Conformation and stability properties of B17: I. Analytical investigations using circular dichroism

Structural characterization of B17, the 17?% N-terminal domain of apo B, was carried out using circular dichroic (CD) spectroscopy, where secondary and tertiary structures were studied as a function of temperature and pH. Mild acidic conditions that correlate with histidine protonation invoked a change in the α-helix and random coil contents of the protein, with no apparent change in the β-sheet structural content. Specific changes in the structure of the protein that occur in response to temperature were also investigated to understand the stability and conformational changes of B17. Far- and near-UV CDs were used to probe the thermal changes in the protein. The protonation of some histidine residues was attributed to underlie the increase in the helical content of the protein.     

Measurement of protein stability and protein denaturation in cells using differential scanning calorimetry

Many methods exist for measuring and studying protein denaturation in vitro. However, measuring protein denaturation in cells under conditions relevant to heat shock presents problems due to cellular complexity and high levels of light scattering that interfere with optical techniques. A general method for measuring protein denaturation in cells using high sensitivity differential scanning calorimetry (DSC) is given. Profiles of specific heat (c(p) vs. temperature) are obtained providing information about transitions in cellular components including the denaturation of proteins. The specific approaches employed with erythrocytes, bacteria, and mammalian cells are described, and an identification of several features of the DSC profiles is given. Protein denaturation on the level of roughly 7-20% occurs for commonly used heat shocks in mammalian cells.     

Thermal stability of Phaseolus vulgaris leucoagglutinin: a differential scanning calorimetry study

Phaseolus vulgaris phytohemagglutinin L is a homotetrameric-leucoagglutinating seed lectin. Its three-dimensional structure shows similarity with other members of the legume lectin family. The tetrameric form of this lectin is pH dependent. Gel filtration results showed that the protein exists in its dimeric state at pH 2.5 and as a tetramer at pH 7.2. Contrary to earlier reports on legume lectins that possess canonical dimers, thermal denaturation studies show that the refolding of phytohemagglutinin L at neutral pH is irreversible. Differential scanning calorimetry (DSC) was used to study the denaturation of this lectin as a function of pH that ranged from 2.0 to 3.0. The lectin was found to be extremely thermostable with a transition temperature around 82 degrees C and above 100 degrees C at pH 2.5 and 7.2, respectively. The ratio of calorimetric to vant Hoff enthalpy could not be calculated because of its irreversible-folding behavior. However, from the DSC data, it was discovered that the protein remains in its compact-folded state, even at pH 2.3, with the onset of denaturation occurring at 60 degrees C.     

Kinetics of solid state stability of glycine derivatives as a model for peptides using differential scanning calorimetry

Kinetics of solid state stability of seven derivatives of 3,5-disubstituted tetrahydro-2H-1,3,5-thiadiazine-2-thione (THTT) of glycine as a model for amino acids and peptide drugs were studied using differential scanning calorimetry (DSC). Each DSC curve for each derivative showed an endothermic peak followed by an exothermic one, which could be attributed to the melting and decomposition, respectively. The decomposition activation energy of each derivative was calculated using the Augis and Bennet, Kissinger equations and Mahadevan approximation. Also, the melting activation energies as well as the thermodynamic parameter (enthalpy) for the investigated derivatives were evaluated. The relative stability of the derivatives in the solid state according to the calculated values of the decomposition activation energy, frequency factors and half-life for each derivative could be determined.     

A differential scanning calorimetry study of tetracycline repressor.

Tetracycline repressor (TetR), which constitutes the most common mechanism of bacterial resistance to an antibiotic, is a homodimeric protein composed of two identical subunits, each of which contains a domain possessing a helix-turn-helix motif and a domain responsible for binding tetracycline. Binding of tetracycline in the protein pocket is accompanied by conformational changes in TetR, which abolish the specific interaction between the protein and DNA. Differential scanning calorimetry (DSC) and CD measurements, performed at pH 8.0, were used to observe the thermal denaturation of TetR in the absence and presence of tetracycline. The DSC results show that, in the absence of tetracycline, the thermally induced transitions of TetR can be described as an irreversible process, strongly dependent on scan rate and indicating that the protein denaturation is under kinetic control described by the simple kinetic scheme: N(2)--->D(2), where k is a first-order kinetic constant, N is the native state, and D is the denatured state. On the other hand, analysis of the scan rate effect on the transitions of TetR in the presence of tetracycline shows that thermal unfolding of the protein can be described by the two-state model: N(2)<--->U(2)--->D. In the proposed model, TetR in the presence of tetracycline undergoes co-operative unfolding, characterized by an enthalpy change (DeltaH(cal) = 1067 kJ x mol(-1)) and an entropy change (DeltaS = 3.1 kJ x mol(-1)).     

