Ligand-induced thermostability in proteins: thermodynamic analysis of ANS-albumin interaction

A comparative thermodynamic study of the interaction of anilinonaphthalene sulfonate (ANS) derivatives with bovine serum albumin (BSA) was performed by using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The chemically related ligands, 1,8-ANS and 2,6-ANS, present a similar affinity for BSA with different binding energetics. The analysis of the binding driving forces suggests that not only hydrophobic effect but also electrostatic interactions are relevant, even though they have been extensively used as probes for non-polar domains in proteins. Ligand association leads to an increase in protein thermostability, indicating that both dyes interact mainly with native BSA. ITC data show that 1,8-ANS and 2,6-ANS have a moderate affinity for BSA, with an association constant of around 1-9x10(5) M(-1) for the high-affinity site. Ligand binding is disfavoured by conformational entropy. The theoretical model used to simulate DSC data satisfactorily reproduces experimental thermograms, validating this approach as one which provides new insights into the interaction between one or more ligands with a protein. By comparison with 1,8-ANS, 2,6-ANS appears as a more "inert" probe to assess processes which involve conformational changes in proteins.     

A comparative analysis of extrinsic fluorescence in nerve membranes and lipid bilayers

Changes in extrinsic fluorescence intensity, associated with step changes in membrane potential, have been studied in intracellularly or extracellularly stained squid axons, and in lipid bilayers, using six different aminonaphthalene dyes: 1,8-TNS; 2,6-TNS; 1,8-MANS; 2,6-MANS; 2,6-ANS and NPN. In all preparations the optical signals were found to be roughly proportional to the voltage applied. All signals had a very fast initial component, which was followed in some case by a slower change in the same direction. The slow component was observed only in intracellularly stained axons, and not for all chromophores studied. 1,8-TNS, 1,8-MANS and 2,6-MANS yielded the largest fluorescence signals in all preparations. The sign of these signals was independent of the type of membrane studied. However, the fluorescence changes of 2,6-MANS were opposite to those of 1,8-TNS and 1,8 MANS. Staining of both sides of the axolemma with 1,8-MANS or 2,6-MANS showed that these dyes yield larger signals when applied to the extracellular face. The changes in fluorescence light intensity of 2,6-TNS, 2,6-ANS and NPN were smaller and their sign depended on the membrane preparation studied. The comparison of the extrinsic fluorescence signals from the nerve membrane and the phosphatidylcholine bilayer suggests strong similarities between the basic structures of the two systems. The variety of observed signals cannot be easily interpreted in terms of changes in membrane structure. A possible alternative interpretation in terms of electrically induced displacements, rotations and changes in partition coefficient of bound chromophores, is discussed.Abbreviations 1,8-TNS 1-toluidinonaphthalene-8-sulfonate, and similarly, 2,6-TNS - 1,8-MANS 1-N-methylanilinonaphthalene-8-sulfonate, and similarly, 2,6-MANS - 1,8-ANS 1-anilinonaphthalene-8-sulfonate, and similarly, 2,6-ANS - NPN N-phenyl-1-naphthylamine     

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.     

Alignment of anilinonaphthalene-sulfonate and related fluorescent probe molecules in squid axon membrane and in synthetic polymers

As a fluorescent probe for the squid axon membrane, the behavior of 1-anilinonaphthalene-8-sulfonate (1,8-ANS) was found to be very different from that of its positional isomer, 2,6-ANS, or of the methylated derivative, 2,6-TNS. The degree of polarization of the fluorescent light contributing to a transient intensity reduction during nerve excitation was larger than about 0.7 for both 2,6-ANS and 2,6-TNS, while the corresponding value for 1,8-ANS in a squid axon was about 0.35.The physicochemical basis of this difference was investigated by measuring the fluorescence polarization of these probe molecules incorporated into poly(vinyl alcohol) sheets. In a stretched sheet of this synthetic polymer, 1,8-ANS showed poor alignment, while the 2,6-derivatives were highly oriented with their transition moments aligned approximately in the direction of stretching. Based on these findings, the experimental results obtained from squid axons were interpreted as an indication of the existence, at or near the membrane, of a longitudinally oriented macromolecular structure, bringing about a high degree of alignment of 2,6-ANS or 2,6-TNS molecules.It is clear that, as a probe for fluorescence polarization studies of macromolecular structures, 2,6-TNS is far superior to 1,8-ANS.     

