Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

Kinetic and structural analysis of the inhibition of adenosine deaminase by acetaminophen

Kinetic and thermodynamic studies have been made on the effect of acetaminophen on the activity and structure of adenosine deaminase in 50 mM sodium phosphate buffer pH 7.5, at two temperatures of 27 and 37 degrees C using UV spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. Acetaminophen acts as a competitive inhibitor at 27 degrees C (Ki = 126 microM) and an uncompetitive inhibitor at 37 degrees C (Ki = 214 microM). Circular dichroism studies do not show any considerable effect on the secondary structure of adenosine deaminase by increasing the temperature from 27 to 37 degrees C. However, the secondary structure of the protein becomes more compact at 37 degrees C in the presence of acetaminophen. Fluorescence spectroscopy studies show considerable change in the tertiary structure of the protein by increasing the temperature from 27 to 37 degrees C. Also, the fluorescence spectrum of the protein incubated with different concentrations of acetaminophen show different inhibition behaviors by the effector at the two temperatures.     

Conformational lability of herpesvirus protein VP22

The herpesvirus protein VP22 traffics between cells, being exported from expressing cells in a non-Golgi-dependent manner and localizing in the nuclei of surrounding cells. This transport is retained in certain VP22 fusion proteins, making VP22 a candidate for use in macromolecular drug delivery. In an effort to understand the physical basis for this activity, we have initiated structural studies of VP22.C1, the C-terminal half of VP22, which possesses the full transport activity of the native protein. CD and Fourier transform infrared analyses indicate a secondary structure consisting of approximately 30% alpha-helix, 17% beta-sheet, and 51% disordered and turn structure. Unfolding studies conducted by CD, differential scanning calorimetry, and fluorescence reveal a series of discrete structural transitions in the range of 20-60 degrees C. CD and fluorescence studies of interactions between VP22.C1 and divalent cations and model polyanions indicate that Mg(2+), Zn(2+), oligonucleotides, and heparin interact with the protein, causing changes in secondary structure and thermal stability. Additionally, the interaction of VP22.C1 with model lipids was examined. Fluorescence titrations of the protein with trans-parinaric acid at various temperatures suggest the binding of one to two molecules of parinaric acid to VP22.C1 at temperatures >40 degrees C, suggesting the possibility of conformation dependent membrane interaction under physiological conditions.     

Calorimetric investigations of the structural stability and interactions of colicin B domains in aqueous solution and in the presence of phospholipid bilayers

The effects of pH and temperature on the stability of interdomain interactions of colicin B have been studied by differential-scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The calorimetric properties were compared with those of the isolated pore-forming fragment. The unfolding profile of the full-length toxin is consistent with two endothermic transitions. Whereas peak A (T(m) = 55 degrees C) most likely corresponds to the receptor/translocation domain, peak B (T(m) = 59 degrees C) is associated with the pore-forming domain. By lowering the pH from 7 to 3.5, the transition temperature of peaks A and B are reduced by 25 and 18 degrees C, respectively, due to proton exchange upon denaturation. The isolated pore-forming fragment unfolds at much higher temperatures (T(m) = 65 degrees C) and is stable throughout a wide pH range, indicating that intramolecular interactions between the different colicin B domains result in a less stable protein conformation. In aqueous solution circular dichroism spectra have been used to estimate the content of helical secondary structure of colicin B ( approximately 40%) or its pore-forming fragment ( approximately 80%). Upon heating, the ellipticities at 222 nm strongly decrease at the transition temperature. In the presence of lipid vesicles the differential-scanning calorimetry profiles of the pore-forming fragment exhibit a low heat of transition multicomponent structure. The heat of transition of membrane-associated colicin B (T(m) = 54 degrees C at pH 3.5) is reduced and its secondary structure is conserved even at intermediate temperatures indicating incomplete unfolding due to strong protein-lipid interactions.     