Study of strong to ultratight protein interactions using differential scanning calorimetry

Data from differential scanning calorimetry (DSC) may be used to estimate very large binding constants that cannot be conveniently measured by more conventional equilibrium techniques. Thermodynamic models have been formulated to describe interacting systems that involve either one thermal transition (protein-ligand) or two thermal transitions (protein-protein) and either 1:1 or higher binding stoichiometry. Methods are described for obtaining binding constants and heats of binding by two different methods: calculation or simulation fitting of data. Extensive DSC data on 2'CMP binding to RNase are presented and analyzed by the two methods. It is found that the methods agree when binding sites are completely saturated, but substantial errors arise in the calculation method when site saturation is incomplete and the transition of liganded molecules overlaps that of unliganded molecules. This arises primarily from an inability to determine TM (i.e., the temperature where concentrations of folded and unfolded protein are equal) under weak-binding conditions. Results from simulation show that the binding constants and heats of binding from the DSC method agree quantitatively with corresponding estimates obtained from equilibrium methods when extrapolated to the same temperature. It was also found from the DSC data that the binding constant decreases with increasing concentration of ligand, which might arise from nonideality effects associated with dimerization of 2'CMP. Simulations show that the DSC method is capable of estimating binding constants for ultratight interactions up to perhaps 10(40) M-1 or higher, while most equilibrium methods fail well below 10(10) M-1. DSC data from the literature on a number of interacting systems (trypsin-soybean trypsin inhibitor, trypsin-ovomucoid, trypsin-pancreatic trypsin inhibitor, chymotrypsin-subtilisin inhibitor, subtilisin BPN-subtilisin inhibitor, RNase S protein-RNase S peptide, avidin-biotin, ovotransferrin-Fe3+, superoxide dismutase-Zn2+, alkaline phosphatase-Zn2+, and assembly of regulatory and catalytic subunits of aspartate transcarbamoylase) were analyzed by simulation fitting or by calculation. Apparent single-site binding constants ranged from ca. 10(5) to 10(20) M-1, while the interaction constant for assembly of aspartate transcarbamoylase was estimated as 10(37) in molarity units. For most of these systems, the DSC interaction constants compared favorably with other literature estimates, for some it did not for reasons unknown, while for still others this represented the first estimate. Simulations show that for proteins having two binding sites for the same ligand within a single cooperative unit, ligand rearrangement will occur spontaneously during a DSC scan as the transition temperature of the unliganded protein is approached.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evaluation of riboflavin binding protein domain interaction using differential scanning calorimetry

Riboflavin binding (or carrier) protein (RfBP) is a monomeric, two-domain protein, originally purified from hens' egg white. RfBP contains nine disulfide bridges; as a result, the protein forms a compact structure and undergoes reversible three-state thermal denaturation. This was demonstrated using a differential scanning calorimetry (DSC) method [Wasylewski M. (2000) J. Prot. Chem. 19(6), 523-528]. It has been shown that the RfBP complex with riboflavin denaturates in a three-state process which may be attributed to sequential unfolding of the RfBP domains. In case of apo RfBP, the ligand binding domain denaturates at a lower temperature than the C-terminal domain. Ligand binding greatly enhances the thermostability of the N-terminal domain, whereas the C-terminal domain thermostability is only slightly affected and, in case of the examined holo RfBPs, the denaturation peaks of both domains merge or cross over. The magnitude of the changes depends on ligand structure. A detailed study of protein concentration effects carried out in this work allowed to estimate not only the thermostability of both domains but also the strength of domain interactions. The DeltaCp, of denaturation was found for C-terminus and N-terminus of RfBP-riboflavin complex to amount to 2.5 and -1.9 kcal mol(-1), respectively. The calculated domain interaction free energy, DeltaGCN, was estimated to be approximately -1580 cal mol(-1) at 67.0 degrees C. This value indicates that the interdomain interaction is of medium strength.     