Protein l-isoaspartyl-O-methyltransferase of Vibrio cholerae: Interaction with cofactors and effect of osmolytes on unfolding

Protein l-isoaspartyl-O-methyltransferase (PIMT) is an ubiquitous enzyme widely distributed in cells and plays a role in the repair of deamidated and isomerized proteins. In this study, we show that this enzyme is present in cytosolic extract of Vibrio cholerae, an enteric pathogenic Gram-negative bacterium and is enzymatically active. Additionally, we focus on the detailed biophysical characterization of the recombinant PIMT from V. cholerae to gain insight into its structure, stability and the cofactor binding. The equilibrium denaturation of PIMT has been studied using tryptophan fluorescence and CD spectroscopy. The far- and near-UV CD, as well as fluorescence experiments reveal the presence of a non-native intermediate in the folding pathway. Binding of the hydrophobic fluorescent probe, bis-ANS, to the intermediate occurs with high affinity because of the exposure of the hydrophobic clusters during the unfolding process. The existence of the probable intermediate has also been confirmed from limited tryptic digestion and DLS experiments. The protein shows higher binding affinity for AdoHcy, in comparison to AdoMet, and the binding increases the midpoint of thermal unfolding by 6 and 5 °C, respectively. Modeling and molecular dynamics simulations also support the higher stability of the protein in presence of AdoHcy.     

The conformational stability and flexibility of insulin with an additional intramolecular cross-link

The conformational stability and flexibility of insulin containing a cross-link between the alpha-amino group of the A-chain to the epsilon-amino group of Lys29 of the B-chain was examined. The cross-link varied in length from 2 to 12 carbon atoms. The conformational stability was determined by guanidine hydrochloride-induced equilibrium denaturation and flexibility was assessed by H2O/D2O amide exchange. The cross-link has substantial effects on both conformational stability and flexibility which depend on its length. In general, the addition of a cross-link enhances conformational stability and decreases flexibility. The optimal length for enhanced stability and decreased flexibility was the 6-carbon link. For the 6-carbon link the Gibbs free energy of unfolding was 8.0 kcal/mol compared to 4.5 kcal/mol for insulin, and the amide exchange rate decreased by at least 3-fold. A very short cross-link (i.e. the 2-carbon link) caused conformational strain that was detectable by a lack of stabilization in the Gibbs free energy of unfolding and enhancement in the amide exchange rate compared to insulin. The effect of the cross-link length on insulin hydrodynamic properties is discussed relative to previously obtained receptor binding results.     

The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL

The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment of GroEL, or minichaperone (residues 191-345), was investigated and compared with that of the GroEL tetradecamer. Although a total of seven molecules of bis-ANS bind cooperatively to this minichaperone, most of the hydrophobic sites were induced following initial binding of one to two molecules of probe. From the equilibrium and kinetics studies at low bis-ANS concentrations, it is evident that the native apical domain is converted to an intermediate conformation with increased hydrophobic surfaces. This intermediate binds additional bis-ANS molecules. Tyrosine fluorescence detected denaturation demonstrated that bis-ANS can destabilize the apical domain. The results from (i) bis-ANS titrations, (ii) urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosine fluorescence studies of the apical domain are consistent with a model in which bis-ANS binds tightly to the intermediate state, relatively weakly to the native state, and little to the denatured state. The results suggest that the conformational changes seen in apical domain fragments are not seen in the intact GroEL oligomer due to restrictions imposed by connections of the apical domain to the intermediate domain and suppression of movement due to quaternary structure.     

Controlling {beta}-amyloid oligomerization by the use of naphthalene sulfonates: trapping low molecular weight oligomeric species