Structure-function studies of Murraya koenigii trypsin inhibitor revealed a stable core beta sheet structure surrounded by alpha-helices with a possible role for alpha-helix in inhibitory function

Structure-function studies of Murraya koenigii trypsin inhibitor revealed a compact structure made of central beta-sheet surrounded by alpha-helices with differences in structure and functional stability. Proteolysis studies, of native and heat-treated protein, demonstrated that inhibitor exhibited strong resistance to proteolysis by many proteases. However, the inhibitory activity gradually decreased with increasing temperature and was completely lost at 90 degrees C. CD studies, under native conditions, showed that inhibitor contains approximately 46% beta-strand, 30.1% alpha-helical, 16.2% turn and 6.9% random coil structure. At increasing temperatures, however, helix to coil transition was observed. The ANS fluorescence study showed linear increase of fluorescence intensity without showing any melting transition. Correlating decrease in inhibitory activity and helical content at increasing temperatures suggest a possible role for alpha-helical structure in inhibitory function of the protein.     

Investigation of apomyoglobin stability depending on urea and temperature at two different pH values

Equilibrium unfolding of apomyoglobin by urea was investigated in the temperature range from 5 to 25 degrees C at two pH values. The thermodynamic parameters of the apomyoglobin native-unfolded state transition were determined. Conformational changes in the protein structure were monitored by tryptophan fluorescence and far UV circular dichroism. Apomyoglobin preserves its native conformation at pH 5.7 and 6.2 in the temperature range used. It was shown that the apomyoglobin stability and its unfolding cooperativity are substantially lower at 5 degrees C than at other temperatures. This fact should be taken in account at the investigation of apomyoglobin.     

DnaK-sigma 32 interaction is temperature-dependent. Implication for the mechanism of heat shock response

The heat shock response in bacteria is a complex phenomenon in which sigma 32 plays the central role. The DnaK/J chaperone system binds and promotes degradation of sigma 32 at lower temperatures. At heat shock temperatures, the DnaK/J-mediated degradation of sigma 32 is largely abolished by a mechanism, which is not yet fully understood. In this article we have shown that interaction of DnaK with sigma 32 is highly temperature-dependent. This interaction is completely abolished at 42 degrees C. To investigate the origin of such strong temperature dependence, we have monitored the structural changes that occur in the sigma 32 protein upon upshift of temperature and attempted to elucidate its functional roles. Upon a shift of temperature from 30 to 42 degrees C, the CD spectrum of sigma 32 becomes significantly more positive without significant change in either tryptophan fluorescence spectra or quenchability to external quenchers. 1,8-Anilinonaphthalene sulfonic acid binding at 42 degrees C is not significantly affected. The equilibrium guanidine hydrochloride denaturation of sigma 32 is biphasic. The first phase shifts to even lower guanidine hydrochloride concentrations at 42 degrees C, whereas the major phase remains largely unchanged. The sigma 32-core interaction remains unchanged as a function of temperature. This suggests that increased temperature destabilizes a structural element. We discuss the possible location of this temperature-sensitive structural element.     

Conformational Stability and Hemolytic Activity of Actinoporin RTX-SII from the Sea Anemone <Emphasis Type="Italic">Radianthus macrodactylus</Emphasis>

The spatial organization of actinoporin RTX-SII from the sea anemone Radianthus macrodactylus on the level of tertiary and secondary structures was studied by UV and CD spectroscopy and intrinsic protein fluorescence. The specific and molar extinction coefficients of RTX-SII were determined. The percentages of canonical secondary structures of actinoporin were calculated. The tertiary structure of the polypeptide is well developed and its secondary structure is highly ordered and contains about 50% antiparallel folded beta-sheets. The irreversible thermal denaturation of RTX-SII was studied by CD spectroscopy; a conformational transition occurs at 53 degrees C. Above this temperature irreversible conformational changes are observed in the secondary and tertiary structures. This is accompanied by redistribution of the content of regular and distorted forms of beta-sheet and also by increase in the content of an unordered form. It is suggested that an intermediate is formed in the process of thermal denaturation. Acid-base titration of RTX-SII results in irreversible conformational changes at pH below 2.0 and above 12.0. As shown by intrinsic protein fluorescence, tyrosine residues of RTX-SII make a fundamental contribution to emission, and the total fluorescence depends more on temperature and ionic strength of the solution than tryptophan fluorescence. The data on conformational stability of actinoporin are correlated with data on its hemolytic activity. Activity of RTX-SII significantly decreases at increased temperature and slightly decreases at low pH. Hemolytic activity drastically increases at high pH. Increase in the actinoporin activity at pH above 10 seems to be caused by ionization of the molecule.     