Physical-chemical characterization and stability study of alpha-trypsin at pH 3.0 by differential scanning calorimetry

alpha-Trypsin is a serine-protease with a polypeptide chain of 223 amino acid residues and six disulfide bridges. It is a globular protein with predominance of antiparallel ss-sheet secondary structure and it has two domains with similar structures. In the present work, a stability study of alpha-trypsin in the acid pH range was performed and some physical-chemical denaturation parameters were measured by using differential scanning calorimetry (DSC). The alpha-trypsin has a shelf-life (t(95%)) of about 10 months at pH 3.0 and 4 degrees C and its hydrolysis into the psi-trypsin isoform is negligible during 6 months. The observed ratio DeltaH(cal)/DeltaH(vH) is close to unity, which suggests the occurrence of a two-state transition. At pH 3.0, alpha-trypsin unfolded with T(m) = 325.9 K and DeltaH = 99.10 kcal mol(-1), and the change in heat capacity between the native and unfolded forms of the protein was estimated to be 1.96+/-0.18 kcal mol(-1)K(-1). The stability of alpha-trypsin calculated at 298 K was DeltaG(U)=6.10 kcal mol(-1) at pH 3.0. These values are in the range expected for a small globular protein. These results show that the thermodynamic parameters of unfolding of beta-trypsin do not change substantially after its conversion to alpha-trypsin.     

Interactions of angiotensin II with membranes using a combination of differential scanning calorimetry and 31P NMR spectroscopy

Summary This study of angiotensin II (ANG II) membrane interactions uses a combination of³¹P NMR spectroscopy and differential scanning calorimetry (DSC), two valuable and complementary techniques which can provide useful information about the thermotropic and dynamic properties of peptide hormones in membranes. The major conclusion from the calorimetric experiments is that ANG II affects the phase properties of hydrated dipalmitoyl-phosphatidylcholine (DPPC) bilayers by mainly broadening the pretransition area. Preliminary³¹P NMR data seem to confirm the DSC results by showing that ANG II produces a lowering of the pretransition temperature but affects only minimally the main phase transition. In combination, the results from the two methods may indicate that the hormone produces its effects on the phospholipid head groups while its effects on the bilayer alkyl chains are not significant. Such results can be interpreted to mean that ANG II closely interacts with the phospholipid head groups perhaps up to the level of the interface, but does not enter deeper into the membrane bilayer.     

Lipid reorganization induced by membrane-active peptides probed using differential scanning calorimetry

The overlapping biological behaviors between some cell penetrating peptides (CPPs) and antimicrobial peptides (AMPs) suggest both common and different membrane interaction mechanisms. We thus explore the capacity of selected CPPs and AMPs to reorganize the planar distribution of binary lipid mixtures by means of differential scanning calorimetry (DSC). Additionally, membrane integrity assays and circular dichroism (CD) experiments were performed. Two CPPs (Penetratin and RL16) and AMPs belonging to the dermaseptin superfamily (Drs B2 and C-terminal truncated analog [1–23]-Drs B2 and two plasticins DRP-PBN2 and DRP-PD36KF) were selected. Herein we probed the impact of headgroup charges and acyl chain composition (length and unsaturation) on the peptide/lipid interaction by using binary lipid mixtures. All peptides were shown to be α-helical in all the lipid mixtures investigated, except for the two CPPs and [1–23]-Drs B2 in the presence of zwitterionic lipid mixtures where they were rather unstructured. Depending on the lipid composition and peptide sequence, simple binding to the lipid surface that occur without affecting the lipid distribution is observed in particular in the case of AMPs. Recruitments and segregation of lipids were observed, essentially for CPPs, without a clear relationship between peptide conformation and their effect in the lipid lateral organization. Nonetheless, in most cases after initial electrostatic recognition between the peptide charged amino acids and the lipid headgroups, the lipids with the lowest phase transition temperature were selectively recruited by cationic peptides while those with the highest phase transition were segregated. Membrane activities of CPPs and AMPs could be thus related to their preferential interactions with membrane defects that correspond to areas with marked fluidity. Moreover, due to the distinct membrane composition of prokaryotes and eukaryotes, lateral heterogeneity may be differently affected by cationic peptides leading to either uptake or/and antimicrobial activities.     

Studies on conformation of soluble and immobilized enzymes using differential scanning calorimetry. 1. Thermal stability of nicotinamide adenine dinucleotide dependent dehydrogenases.