Aggregation of proteins and peptides has been shown to be responsible for several diseases known as amyloidoses, which include Alzheimer disease (AD), prion diseases, among several others. AD is a neurodegenerative disorder caused primarily by the aggregation of beta-amyloid peptide (Abeta). Here we describe the stabilization of small oligomers of Abeta by the use of sulfonated hydrophobic molecules such as AMNS (1-amino-5-naphthalene sulfonate); 1,8-ANS (1-anilinonaphthalene-8-sulfonate) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate). The experiments were performed with either Abeta-1-42 or with Abeta-13-23, a shorter version of Abeta that is still able to form amyloid fibrils in vitro and contains amino acid residues 16-20, previously shown to be essential to peptide-peptide interaction and fibril formation. All sulfonated molecules tested were able to prevent Abeta aggregation in a concentration dependent fashion in the following order of efficacy: 1,8-ANS < AMNS < bis-ANS. Size exclusion chromatography revealed that in the presence of bis-ANS, Abeta forms a heterogeneous population of low molecular weight species that proved to be toxic to cell cultures. Since the ANS compounds all have apolar rings and negative charges (sulfonate groups), both hydrophobic and electrostatic interactions may contribute to interpeptide contacts that lead to aggregation. We also performed NMR experiments to investigate the structure of Abeta-13-23 in SDS micelles and found features of an alpha-helix from Lys(16) to Phe(20). 1H TOCSY spectra of Abeta-13-23 in the presence of AMNS displayed a chemical-shift dispersion quite similar to that observed in SDS, which suggests that in the presence of AMNS this peptide might adopt a conformation similar to that reported in the presence of SDS. Taken together, our studies provide evidence for the crucial role of small oligomers and their stabilization by sulfonate hydrophobic compounds.     

Four-state equilibrium unfolding of an scFv antibody fragment

The conformational stability of a single-chain Fv antibody fragment against a hepatitis B surface antigen (anti-HBsAg scFv) has been studied by urea and temperature denaturation followed by fluorescence and circular dichroism. At neutral pH and low protein concentration, it is a well-folded monomer, and its urea and thermal denaturations are reversible. The noncoincidence of the fluorescence and circular dichroism transitions indicates the accumulation in the urea denaturation of an intermediate (I(1)) not previously described in scFv molecules. In addition, at higher urea concentrations, a red-shift in the fluorescence emission maximum reveals an additional intermediate (I(2)), already reported in the denaturation of other scFvs. The urea equilibrium unfolding of the anti-HBsAg scFv is thus four-state. A similar four-state behavior is observed in the thermal unfolding although the intermediates involved are not identical to those found in the urea denaturation. Global analysis of the thermal unfolding data suggests that the first intermediate displays substantial secondary structure and some well-defined tertiary interactions while the second one lacks well-defined tertiary interactions but is compact and unfolds at higher temperature in a noncooperative fashion. Global analysis of the urea unfolding data (together with the modeled structure of the scFv) provides insights into the conformation of the chemical denaturation intermediates and allows calculation of the N-I(1), I(1)-I(2), and I(2)-D free energy differences. Interestingly, although the N-D free energy difference is very large, the N-I(1) one, representing the "relevant" conformational stability of the scFv, is small.     

Folding intermediates of the prion protein stabilized by hydrostatic pressure and low temperature

Prion diseases are associated with conformational conversion of the cellular prion protein, PrPC, into a misfolded form, PrPSc. We have investigated the equilibrium unfolding of the structured domain of recombinant murine prion protein, comprising residues 121-231 (mPrP-(121-231)). The equilibrium unfolding of mPrP-(121-231) by urea monitored by intrinsic fluorescence and circular dichroism (CD) spectroscopies indicated a two-state transition, without detectable folding intermediates. The fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5-disulfonic acid (bis-ANS) binds to native mPrP-(121-231), indicating exposure of hydrophobic domains on the protein surface. Increasing concentrations of urea (up to 4 M) caused the release of bound bis-ANS, whereas changes in intrinsic fluorescence and CD of mPrP took place only above 4 M urea. This indicates the existence of a partially unfolded conformation of mPrP, characterized by loss of bis-ANS binding and preservation of the overall structure of the protein, stabilized at low concentrations of urea. Hydrostatic pressure and low temperatures were also used to stabilize partially folded intermediates that are not detectable in the presence of chemical denaturants. Compression of mPrP to 3.5 kbar at 25 degrees C and pH 7 caused a slight decrease in intrinsic fluorescence emission and an 8-fold increase in bis-ANS fluorescence. Lowering the temperature to -9 degrees C under pressure reversed the decrease in intrinsic fluorescence and caused a marked (approximately 40-fold) increase in bis-ANS fluorescence. The increase in bis-ANS fluorescence at low temperatures was similar to that observed for mPrP at 1 atm at pH 4. These results suggest that pressure-assisted cold denaturation of mPrP stabilizes a partially folded intermediate that is qualitatively similar to the state obtained at acidic pH. Compression of mPrP in the presence of a subdenaturing concentration of urea stabilized another partially folded intermediate, and cold denaturation under these conditions led to complete unfolding of the protein. Possible implications of the existence of such partially folded intermediates in the folding of the prion protein and in the conversion to the PrPSc conformer are discussed.     

Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel

The insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis undergo several conformational changes from crystal inclusion protoxins to membrane-inserted channels in the midgut epithelial cells of the target insect. Here we analyzed the stability of the different forms of Cry1Ab toxin, monomeric toxin, pre-pore complex, and membrane-inserted channel, after urea and thermal denaturation by monitoring intrinsic tryptophan fluorescence of the protein and 1-anilinonaphthalene-8-sulfonic acid binding to partially unfolded proteins. Our results showed that flexibility of the monomeric toxin was dramatically enhanced upon oligomerization and was even further increased by insertion of the pre-pore into the membrane as shown by the lower concentration of chaotropic agents needed to achieve unfolding of the oligomeric species. The flexibility of the toxin structures is further increased by alkaline pH. We found that the monomer-monomer interaction in the pre-pore is highly stable because urea promotes oligomer denaturation without disassembly. Partial unfolding and limited proteolysis studies demonstrated that domains II and III were less stable and unfold first, followed by unfolding of the most stable domain I, and also that domain I is involved in monomer-monomer interaction. The thermal-induced unfolding and analysis of energy transfer from Trp residues to bound 1-anilinonaphthalene-8-sulfonic acid dye showed that in the membrane-inserted pore domains II and III are particularly sensitive to heat denaturation, in contrast to domain I, suggesting that only domain I may be inserted into the membrane. Finally, the insertion into the membrane of the oligomeric pre-pore structure was not affected by pH. However, a looser conformation of the membrane-inserted domain I induced by neutral or alkaline pH correlates with active channel formation. Our studies suggest for the first time that a more flexible conformation of Cry toxin could be necessary for membrane insertion, and this flexible structure is induced by toxin oligomerization. Finally the alkaline pH found in the midgut lumen of lepidopteran insects could increase the flexibility of membrane-inserted domain I necessary for pore formation.     

Comparison of the stability of fish light meromyosins by guanidine hydrochloride denaturation

The guanidine hydrochloride denaturation of light meromyosins (LMMs) of fish (carp, sardine and greenling) and rabbit was investigated to determine their structural stability quantitatively. The circular dichroism (CD) and fluorescence spectroscopies were applied to monitor denaturation. The CD results indicate that the LMM α-helix undergoes a two-step unfolding. The free energy of denaturation was calculated based on the linear extrapolation method and the denaturant binding model. Total free energies of the two-step unfolding of the α-helix are related to the water temperatures in which the fish live and the body temperature of rabbit. The stability of α-helical structure of LMM was in the following descending order: rabbit>carp>sardine>greenling. The free energies of denaturation obtained by tryptophan fluorescence differ from the free energies of the unfolding α-helix. The data from the two spectroscopic measurements are discussed along with the conformational changes of LMMs.     

Conformational flexibility of avidin: the influence of biotin binding

Ligand binding to proteins is a key process in cell biochemistry. The interaction usually induces modifications in the unfolding thermodynamic parameters of the macromolecule due to the coupling of unfolding and binding equilibria. In addition, these modifications can be attended by changes in protein structure and/or conformational flexibility induced by ligand binding. In this work, we have explored the effect of biotin binding on conformation and dynamic properties of avidin by using infrared spectroscopy including kinetics of hydrogen/deuterium exchange. Our results, along with previously thermodynamic published data, indicate a clear correlation between thermostability and protein compactness. In addition, our results also help to interpret the thermodynamic binding parameters of the exceptionally stable biotin:AVD complex.     

