility changes despite differences in sequence and conformation. Thus, insofar as thermodynamic properties are concerned, when comparing duplexes, the temperature under consideration is as important as, if not more important than, the duplex type, the base composition, or the base sequence. This general behavior has significant implications for our basic understanding of the forces that stabilize nucleic acid duplexes. This behavior also is of practical significance in connection with the use of thermodynamic databases for designing probes and for assessing the affinity and specificity associated with hybridization-based protocols used in a wide range of sequencing, diagnostic, and therapeutic applications.

Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large deltaCp's.

Maltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8-anilino-1-naphthalene sulfonate (ANS). The unfolding thermodynamics of MBP at both pHs were measured by carrying out a series of isothermal urea melts at temperatures ranging from 274-329 K. At 298 K, values of deltaGdegrees , deltaCp, and Cm were 3.1+/-0.2 kcal mol(-1), 5.9+/-0.8 kcal mol(-1) K(-1) (15.9 cal (mol-residue)(-1) K(-1)), and 0.8 M, respectively, at pH 3.0 and 14.5+/-0.4 kcal mol(-1), 8.3+/-0.7 kcal mol(-1) K(-1) (22.4 kcal (mol-residue)(-1) K(-1)), and 3.3 M, respectively, at pH 7.1. Guanidine hydrochloride denaturation at pH 7.1 gave values of deltaGdegrees and deltaCp similar to those obtained with urea. The m values for denaturation are strongly temperature dependent, in contrast to what has been previously observed for small globular proteins. The value of deltaCp per mol-residue for the molten globule is comparable to corresponding values of deltaCp for the unfolding of typical globular proteins and suggests that it is a highly ordered structure, unlike molten globules of many small proteins. The value of deltaCp per mol-residue for the unfolding of the native state is among the highest currently known for any protein.

Thermodynamics of replacing an alpha-helical Pro residue in the P40S mutant of Escherichia coli thioredoxin.

Escherichia coli thioredoxin is a 108 amino acid oxidoreductase and contains a single Met residue at position 37. The protein contains a long alpha-helical stretch between residues 32 and 49. The central residue of this helix, Pro40, has been replaced by Ser. The stabilities of the oxidized states of two proteins, the single mutant M37L and the double mutant M37L,P40S, have been characterized by differential scanning calorimetry (DSC) and also by a series of isothermal guanidine hydrochloride (GuHCl) melts in the temperature range of 277 to 333 K. The P40S mutation was found to stabilize the protein at all temperatures upto 340 K though both proteins had similar Tm values of about 356 K. At 298 K, the M37L,P40S mutant was found to be more stable than M37L by 1.5 kcal/mol. A combined analysis of GuHCl and calorimetric data was carried out to determine the enthalpy, entropy, and heat capacity change upon unfolding. At 298 K there was a large, stabilizing enthalpic effect in P40S though significant enthalpy-entropy compensation was observed and the two proteins had similar values of deltaCp. Thus, replacement of a Pro in the interior of an alpha helix can have substantial effects on protein stability.

Denaturation of type I collagen fibrils is an endothermic process accompanied by a noticeable change in the partial heat capacity.

Thermal transitions of type I collagen fibrils were investigated by differential scanning calorimetry and spectrophotometry of turbidity within a wide range of external conditions. The advanced microcalorimeter allowed us to carry out the measurements at low concentrations of collagen (0.15-0.3 mg/mL). At these concentrations of collagen and under fibril-forming conditions, the melting curves display two pronounced heat adsorption peaks (at 40 and 55 degreesC). The low-temperature peak was assigned to the melting of monomeric collagen, while the high-temperature peak was assigned to the denaturation of collagen fibrils. It was shown that the denaturation of fibrils, in contrast to the monomeric collagen, is accompanied by a noticeable change in the partial specific heat capacity. Surprisingly, comparison of the collagen calorimetric curves in the fibril-forming and nonforming conditions revealed that DeltaCp of fibril denaturation is caused by a decrease in the Cp of collagen at premelting temperatures. This suggests the existence of an intermediate structural state of collagen in a transparent solution preceding fibril formation. Our study also shows that collagen fibrils formed prior to heating have thermodynamic parameters different from those of fibrils formed and denatured during heating in the calorimeter. Analysis of the data allowed us to determine the denaturation enthalpy of the mature fibrils and to conclude that the enthalpy plays a more important role in fibril stabilization than was previously assumed. The observed large DeltaCp value of fibril denaturation as well as the difference between thermodynamic parameters of the mature and newly formed fibrils is readily explained by the presence of water molecules in the fibril structure.

Thermodynamic Analysis of the Interaction between the 0.5β Fv Fragment and the RP135 Peptide Antigen Derived from the V3 Loop of HIV-1 gp120

The Fv fragment of the 0.5beta monoclonal antibody has recently been constructed, expressed, and purified. It binds with nanomolar affinity to the immunogenic RP135 peptide that is derived from the principal neutralizing determinant of HIV-1 in the third hypervariable region of gp120. Here, we analyzed the temperature-dependence of binding of the 0.5beta Fv fragment to the RP135 peptide and a series of mutants thereof. Our results show that there is almost complete enthalpy-entropy compensation in the effects of mutations in the peptide on binding to the Fv, indicating that the mutations do not change the binding mechanism. There is good correlation, for residues within the antigenic epitope, between mutational effects on DeltaCp and calculated values of DeltaDeltaCp based on the extent of burial of polar and non-polar surface areas of amino acids. The value of DeltaCp for the binding of the 0.5beta Fv fragment to the wild-type RP135 peptide is found to be -5.0 (+/- 0.9) kcal K-1 mol-1 in the presence of 0.1% Tween-20 but only -0.1 (+/- 0.9) kcal K-1 mol-1 in its absence. This result has important implications for the successful application of the structural parameterization approach to predicting changes in heat capacity that accompany binding reactions carried out in the presence of detergent or protein-stabilizing agents.