Comparative thermodynamic analyses of the Fv, Fab* and Fab and Fab fragments of anti-dansyl mouse monoclonal antibody

In order to investigate the role of the constant domainson the antigen-binding property of the variable domains, we have carried out a comparative thermodynamic study of the anti-dansyl Fv, Fab* and Fab fragments that possess the identical amino acid sequence of the variable domains. The thermodynamic analyses have shown that binding constants, enthalphy changes and entropy changes are similar for the three antigen-binding fragments, whereas the thermal stability of Fab is much higher than that of Fv and Fab*. We have concluded that (i) the variable domains of the three antigen-binding fragments possess identical intrinsic capability for antigen binding and (ii) the two constant domains serve to improve the stability of the variable domains.     

Thermotropic lipid phase transition and the behavior of hydrolytic enzymes in the kidney cortex brush border membrane

Functional interactions of lipids and proteins were examined in brush-border membranes isolated from the kidney cortex by studying the temperature dependence of the hydrolytic enzyme activities. A close relationship was observed for the membrane proteins and the thermotropic lipid phase transitions. Three lines of evidences were provided for such dependence: a) Arrhenius relationship of the membrane-bound enzyme activities, and the effect of temperature in native and partially delipidated membranes, b) differential scanning calorimetric study of the membrane lipid phase transitions in the native and delipidated membranes, multilamellar vesicles prepared from the membrane extracted lipids, and in vesicles from dimyristoyl phosphatidylcholine, and c) the excimer (dimer)-formation studies of the membrane extrinsic fluorescent probe, pyrene, and the resultant membrane microviscosity. The brush-border membranes were partially delipidated with BuOH and 2,2,2-trifluoroethanol. The functional interactions of the delipidated membranes, which were greatly lost on lipid removal, were largely restored by the addition of exogenous lipids in the reconstitution process, which indicate the critical dependence of the membrane integral proteins on the neighboring lipid molecules in the bulk lipid phase.     

Effect of surfactants on the thermal, conformational and rheological properties of collagen

Collagen is an important biomaterial and its interaction with surfactant is important in light of its use in various cosmetics and dermatological applications. Presently, the effect of surfactants on the physico-chemical properties of collagen has been studied. The thermal stability of collagen is reduced by sodium dodecyl sulfate and hexadecyltrimethylammmonium bromide, whereas Triton X-100 does not. The viscosity of collagen is influenced greatly depending on the surfactant concentrations. The secondary structure of collagen shows changes only in the molar ellipticity. The role of charge and concentration of surfactants in influencing the various physico-chemical properties of collagen has been elucidated.     

Calorimetric study of a series of designed repeat proteins: modular structure and modular folding

Repeat proteins comprise tandem arrays of a small structural motif. Their structure is defined and stabilized by interactions between residues that are close in the primary sequence. Several studies have investigated whether their structural modularity translates into modular thermodynamic properties. Tetratricopeptide repeat proteins (TPRs) are a class in which the repeated unit is a 34 amino acid helix-turn-helix motif. In this work, we use differential scanning calorimetry (DSC) to study the equilibrium stability of a series of TPR proteins with different numbers of an identical consensus repeat, from 2 to 20, CTPRa2 to CTPRa20. The DSC data provides direct evidence that the folding/unfolding transition of CTPR proteins does not fit a two-state folding model. Our results confirm and expand earlier studies on TPR proteins, which showed that apparent two-state unfolding curves are better fit by linear statistical mechanics models: 1D Ising models in which each repeat is treated as an independent folding unit.     

On the stability of the ripple phase in the DPPC/PLPC/water ternary system

The effect of incorporation of 1-palmitoyl-sn-glycero-3-phosphocholine (PLPC) on the structure of the P_β′ ripple mesophase in aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) has been studied by differential scanning calorimetry (DSC) and scanning dilatometry (SD). For samples containing 34 wt. % ²H₂O and 0–15 wt. % PLPC, a pretransition was observed by DSC. The pretransition disappears at 15 wt. % PLPC. The behavior of thermodynamic functions at the pretransition and main transition gives new insights on the structural changes produced by PLPC on bilayers of DPPC.     

The abasic site lesions in the human telomeric sequence d[TA(G3T2A)3G3]: A thermodynamic point of view

Background

The abasic sites represent one of the most frequent lesions of DNA and most of the events able to generate such modifications involve guanine bases. G-rich sequences are able to form quadruplex structures that have been proved to be involved in several important biological processes.

Methods

In this paper, we report investigations, based on calorimetric, UV, CD and electrophoretic techniques, on 12 oligodeoxynucleotides analogues of the quadruplex forming human telomere sequence d[TA(G₃T₂A)₃G₃], in which each guanine has been replaced, one at a time, by an abasic site mimic.