The role of calcium in the conformational dynamics and thermal stability of the D-galactose/D-glucose-binding protein from Escherichia coli

We have characterized stability and conformational dynamics of the calcium depleted D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli. The structural stability of the protein was investigated by steady state and time resolved fluorescence, and far-UV circular dichroism in the temperature range from 20 degrees C to 70 degrees C. We have found that the absence of the Ca(2+) ion results in a significant destabilization of the C-terminal domain of the protein. In particular, the melting temperature decreases by about 10 degrees C with the simultaneous loss of the melting cooperativity. Time resolved fluorescence quenching revealed significant loosening of the protein when highly shielded Trp residue(s) became accessible to acrylamide at higher temperatures. We have documented a significant stabilizing effect of glucose that mostly reverts the effect of calcium, that is, the thermal stability of the protein increases by about 10 degrees C and the melting cooperativity is restored. Moreover, the protein structure remains compact with low amplitude of the segmental mobility up to high temperatures. We have used molecular dynamics to identify the structural feature responsible for changes in the temperature stability. Disintegration of the Ca(2+)-binding loop seems to be responsible for the loss of the stability in the absence of calcium. The new insights on the structural properties and temperature stability of the calcium depleted GGBP contribute to better understanding of the protein function and constitute important information for the development of new biotechnological applications of this class of proteins.     

Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.     

Regulation of the electrogenic (Na+ + K+)-pump of Ehrlich cells by intracellular cation levels.

Dihydrofolate reductase and its complexes have been studied by fluorescence and circular dichroism. NADPH, trimethoprim, pyrimethamine, or Methotrexate binding causes small changes in the enzyme far ultraviolet CD which possibly arise from alterations in polypeptide backbone of the enzyme; however, their effects on enzyme far ultraviolet CD are also explained as the result of ligand interactions with enzyme aromatic groups. In ternary complexes of the enzyme, fluorescence properties of bound NADPH are surprisingly sensitive to the type of inhibitor bound nearby. The effect of temperature on the enzyme and its complexes is clearly shown by changes in enzyme fluorescence and CD. At temperatures near 45 degrees C, the enzyme undergoes an irreversible denaturation, as shown by major alterations in enzyme far ultraviolet CD and by an increased rate of fluorescence quenching. Binary complexes with NADPH or Methotrexate stabilize the enzyme towards this heat denaturation, whereas bound trimethoprim and pyrimethamine do not. Ternary complexes with NADPH and any of the ligands are more stable than the enzyme itself toward heat denaturation. Fluorescence-temperature and fluorescence polarization studies show that near 30 degrees C the enzyme undergoes a reversible transition that is modified by NADPH or methotrexate.     