The technique of differential scanning calorimetry (DSC) has been applied to the study of temperature-induced irreversible denturation and thus to the heat stability of soluble and Sepharose-bound liver alcohol dehydrogenase (LADH, EC 1.1.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) in the presence of various coenzymes or coenzyme fragments. The transition temperature (Ttr) of 82.5 degrees C obtained for soluble LADH was increased by 12.5 degrees C in the presence of a saturating concentration of NACH. In the presence of NAD+, Ttr increased by 8.5 degrees C, whereas ADP-ribose and AMP caused an increase in Ttr of only 2 and 1 degree C, respectively. The Ttr of 85.5 degrees C obtained for Sepharose-bound LADH was increased by about 12 degrees C after the addition of free NADH. However, when the enzyme was immobilized simultaneously with a NADH analogue (which also binds to the matrix), a broad endotherm with a Ttr of 91.5 degrees C was obtained, indicating the presence of immobilized enzyme molecules both with, and without, associated NADH. Corresponding increases in heat stability were observed for LDH in solution in the presence of NADH, NAD+, and AMP, leading to increases in Ttr from 72 to 79.5 and 74 and 73 degrees C, respectively. The addition of pyruvate and NAD+ to the enzyme to form an abortive ternary complex led to the same stabilization as that observed with NADH, attendant with a large increase in the enthalpy of transition, deltaHtr. In these studies the technique of DSC was utilized because it is applicable both to soluble and immobilized enzymes and (1) provides rapid information about Ttr and thus thermal stability of enzymes, (2) different energetic states of an enzyme molecule can be identified, and (3) an overall picture of the thermal process is rapidly obtained.     

Thermal stability and mode of oligomerization of the tetrameric peanut agglutinin: a differential scanning calorimetry study

Peanut agglutinin is a homotetrameric legume lectin. The crystal structure of peanut agglutinin shows that the four subunits associate in an unusual manner, giving rise to open quaternary structure [Banarjee, R., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 227-231]. The thermal unfolding of peanut agglutinin has been characterized by differential scanning calorimetry and gel filtration to elucidate its thermal stability and its mode of oligomerization. The unfolding process is reversible and could be described by a three-state model with two transitions occurring at around 331 and 336 K. For the tetramer, the ratio of DeltaHc/DeltaHv for the first transition is close to 4 and for the second transition is close to 0.25, suggesting that 4 and 0.25 cooperative unit(s) of the tetramer are involved in the first and second transitions, respectively. The agreement between the model-independent DeltaHv(S) determined from the values of the temperatures of the peak maximum (Tp1) with the protein concentration with the values of DeltaHv obtained from the fit of the data to the transition confirms that the first peak is associated with the dissociation of peanut agglutinin tetramers (A4) to "folded" monomers (4A), whereas the second peak describes the unfolding (4U) of these monomers. The overall process for the thermal unfolding of peanut agglutinin could therefore be summarized as A4 <==> 4A <==> 4U. Gel filtration studies confirm this process, as peanut agglutinin elutes as a tetramer up to 50 degrees C, and at and above 56 degrees C (Tm of first transition), it elutes at a position commensurate with that of the folded monomer of peanut agglutinin. The unfolding behavior of peanut agglutinin in the presence of saturating amounts of carbohydrate ligands is similar to that observed for the unligated form. The temperature of maximal stability of the peanut agglutinin tetramer at pH 7.4 is calculated to be around 33 degrees C with a maximal free energy of stabilization of 8.70 kcal/mol. The results demonstrate that unfolding of peanut agglutinin goes through two distinct phases with folded monomer being the intermediate.     

Thermal stability of PNA/DNA and DNA/DNA duplexes by differential scanning calorimetry

Thermodynamics of the thermal dissociation transitions of 10 bp PNA/DNA duplexes and their corresponding DNA/DNA duplexes in 10 mM sodium phosphate buffer (pH 7.0) were determined from differential scanning calorimetry (DSC) measurements. The PNA/DNA transition temperatures ranged from 329 to 343 K and the calorimetric transition enthalpies ranged from 209 +/- 6 to 283 +/- 37 kJ mol(-1). The corresponding DNA/DNA transition temperatures were 7-20 K lower and the transition enthalpies ranged from 72 +/- 29 to 236 +/- 24 kJ mol(-1). Agreement between the DSC and UV monitored melting (UVM) determined transition enthalpies validated analyzing the UVM transitions in terms of a two-state transition model. The transitions exhibited reversibility and were analyzed in terms of an AB = A + B two-state transition model which yielded van't Hoff enthalpies in agreement with the transition enthalpies. Extrapolation of the transition enthalpies and free energy changes to ambient temperatures yielded more negative values than those determined directly from isothermal titration calorimetry measurements on formation of the duplexes. This discrepancy was attributed to thermodynamic differences in the single-strand structures at ambient and at the transition temperatures, as indicated by UVM measurements on single DNA and PNA strands.     