Results

Although all data show that the modified sequences preserve their ability to form quadruplex structures, the thermodynamic parameters clearly indicate that the presence of an abasic site decreases their thermal stability compared to the parent unmodified sequence, particularly if the replacement concerns one of the guanosines involved in the formation of the central G-tetrad.

Conclusions

The collected data indicate that the effects of the presence of abasic site lesions in telomeric quadruplex structures are site-specific. The most dramatic consequences come out when this lesion involves a guanosine in the centre of a G-run.

General significance

Abasic sites, by facilitating the G-quadruplex disruption, could favour the formation of the telomerase primer. Furthermore they could have implications in the pharmacological approach targeting telomere.     

Phase equilibria of model milk membrane lipid systems

The phase behaviour of mixtures of recombined milk membrane lipids dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM), dioleoylphosphatidylethanolamine (DOPE), phosphatidylinositol (PI) and dioleoylphosphatidylserine (DOPS) in 60% water was examined as a function of temperature between 5 and 90 degrees C. The aim was to examine under which lipid composition the average properties turn from balanced over to hydrophobic. The phase boundaries were determined by small angle X-ray diffraction (SAXD) and differential scanning calorimetry (DSC). The lamellar phase was dominating in the DOPC/SM/DOPE system. The phase boundary for the reversed hexagonal phase was only observed at high DOPE content within the examined temperature interval. The anionic phospholipids PI and DOPS induced a swollen lamellar phase, but no significant change of the transition between the lamellar phase and the reversed hexagonal phase was observed.     

Consequences of ions and pH on the supramolecular organization of sphingomyelin and sphingomyelin/cholesterol bilayers

For drug delivery purpose the anticancer drug S12363 was loaded into ESM/Chol-liposomes using either a pH or an ammonium gradient. Association between the drug and the liposome depends markedly on the liposome membrane structure. Thus, ESM and ESM/Chol bilayer organization had been characterized by coupled DSC and XRDT as a function of both cholesterol concentration and aqueous medium composition. ESM bilayers exhibited a ripple lamellar gel phase P(beta') below the melting temperature and adopted a L(beta)-like gel phase upon Chol insertion. Supramolecular organization of ESM and ESM/Chol bilayers was not modified by citrate buffer or ammonium sulfate solution whatever the pH (3< or = pH < or =7). Nevertheless, in ESM bilayer, ammonium sulfate salt induced a peculiar organization of head groups, leading to irregular d-spacing and weakly correlated bilayers. Moreover, in the presence of salts, a weakening of van der Waals attraction forces was seen and led to a swelling of the water layer.     

The role of water in the reversibility of thermal denaturation of lysozyme in solid and liquid states

Although unfolding of protein in the liquid state is relatively well studied, its mechanisms in the solid state, are much less understood. We evaluated the reversibility of thermal unfolding of lysozyme with respect to the water content using a combination of thermodynamic and structural techniques such as differential scanning calorimetry, synchrotron small and wide-angle X-ray scattering (SWAXS) and Raman spectroscopy. Analysis of the endothermic thermal transition obtained by DSC scans showed three distinct unfolding behaviors at different water contents. Using SWAXS and Raman spectroscopy, we investigated reversibility of the unfolding for each hydration regime for various structural levels including overall molecular shape, secondary structure, hydrophobic and hydrogen bonding interactions. In the substantially dehydrated state below 37 wt% of water the unfolding is an irreversible process and can be described by a kinetic approach; above 60 wt% the process is reversible, and the thermodynamic equilibrium approach is applied. In the intermediate range of water contents between 37 wt% and 60 wt%, the system is phase separated and the thermal denaturation involves two processes: melting of protein crystals and unfolding of protein molecules. A phase diagram of thermal unfolding/denaturation in lysozyme - water system was constructed based on the experimental data.     