A spectroscopic analysis of thermal stability of the Chromobacterium viscosum lipase

The thermal stability of the lipase from Chromobacterium viscosum was assessed by deactivation (loss of activity), fluorescence, circular dichroism (CD) and static light scattering (SLS) measurements. Lipase fluorescence emission is dominated by the tryptophyl contribution. An increase in the tyrosyl contribution from 2 to 16% was only observed upon prolonged incubation at 60 degrees C. The effect of temperature on the tryptophyl quantum yield was studied and two activation energies were calculated. Tryptophan residues in the native structure have an activation energy of 1.9 kcal mol(-1) for temperature-dependent non-radiative deactivation of the excited state. A structural change occurs at approximately 66.7 degrees C and the activation energy increases to 10.2 kcal mol(-1). This structural change is not characterized by tryptophan exposure on the surface of the protein. The deactivation and the evolution of structural changes with time after lipase incubation at 60 degrees C were assessed by fluorescence, CD and SLS measurements. CD spectra show that both secondary and tertiary structures remain native-like after incubation at 60 degrees C in spite of the fluorescence changes observed (red-shift from 330 to 336 nm on the trytophyl emission). SLS measurements together with the CD data show that deactivation may be due to protein association between native molecules. Deactivation and the decrease on the fraction of non-associated native lipase evaluated by changes in fluorescence intensity with time, show apparent first order kinetics. According to the rate constants, fluorescence changes precede deactivation pointing to an underestimation of the deactivation. Reactivation upon dilution during the activity assay and substrate-induced reactivation due to lipase interfacial adsorption are possible causes for this underestimation.     

Insulin amyloid fibrillation at above 100 degrees C: new insights into protein folding under extreme temperatures

To investigate the folding behavior of amyloidogenic proteins under extreme temperatures, the kinetics of fibrillation and accompanying secondary structure transitions of bovine insulin were studied for temperatures ranging up to 140 degrees C. The presence of extreme heat stress had traditionally been associated with irreversible denaturation of protein while the initial steps of such a denaturation process may be common with a fibril formation pathway of amyloidogenic proteins. The present work demonstrates the ability of insulin to form amyloid fibrils at above 100 degrees C. Amyloid formation was gradually replaced by random coil generation after approximately 80 degrees C until no amyloid was detected at 140 degrees C. The morphology of insulin amyloid fibrils underwent sharp changes with increasing the temperature. The dependence of amyloid formation rate on incubation temperature followed non-Arrhenius kinetics, which is explained by temperature-dependent enthalpy change for amyloid formation. The intermediate stage of amyloid formation and random coil generation consisted of a partially folded intermediate common to both pathways. The fully unfolded monomers in random coil conformation showed partial reversibility through this intermediate by reverting back to the amyloid pathway when formed at 140 degrees C and incubated at 100 degrees C. This study highlights the non-Arrhenius kinetics of amyloid fibrillation under extreme temperatures, and elucidates its intermediate stage common with random coil formation.     

Conformation, pH-induced conformational changes, and thermal unfolding of anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab and Fc fragments

Conformation, acid-induced conformational changes and stability of the murine monoclonal antibody CB4-1 directed against the human immunodeficiency virus type 1 capsid protein p24, and its Fab and Fc fragments, were analysed by circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) measurements. CD spectra show the characteristics expected for beta-proteins. Lowering the pH to 3.5 reduces the stability, but does not change the conformation. Between pH 3.5 and 2.0 conformational changes and the formation of new structures are indicated. Deconvolution of the bimodal DSC curves of CB4-1 reveals five 'two-state' transitions at pH 7.5. At pH 5 and below, only four transitions are found. Half transition temperatures Tm and molar enthalpy changes DeltaHm gradually decrease at pH 4 and 3.4. At pH 2.1, two low-temperature (Tm=36.9 and 44.1 degrees C) and two high-temperature (Tm=74.6 and 76.8 degrees C) transitions are identified. The Fab and Fc fragments behave similarly. Deconvolution of their monophasic DSC curves yields two 'two-state' transitions for each fragment. Tm and DeltaHm values gradually decrease at pH 4.0 and 3.4; and at pH 2.1 and 2.8 for Fab and Fc, respectively, one of the transitions is found at high temperature (Tm=67.2 and 75.9 degrees C for Fab and Fc, respectively).     