High-throughput measurement of protein stability in microtiter plates

The direct determination of protein stability at high throughput has applications in proteomics, directed evolution, and formulation. Each application places different requirements on the accuracy of stability or transition midpoint determination. The measurement of protein stability by chemical denaturation has been previously performed at medium throughput and high accuracy using autotitrating fluorometers, after removal of proteins from the 96-well plate format in which they were expressed and purified. Herein we present a higher-throughput method for measuring and indexing the stability of proteins maintained within the 96-well format using a fluorescence microplate reader. Protein unfolding transitions were monitored by tryptophan fluorescence at 340 nm and assessed using bovine and equine cytochrome c (cyt c), as well as bovine serum albumin (BSA) stabilized with various amounts of palmitic acid. Two different approaches for generating unfolding curves in microtiter plates have been evaluated for their accuracy and applicability. Unfolding curves generated by the serial addition of denaturant into single wells allowed high-throughput stability screens capable of identifying protein variants with unfolding midpoint differences of 0.15 M denaturant concentration or larger. Such a method would be suitable for screening large numbers of proteins, as typically generated for directed evolution. Unfolding curves generated using one well per denaturant concentration allowed for medium-throughput stability screening and generated more accurate and precise stability values (C(1/2) +/- 0.05 M, m(G), and DeltaG(H2O)) for cyt c that are similar to values reported in literature. This method is suitable for screening the smaller numbers of proteins generated in proteomic research programmes. By using BSA stabilized with various palmitate concentrations and simple numerical indexing, it was shown that both experimental methods can successfully rank the order of protein stability.     

Use of fluorescence decay times of 8-ANS-protein complexes to study the conformational transitions in proteins which unfold through the molten globule state

The conformational transitions starting with the native protein, passing the molten globule state and finally approaching the unfolded state of proteins was investigated for bovine carbonic anhydrase B (BCAB) and human -lactalbumin (-HLA) by means of fluorescence decay time measurements of the dye 8-anilinonaphthalene-1-sulphonic acid (8-ANS). Stepwise denaturation was realized by using the denaturant guanidinium chloride (GdmCl). It was shown that 8-ANS bound with protein yields a double-exponential fluorescence decay, where both decay times considerably exceed the decay time of free 8-ANS in water. This finding reflects the hydrophobic environment of the dye molecules attached to the proteins.

The fluorescence lifetime of the short-time component is affected by protein association and can be effectively quenched by acrylamide, indicating that 8-ANS molecules preferentially bind at the protein surface. The fluorescence lifetime of the long-time component is independent of the protein and acrylamide concentration and may be related to protein-embedded dye molecules.

Changes of the long lifetime component upon GdmCl-induced denaturation and unfolding of BCAB and -HLA correlate well with overall changes of the protein conformation. The transition from native protein to the molten globule state is accompanied by an increase of the number of protein-embedded 8-ANS molecules, while the number of dye molecules located at the protein surface decreases. For the transition from the molten globule to the unfolded state was the opposite behaviour observed.     

A study of the interaction of avidin with 2-anilinonaphthalene-6-sulfonic acid as a probe of the biotin binding site

The environment of the biotin binding site on avidin was investigated by determining the fluorescence enhancement of a series of fluorescent probes that are anilinonaphthalene sulfonic acid derivatives. Of the compounds tested, 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) exhibited the greatest enhancement under the conditions used (which would reflect both molar fluorescence enhancement and binding affinity) and exhibited more than 95% reversal upon addition of biotin. Thus, 2,6-ANS was chosen for more detailed characterization of the interaction with avidin. Only a single class of binding sites for 2,6-ANS was identified; the mean value for the Kd was 203 +/- 16 microM (X +/- 1 S.D.), and the molar ratio of 2,6-ANS binding sites to biotin binding sites was approx. 1. These results provide evidence that the biotin binding site and the 2,6-ANS binding site are at least partially overlapping, but the possibility that the probe binding site is altered by a conformational change induced by biotin binding cannot be excluded. At excitation = 328 nm and emission = 408 nm, the molar fluorescence of the bound probe was 6.8 +/- 1.0 microM-1 and that of the free probe was 0.061 +/- 0.008 microM-1 giving an enhancement ratio (molar fluorescence of bound probe/molar fluorescence of free probe) of 111 +/- 22. Upon binding, the wavelength of maximum fluorescence decreases. These findings also provide evidence that the fluorescence enhancement associated with the interaction of 2,6-ANS and avidin reflects the environment of the biotin binding site. The Kosower's Z factor, an empirical index of apolarity, was 82.1 for the 2,6-ANS binding site on avidin. This value reflects a degree of apolarity that is similar to apolar environments observed for substrate binding sites on several enzymes; although not the dominant factor, this environment may contribute to the strong binding of biotin.     

Conformational stability of apoflavodoxin.