Small angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) studies of amide phospholipids

Varying chemically the structure of phospholipids in the region between hydrophobic and hydrophilic segments is expected to have a strong influence on the interaction with water and the phase behavior. This is studied in this work with the motivation to investigate these lipids as potential inhibitors of phospholipase A2. Thus the amide phospholipids L-ether-amide-PC (1-O-hexadecyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine), L-ester-amide-PC (1-palmitoyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine) and L-ether-amide-PE (1-O-hexadecyl-2-N-palmitoyl-2-deoxy-sn-glycero-3-phosphoethanolamine) have been synthesized and characterized. The phase behavior and thermal transitions in buffer dispersions are examined by a combination of high-sensitivity differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS) experiments between 10 and 80 degrees C at pH 8.9. The onset temperatures determined from DSC measurements agree well with the starting temperatures of changes in the repeat distance obtained by SAXS measurements. The phases observed are lamellar both below and above the main phase transition. The phase transition temperatures and enthalpies depend strongly on the substitutions in sn-1 position and head group structure. The lamellar repeat distance in gel and liquid-crystalline phases increases with increasing temperature for L-ester-amide-PC and L-ether-amide-PC, whereas the temperature dependence is opposite for the L-ether-amide-PE. The observed behavior is discussed and compared with that of DPPC and DPPE, indicating the strong dependence of hydration and phase behavior on head group structure.     

Intermolecular interaction of voriconazole analogues with model membrane by DSC and NMR,and their antifungal activity using NMR based metabolic profiling

The development of novel antifungal agents with high susceptibility and increased potency can be achieved by increasing their overall lipophilicity. To enhance the lipophilicity of voriconazole, a second generation azole antifungal agent, we have synthesized its carboxylic acid ester analogues, namely p-methoxybenzoate (Vpmb), toluate (Vtol), benzoate (Vbz) and p-nitrobenzoate (Vpnb). The intermolecular interactions of these analogues with model membrane have been investigated using nuclear magnetic resonance (NMR) and differential scanning calorimetric (DSC) techniques. The results indicate varying degree of changes in the membrane bilayer’s structural architecture and physico-chemical characteristics which possibly can be correlated with the antifungal effects via fungal membrane. Rapid metabolite profiling of chemical entities using cell preparations is one of the most important steps in drug discovery. We have evaluated the effect of synthesized analogues on Candida albicans. The method involves real time ¹H NMR measurement of intact cells monitoring NMR signals from fungal metabolites which gives Metabolic End Point (MEP). This is then compared with Minimum Inhibitory Concentration (MIC) determined using conventional methods. Results indicate that one of the synthesized analogues, Vpmb shows reasonably good activity.     

Native fat globules of different sizes selected from raw milk: thermal and structural behavior

The aim of this study was to characterize differences in the thermal and structural behavior between different sized native milk fat globules. A novel microfiltration process permits the selection of native small fat globules (SFG, 1-3 microm) and large fat globules (LFG, >5 microm) in raw milk, that were analyzed by X-ray diffraction (XRD) coupled to differential scanning calorimetry (DSC). There were no major differences in triglyceride crystalline structures between SFG and LFG, after eliminating thermal history and the influence of cooling rates. The three main 3L and 2L crystalline structures appearing under slow cooling existed regardless of globule size. The supercooling increased for the SFG, mainly due to heterogeneous nucleation in winter milk, and also to compositional variations in spring milk. Differences appeared regarding stabilized crystalline forms at 20 degrees C and subsequent cooling: the SFG contained less 2L triglyceride structures than the LFG. These results can be important in dairy manufactures using tempering periods.     

Identification and characterization of the pore-forming protein in the outer membrane of rat liver mitochondria

The proteins of the outer membrane from rat liver mitochondria have been subfractionated by means of density gradient centrifugation. The different polypeptides of the membrane were incorporated into asolectin vesicles and black lipid membranes. It was observed that a polypeptide of M_r 32 000 renders asolectin vesicles permeable to ADP and forms pores in bilayer membrane. These pores showed the same properties as the channels which are formed in the lipid membrane after addition of Triton X-100 solubilized complete outer membrane. The properties of the pore are as follows: (1) The formation of pores depends on the type of phospholipid used for the preparation of the black membranes. (2) The pore is inserted asymmetrically into the membrane. (3) The pore is voltage gated but does not switch off completely at higher voltages. The pore seems to show different conductance states decreasing conductance being observed at increasing voltage. The implications of these findings for the regulation of transport processes across the outer membrane are discussed.     

Structure and Energetics of Encapsidated DNA in Bacteriophage HK97 Studied by Scanning Calorimetry and Cryo-electron Microscopy

Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis. We found that, as in other phages, HK97 DNA is organized in coaxially wound nested shells. When DNA-filled capsids (heads) are scanned in buffer containing 1 mM Mg²⁺, DNA melting and capsid denaturation both contribute to the complex thermal profile between 82 °C and 96 °C. In other conditions (absence of Mg²⁺ and lower ionic strength), DNA melting shifts to lower temperatures and the two events are resolved. Heads release their DNA at temperatures well below the onset of DNA melting or capsid denaturation. We suggest that, on heating, the internal pressure increases, causing the DNA to exit—probably via the portal vertex-while the capsid, although largely intact, sustains local damage that leads to an earlier onset of thermal denaturation. Heads differ structurally from empty capsids in the curvature of their protein shell, a change attributable to outwards pressure exerted by the DNA. We propose that this transition is sensed by the portal that is embedded in the capsid wall, whereupon the structure of the portal and its interactions with terminase, the packaging enzyme, are altered, thus signaling that packaging is at or approaching completion.     