Determination of hydration properties and thermal behavior of Paecilomyces variotii by differential scanning calorimetry

Due to the structure and the composition of Paecilomyces variotii, the mycelia of this fungus could have potential applications as ingredients in wettable foods. For this use, drying could be employed, justifying the study of thermal behavior of P. variotii. The objectives of this work were to perform a study of thermal behavior of P. variotii isolates, to evaluate the hydration properties of these mycelia and to analyze the effect of different technological parameters on the latter properties. Wet cultures exhibited a wide endothermic transition, with mean values of peak temperature of 61°C and denaturation enthalpy of 4 J/g dry matter. Initial (50°C) and final (80°C) temperatures of the endothermic transition were used to dry the mycelia. Freeze-drying was also assayed. For all dried mycelia, a decrease in denaturation enthalpy between 40 and 50% was observed for drying at 50°C and freeze-drying, and a drastic decrease of almost 100% for drying at 80°C. According to the hydration properties, wet mycelia exhibited water holding capacity (WHC) value of 45 g water/g dry matter. Significant differences among dried mycelia, resulting WHC values in order: 50°C > freeze-dried > 80°C (p < 0.05) were revealed for each P. variotii strain. Fungi obtained by drying at 50 C and by freeze-drying, showed a rapid water absorption (t _1/2 < 0.1 min). Ionic strength, pH and particle size of dried mycelia influenced the hydration properties.     

DNA melting investigated by differential scanning calorimetry and Raman spectroscopy.

Thermal denaturation of the B form of double-stranded DNA has been probed by differential scanning calorimetry (DSC) and Raman spectroscopy of 160 base pair (bp) fragments of calf thymus DNA. The DSC results indicate a median melting temperature Tm = 75.5 degrees C with calorimetric enthalpy change delta Hcal = 6.7 kcal/mol (bp), van't Hoff enthalpy change delta HVH = 50.4 kcal/mol (cooperative unit), and calorimetric entropy change delta Scal = 19.3 cal/deg.mol (bp), at the experimental conditions of 55 mg DNA/ml in 5 mM sodium cacodylate at pH 6.4. The average cooperative melting unit (nmelt) comprises 7.5 bp. The Raman signature of 160 bp DNA is highly sensitive to temperature. Analyses of several conformation-sensitive Raman bands indicate the following ranges for thermodynamic parameters of melting: 43 < delta HVH < 61 kcal/mol (cooperative unit), 75 < Tm < 80 degrees C and 6 < (nmelt) < 9 bp, consistent with the DSC results. The changes observed in specific Raman band frequencies and intensities as a function of temperature reveal that thermal denaturation is accompanied by disruption of Watson-Crick base pairs, unstacking of the bases and disordering of the B form backbone. These three types of structural change are highly correlated throughout the investigated temperature range of 20 to 93 degrees C. Raman bands diagnostic of purine and pyrimidine unstacking, conformational rearrangements in the deoxyribose-phosphate moieties, and changes in environment of phosphate groups have been identified. Among these, bands at 834 cm-1 (due to a localized vibration of the phosphodiester group), 1240 cm-1 (thymine ring) and 1668 cm-1 (carbonyl groups of dT, dG and dC), are shown by comparison with DSC results to be the most reliable quantitative indicators of DNA melting. Conversely, the intensities of Raman marker bands at 786 cm-1 (cytosine ring), 1014 cm-1 (deoxyribose ring) and 1092 cm-1 (phosphate group) are largely invariant to melting and are proposed as appropriate standards for intensity normalizations.     

Features of distamycin preferential binding sites on natural DNA predicted using differential scanning calorimetry.

The interaction of distamycin with ColE1 DNA was examined by using differential scanning calorimetry (DSC) taking the helix-coil transition theory of DNA into consideration. Our results here strongly indicate that the affinity of distamycin to DNA, at a low distamycin concentration, depends highly on the DNA sequence, and preferential binding occurs to the sites of four to six successive A-T pairs having two or more successive G-C pairs on both their ends.     

Studies on conformation of soluble and immobilized enzymes using differential scanning calorimetry. 2. Specific activity and thermal stability of enzymes bound weakly and strongly to Sepharose CL 4B

Ribonuclease A (EC 3.1.4.22) and alpha-chymotrypsin (EC 3.4.21.1) have been covalently coupled, by a varying number of bonds, to Sepharose CL 4B which was activated with different amounts of CNBr. Upon increasing the number (1-8) of points of attachment between the enzyme and the matrix, the specific activities of immobilized ribonuclease A relative to its soluble counterpart decreased from 60 to 15% while the amount of protein coupled increased from 5 to 37 mg per g of sucked gel. Differential scanning calorimetry was used to determine whether the immobilization caused any changes in the physicochemical properties of the enzyme. Ribonuclease A, weakly bound to the matrix, showed almost the same behavior as the soluble enzyme. By contrast strongly immobilized enzyme exhibited a higher transition temperature (by about 5 degrees C) and a broader endotherm. Similar results were found for alpha-chymotrypsin.