Spectroscopic study on the structure and stability of beef liver arginase

The secondary and tertiary structure of the oligomeric arginase (EC 3.5.3.1) from beef liver was investigated by circular dichroism (CD) and fluorescence measurements. The far-ultraviolet CD spectrum of the enzyme at neutral pH is indicative of high helical content. The intrinsic fluorescence emission of the protein is due to tryptophan, the contribution of tyrosine being small. Upon excitation at 295 nm, the maximum of emission occurs at 330 nm, implying that the tryptophan residues are rather buried in a hydrophobic interior of the protein. Ethylenediaminetetraacetic acid (EDTA), which inactivates the enzyme by removing the functional Mn2+-ion from the enzyme, does not dissociate the enzyme into subunits, nor affect noticeably its secondary and tertiary structure. Inactivation occurs in the acid pH range, being complete at pH below 4. However, acidification up to pH 1.5 produced only limited changes in the far-ultra-violet CD spectrum and intrinsic fluorescence emission properties. The enzyme shows noteworthy thermal stability, as shown by measuring the residual activity after heating and by evaluating the temperature dependence of the CD signal at 220 nm and the intensity of emission fluorescence. A temperature of half inactivation (Tm) of 77 degrees was determined upon heating the enzyme at pH 7.5 in the presence of Mn2+-ions for 10 min; in the presence of EDTA, Tm is shifted to 55 degrees. Taken together, these observations indicate that the structural stability of beef liver arginase arises from a clustering of hydrophobic amino acids and from Mn2+-ion binding.     

D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis: the binding of trehalose and maltose results in different protein conformational states

In this work, we used fluorescence spectroscopy, molecular dynamics simulation, and Fourier transform infrared spectroscopy for investigating the effect of trehalose binding and maltose binding on the structural properties and the physical parameters of the recombinant D-trehalose/D-maltose binding protein (TMBP) from the hyperthermophilic archaeon Thermococcus litoralis. The binding of the two sugars to TMBP was studied in the temperature range 20 degrees-100 degrees C. The results show that TMBP possesses remarkable temperature stability and its secondary structure does not melt up to 90 degrees C. Although both the secondary structure itself and the sequence of melting events were not significantly affected by the sugar binding, the protein assumes different conformations with different physical properties depending whether maltose or trehalose is bound to the protein. At low and moderate temperatures, TMBP possesses a structure that is highly compact both in the absence and in the presence of two sugars. At about 90 degrees C, the structure of the unliganded TMBP partially relaxes whereas both the TMBP/maltose and the TMBP/trehalose complexes remain in the compact state. In addition, Fourier transform infrared results show that the population of alpha-helices exposed to the solvent was smaller in the absence than in the presence of the two sugars. The spectroscopic results are supported by molecular dynamics simulations. Our data on dynamics and stability of TMBP can contribute to a better understanding of transport-related functions of TMBP and constitute ground for targeted modifications of this protein for potential biotechnological applications.     

Thermal stability of wild type and disulfide bridge containing mutant of poplar plastocyanin

A comparative study of the thermal stability of wild type poplar plastocyanin and of a mutant form containing a disulfide bridge between residues 21 and 25 was performed using differential scanning calorimetry and optical spectroscopic techniques. For wild type plastocyanin the transition temperature, determined from the calorimetric profiles, is 62.7 degrees C at the scan rate of 60 degrees C/h, whereas for the mutant it is reduced to 58.0 degrees C. In both cases, the endothermic peak is followed by an exothermic one at higher temperatures. The unfolding process monitored by optical absorption at 596 nm also reveals a reduced thermal stability of the mutated plastocyanin compared to the wild type protein, with transition temperatures of 54.8 and 58.0 degrees C, respectively. For both proteins, the denaturation process was found to be irreversible and dependent on the scan rate preventing the thermodynamic analysis of the unfolding process. In parallel, small conformational changes between wild type and mutant plastocyanin emerge from fluorescence spectroscopy measurements. Here, a difference in the interaction of the two proteins between the microenvironment surrounding the fluorophores and the solvent was proposed. The destabilization observed in the disulfide containing mutant of plastocyanin suggests that the double mutation, Ile21Cys and Glu25Cys, introduces strain into the protein which offsets the stabilizing effect expected from the formation of a covalent crosslink.     