Comparison of methods to determine the degree of gelatinisation for both high and low starch concentrations

A general procedure was developed to measure the degree of gelatinisation in samples over a broad concentration range. Measurements based on birefringence, DSC (Differential scanning calorimetry), X-ray and amylose–iodine complex formation were used. If a 10 w/w % wheat starch–water mixture was used, each method resulted in approximately the same degree of gelatinisation vs. temperature curve. In case the gelatinisation of a 60 w/w % wheat starch–water mixture was followed as a function of the temperature, each method resulted in a different degree of gelatinisation vs. temperature curve. DSC and X-ray measurements are preferred, because they can be used to determine when the final stage of the gelatinisation process has been completed. Birefringence and amylose–iodine complex formation measurements are suitable alternatives if DSC and X-ray equipment is not available, but will lead to different results. The differences between the methods can be explained by considering the phenomena that take place during the gelatinisation at limiting water conditions.

Based on the experimental data obtained with DSC and X-ray measurements, the gelatinisation of 10 w/w % and 60 w/w % wheat starch–water mixtures started at the same temperature (approximately 50 °C). However, complete gelatinisation was reached at different temperatures (approximately 75 °C and 115 °C for, respectively, 10 w/w % and 60 w/w % wheat starch–water mixtures) according to the experimental DSC and X-ray data. These results are in accordance with independent DSC measurements that were carried out.

The Flory equation was adapted to provide a quantitative explanation for the curves describing the degree of starch gelatinisation as a function of the starch–water ratio and the temperature. The gelatinisation curves that were obtained with the model are in good agreement with the experimentally determined curves. The parameters , ΔH_u and χ₁₂ that resulted in the lowest sum of the squared residuals are 291 ± 63 °C, 29.2 ± 3.9 kJ/mol and 0.53 ± 0.05 (95% confidence interval). These values agree with other values reported in literature.     

Hydration of phospholipid bilayers in the presence and absence of cholesterol

The number of water molecules bound (unfreezable) by a molecule of dipalmitoyl phosphatidylserine (DPPS) or by a molecule of dipalmitoyl phosphatidylcholine (DPPC) alone or in mixtures with cholesterol was determined by differential scanning calorimetry (DSC). When the phospholipids are in the gel state and in the absence of cholesterol, molecule of DPPS binds about 3.5 molecules of water and molecule of DPPC binds about 6 molecules of water. Number of water molecules bound increases when cholesterol crystallites are formed in the bilayer. For DPPS-cholesterol mixture at X(chol) -0.5, as well as for DPPC-cholesterol mixture at X(chol) -0.5 about 7 water molecules are bound.     

The interrelationship between ligand binding and thermal unfolding of the folate binding protein. The role of self-association and pH

The present study utilized a combination of DLS (dynamic light scattering) and DSC (differential scanning calorimetry) to address thermostability of high-affinity folate binding protein (FBP), a transport protein and cellular receptor for the vitamin folate. At pH 7.4 (pI = 7–8) ligand binding increased concentration-dependent self-association of FBP into stable multimers of holo-FBP. DSC of 3.3 μM holo-FBP showed Tm (76 °C) and molar enthalpy (146 kcal M^− 1) values increasing to 78 °C and 163 kcal M^− 1 at 10 μM holo-FBP, while those of apo-FBP were 55 °C and 105 kcal M^− 1. Besides ligand binding, intermolecular forces involved in concentration-dependent multimerization thus contribute to the thermostability of holo-FBP. Hence, thermal unfolding and dissociation of holo-FBP multimers occur simultaneously consistent with a gradual decrease from octameric to monomeric holo-FBP (10 μM) in DLS after a step-wise rise in temperature to 78 °C ≈ Tm. Stable holo-FBP multimers may protect naturally occurring labile folates against decomposition or bacterial utilization. DSC established an interrelationship between diminished folate binding at pH 5, especially in NaCl-free buffers, and low thermostability. Positively charged apo-FBP was almost completely unfolded and aggregated at pH 5 (Tm 38 °C) and holo-FBP, albeit more thermostable, was labile with aggregation tendency. Addition of 0.15 M NaCl increased thermostability of apo-FBP drastically, and even more so that of holo-FBP. Electrostatic forces thus seem to contribute to a diminished thermostability at low pH. Fluorescence spectroscopy after irreversible thermal unfolding of FBP revealed a weak-affinity folate binding.     