Flavodoxins are alpha/beta proteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin from Anaboena PCC 7119 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 11 and pH 6, the apoprotein is folded properly as judged from near-ultraviolet (UV) circular dichroism (CD) and high-field 1H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two-state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5-2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far-UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two-state process at acidic pH. Apoflavodoxin constitutes a promising system for the analysis of the stability and folding of alpha/beta proteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors.     

Conformational stability of the covalent complex between elastase and alpha 1-proteinase inhibitor

The equilibrium unfolding-refolding process of the elastase-alpha 1-proteinase inhibitor complex, induced by guanidinium chloride, was followed by spectroscopic methods. A reversible transition with a midpoint at 2.04 +/- 0.04 M guanidinium chloride was observed by fluorescence. This transition was attributed to elastase on the basis of circular dichroism and uv absorption difference data obtained for the covalent complex and for the free proteins. The conformational stability of elastase in the complex was analyzed considering the approximation of a two-state transition. The free energy of denaturation delta GH2O was 4.2 kcal.mol-1 for complexed elastase compared to 10.5 kcal.mol-1 for the free enzyme. Such a decrease in the stability of elastase suggests that, after forming the covalent complex with the inhibitor, the enzyme undergoes not only the expected local modifications of the active site, but also an extensive structural reorganization.     

Conformation and stability of elastase from Atlantic cod, Gadus morhua

Metal binding and conformational stability characteristics of psychrophilic elastase (ACE) from Atlantic cod (Gadus morhua) has been investigated. Chelation to Ca(2+) was found to be important for maintaining the biologically active conformation and for the thermal stability of the enzyme. However, presence of metal ions such as Zn(2+), Fe(3+) and Cu(2+) was found to inhibit its hydrolytic activity and so did the chelating agent EDTA. Both pH and guanidinium chloride induced denaturation of the enzyme was followed by monitoring the changes in the tryptophan fluorescence. ACE exhibited a simple two-state unfolding pattern in both acidic and basic conditions with the midpoint of transition at pH values 4.08 and 10.29, respectively. Guanidinium chloride and heat induced denaturation of the enzyme was investigated at two pH values, 5.50 and 8.00, wherein the enzyme possesses similar tertiary structure but differ in its hydrolytic activity. Guanidinium chloride induced denaturation indicated that the enzyme unfolds with a C(m) of 1.53 M at pH 8.0 and a DeltaG(H2O) of 6.91 kJ mol(-1) (28.65 J mol(-1) residue(-1)) which is the lowest reported for psychrophilic enzymes investigated till-date. However, at pH 5.50, DeltaG(H2O) value is slightly lowered by 0.65 kJ mol(-1) consistent with the observed increase in the apparent quenching constant obtained with acrylamide. On the other hand, increase in T(m) by 38.45 degrees C was observed for the enzyme at acid pH (5.50) in comparison to the heat induced unfolding at pH 8.0. The increase in the apparent T(m) has been attributed to the possible weak intermolecular association of the enzyme molecules at moderately high temperatures that is favoured by the increase in the accessible surface area / dynamics under acidic conditions. The stability characteristics of ACE have been compared with the available data for mesophilic porcine pancreatic elastase and possible mechanism for the low temperature adaptation of ACE has been proposed.     

Conformational changes and inactivation of rabbit muscle creatine kinase in dimethyl sulfoxide solutions.

The effects of dimethyl sulfoxide (DMSO) on creatine kinase (CK) conformation and enzymatic activity were studied by measuring activity changes, aggregation, and fluorescence spectra. The results showed that at low concentrations (< 65% v/v), DMSO had little effect on CK activity and structure. However, higher concentrations of DMSO led to CK inactivation, partial unfolding, and exposure of hydrophobic surfaces and thiol groups. DMSO caused aggregation during CK denaturation. A 75% DMSO concentration induced the most significant aggregation of CK. The CK inactivation and unfolding kinetics were single phase. The unfolding of CK was an irreversible process in the DMSO solutions. The results suggest that to a certain extent, an enzyme can maintain catalytic activity and conformation in water-organic mixture environments. Higher concentrations of DMSO affected the enzyme structure but not its active site. Inactivation occurred along with noticeable conformational change during CK denaturation. The inactivation and unfolding of CK in DMSO solutions differed from other denaturants such as guanidine, urea, and sodium dodecyl sulfate. The exposure of hydrophobic surfaces was a primary reason for the protein aggregation.