Spectroscopic and differential scanning calorimetric studies on the unfolding of <Emphasis Type="Italic">Trichosanthes dioica</Emphasis> seed lectin. Similar modes of thermal and chemical denaturation

Physico-chemical and unfolding studies have been carried out on Trichosanthes dioica seed lectin (TDSL). The lectin exhibited maximum activity between pH 7.0 and 10.0, which decreased steeply at lower pH. The hemagglutination activity of TDSL was unaffected in the temperature range 4–50°C, but decreased rapidly at higher temperatures. Differential scanning calorimetric studies indicate that thermal unfolding of TDSL is an irreversible process, which could be described by a three-state model. The calorimetric scan recorded at pH 7.0 consists of two transitions, occurring at around 338.6 K, and 342.8 K. In the presence of carbohydrate ligands both these transitions shifted to higher temperatures, suggesting that ligand binding stabilizes the native conformation of the protein. The unfolding temperature was highest at pH 5.0 indicating that TDSL is more stable at acidic pH. Gdn.HCl induced unfolding, monitored by following changes in the intrinsic fluorescence properties of the protein, was also observed to be a three-state process involving an intermediate. CD spectroscopy indicates that the secondary and tertiary structures of TDSL are rather similar at different pH values, indicating that the lectin structure remains essentially unchanged over a wide range of pH.     

Changes in structure and stability of calbindin-D(28K) upon calcium binding

Calbindin-D(28K) is a biologically important protein required for normal neural function and for the transport of calcium in epithelial cells of the intestine and kidney. We have used fluorescence and circular dichroism (CD) spectroscopy to characterize the effects of calcium binding on the structure and stability of calbindin. Ca(2+) titration monitored by fluorescence spectroscopy reveals the presence of two classes of calcium-binding sites with association constants approximately 10(7.5) and approximately 10(8.9)M(-1). CD spectra in the far-UV spectral range show minor changes upon Ca(2+) titration, implying that the secondary structure of calbindin-D(28K) is not greatly affected. On the basis of the CD spectra in the near-UV spectral range, we conclude that the tertiary structure is more sensitive to Ca(2+) addition. The most significant change occurs between pCa 7.0 and pCa 8.0. The variations in the protein thermostability are correlated with those in the near-UV CD spectra. The enthalpy changes upon heat denaturation of calbindin in the apo-state are characteristic of proteins containing several weakly interacting domains with similar thermodynamical properties. Thus, calcium binding by calbindin-D(28K) largely affects the local structure around the aromatic residues and the thermal stability of the protein; the changes in the secondary structure are insignificant.     

Characterization of thermal stability of the Escherichia coli Fapy-DNA glycosylase

Thermal stability of Escherichia coli Fpg protein was studied using far-UV circular dichroism and intrinsic fluorescence. Experimental data indicate that Fpg irreversibly aggregates under heating above 35 degrees C. Heat aggregation is preceded by tertiary conformational changes of Fpg. However, the secondary structure of the fraction that does not aggregate remains unchanged up to approximately 60 degrees C. The kinetics of heat aggregation occurs with an activation enthalpy of approximately 21 kcal/mol. The fraction of monomers forming aggregates decreases with increasing urea concentration, with essentially no aggregation observed above approximately 3 M urea, suggesting that heat aggregation results from hydrophobic association of partially unfolded proteins. With increasing urea concentration, Fpg unfolds in a two-state reversible transition, with a stability of approximately 3.6 kcal/mol at 25 degrees C. An excellent correlation is observed between the unfolded fraction and loss of activity of Fpg. A simple kinetic scheme that describes both the rates and the extent of aggregation at each temperature is presented.