Lipid oxidation and gel to liquid-crystalline transition temperatures of synthetic polyunsaturated mixed-acid phosphatidylcholines

Synthetic preparations of the polyunsaturated phosphatidylcholines 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC) and 1-stearoyl-2-α-linolenoyl-sn-glycero-3-phosphocholine (SLnPC) were observed to undergo autooxidation sometimes during synthesis and also on storage. Oxidation was also induced by treatment of unoxidized SLPC with ultraviolet irradiation. Oxidation was estimated by thin layer chromatographic, fatty acid and ultraviolet spectral analyses. With limited oxidation, the gel to liquid-crystalline transition temperatures of aqueous dispersions of these lipids were seen to increase. Extensive oxidation led to a reduction in the enthalpies of the transitions. The increases in transition temperatures were consistent with the presence of conjugated double bonds, as shown by increased absorption at 230 nm, and trans double bonds, in the oxidized lipids leading to the creation of more rigid domains within the bilayer. Some of the changes in the transitions, especially the decreasing enthalpy after extensive oxidation, may have occurred because of the presence of small amounts of lysophosphatidylcholine and other oxidation intermediates or breakdown products seen by thin layer analysis. Thermograms of mixtures of unoxidized SLPC with amounts of lysostearoylPC found in the oxidized samples showed, however, that lysoPC likely did not contribute significantly to the increase in transition temperatures. Thin layer analysis suggested that the presence of cross-linked products could have contributed to the observed thermotropic properties.     

Biophysical characterization and mutational analysis of the antibiotic resistance protein NimA from Deinococcus radiodurans

Metronidazole (MTZ) is an antibiotic commonly used to treat anaerobic bacterial infections in humans and animals. Antibiotic resistance toward this class of 5-nitroimidazole (5-Ni) drug derivatives has been related to the Nim genes thought to encode a reductase. Here we report the biophysical characteristics of the NimA protein from Deinococcus radiodurans (DrNimA) binding to MTZ and three other 5-Ni drugs. The interaction energies of the protein and antibiotic are studied by isothermal titration calorimetry (ITC) and with free energy and linear interaction energy (LIE) calculations, where the latter method revealed that the antibiotic binding is mainly of hydrophobic character. ITC measurements further found that one DrNimA dimer has two antibiotic binding sites which were not affected by mutation of the reactive His71. The observed association constants (K_a) were in the range of 5.1–49 ? 10⁴ M^− 1 and the enthalpy release upon binding to DrNimA for the four drugs studied was relatively low (∼ − 1 kJ/mol) but still measurable. The drug binding is mainly entropy driven and along with the hydrophobic drug binding site found by crystallography, this possibly explains the low observed enthalpy values. The effect of the His71 mutation and the presence of MTZ were studied by differential scanning calorimetry (DSC). Native DrNimA is a yellow colored protein where the interaction from His71 to the cofactor is thought to be responsible for the coloring. Mutations of His71 to Ala, Ser, Leu or Asp all gave transparent, colorless protein solutions, and the two mutant crystal structures of DrNimA-H71A and DrNimA-H71S presented revealed no cofactor binding.     

Herbicide binding and thermal stability of photosystem II isolated from Thermosynechococcus elongatus

Binding of herbicides to photosystem II inhibits the electron transfer from Q_A to Q_B due to competition of herbicides with plastoquinone bound at the Q_B site. We investigated herbicide binding to monomeric and dimeric photosystem II core complexes (PSIIcc) isolated from Thermosynechococcus elongatus by a combination of different methods (isothermal titration and differential scanning calorimetry, CD spectroscopy and measurements of the oxygen evolution) yielding binding constants, enthalpies and stoichiometries for various herbicides as well as information regarding stabilization/destabilization of the complex. Herbicide binding to detergent-solubilized PSIIcc can be described by a model of single independent binding sites present on this important membrane protein. Interestingly, binding stoichiometries herbicide:PSIIcc are lower than 1:1 and vary depending on the herbicide under study. Strong binding herbicides such as terbutryn stabilize PSIIcc in thermal unfolding experiments and endothermically binding herbicides like ioxynil probably cause large structural changes accompanied with the binding process as shown by differential scanning calorimetry experiments of the unfolding reaction of PSIIcc monomer in the presence of ioxynil. In addition we studied the occupancy of the Q_B sites with plastoquinone (PQ9) by measuring flash induced fluorescence relaxation yielding a possible explanation for the deviations of herbicide binding from a 1:1 herbicide/binding site model.