Effects of troponin on thermal unfolding of actin-bound tropomyosin

Differential scanning calorimetry (DSC) was used to study the effect of troponin (Tn) and its isolated components on the thermal unfolding of skeletal muscle tropomyosin (Tm) bound to F-actin. It is shown that in the absence of actin the thermal unfolding of Tm is expressed in two well-distinguished thermal transitions with maxima at 42.8 and 53.8°C. Interaction with F-actin affects the character of thermal unfolding of Tm leading to appearance of a new Tm transition with maximum at about 48°C, but it has no influence on the thermal denaturation of F-actin stabilized by aluminum fluoride, which occurs within the temperature region above 70°C. Addition of troponin leads to significant increase in the cooperativity and enthalpy of the thermal transition of the actin-bound Tm. The most pronounced effect of Tn was observed in the absence of calcium. To elucidate how troponin complex affects the properties of Tm, we studied the influence of its isolated components, troponin I (TnI) and troponin T (TnT), on the thermal unfolding of actin-bound Tm. Isolated TnT and TnI do not demonstrate cooperative thermal transitions on heating up to 100°C. However, addition of TnI, and especially of TnT, to the F-actin–Tm complex significantly increased the cooperativity of the thermal unfolding of actin-bound tropomyosin.     

Specific sequences determine the stability and cooperativity of folding of the C-terminal half of tropomyosin

Tropomyosin is a flexible 410 A coiled-coil protein in which the relative stabilities of specific regions may be important for its proper function in the control of muscle contraction. In addition, tropomyosin can be used as a simple model of natural occurrence to understand the inter- and intramolecular interactions that govern the stability of coiled-coils. We have produced eight recombinant tropomyosin fragments (Tm(143-284(5OHW),) Tm(189-284(5OHW)), Tm(189-284), Tm(220-284(5OHW)), Tm(220-284), Tm(143-235), Tm(167-260), and Tm(143-260)) and one synthetic peptide (Ac-Tm(215-235)) to investigate the relative conformational stability of different regions derived from the C-terminal region of the protein, which is known to interact with the troponin complex. Analytical ultracentrifugation experiments show that the fragments that include the last 24 residues of the molecule (Tm(143-284(5OHW)), Tm(189-284(5OHW)), Tm(220-284(5OHW)), Tm(220-284)) are completely dimerized at 10 microm dimer (50 mm phosphate, 100 mm NaCl, 1.0 mm dithiothreitol, and 0.5 mm EDTA, 10 degrees C), whereas fragments that lack the native C terminus (Tm(143-235),Tm(167-260), and Tm(143-260)) are in a monomer-dimer equilibrium under these conditions. The presence of trifluoroethanol resulted in a reduction in the [theta](222)/[theta](208) circular dichroism ratio in all of the fragments and induced stable trimer formation only in those containing residues 261-284. Urea denaturation monitored by circular dichroism and fluorescence revealed that residues 261-284 of tropomyosin are very important for the stability of the C-terminal half of the molecule as a whole. Furthermore, the absence of this region greatly increases the cooperativity of urea-induced unfolding. Temperature and urea denaturation experiments show that Tm(143-235) is less stable than other fragments of the same size. We have identified a number of factors that may contribute to this particular instability, including an interhelix repulsion between g and e' positions of the heptad repeat, a charged residue at the hydrophobic coiled-coil interface, and a greater fraction of beta-branched residues located at d positions.     

The conformational stabilities of tropomyosins.

The stability to denaturation by heat and guanidine hydrochloride of seven vertebrate (including skeletal, cardiac and smooth muscle) tropomyosins and three invertebrate tropomyosins was examined. The transition profiles were discontinuous and in many cases distinct plateaux were observed which indicated the presence of unique partially unfolded states at intermediate temperatures and guanidine hydrochloride concentrations. The denaturation by guanidine hydrochloride could be described in the majority of cases by a model in which the native state unfolds to a partially unfolded stable intermediate which then unfolds to the completely denatured state. On this basis it was possible to estimate the free energies of unfolding in water. It was shown that part of the alpha-helical structure of tropomyosin is only marginally stable and the free energy of unfolding in water of this segment is less than values found for globular proteins, whereas another segment (or segments) has a stability comparable to that found for globular proteins. The stepwise unfolding may be explained in terms of the coiled-coil interactions in tropomyosin. Differences in stability were found between tropomyosins from different muscles of the same species as well as between species, no two tropomyosins giving the same denaturation profiles. The invertebrate tropomyosins showed a wider range of stabilities, that from scallop striated muscle being far more easily denatured than all the others. No correlation was found between the stability of tropomyosin and the type of regulatory system of the muscle. A comparison of the results from vertebrate and invertebrate species suggests that there has been no selection for proteins of higher or lower stability during the evolutionary time scale.     

Pressure denaturation of the yeast prion protein Ure2

Denaturation of the Saccharomyces cerevisiae prion protein Ure2 was investigated using hydrostatic pressure. Pressures of up to 600 MPa caused only limited perturbation of the structure of the 40-kDa dimeric protein. However, nondenaturing concentrations of GdmCl in combination with high pressure resulted in complete unfolding of Ure2 as judged by intrinsic fluorescence. The free energy of unfolding measured by pressure denaturation or by GdmCl denaturation is the same, indicating that pressure does not induce dimer dissociation or population of intermediates in 2 M GdmCl. Pressure-induced changes in 5 M GdmCl suggest residual structure in the denatured state. Cold denaturation under pressure at 200 MPa showed that unfolding begins below -5 degrees C and Ure2 is more susceptible to cold denaturation at low ionic strength. Results obtained using two related protein constructs, which lack all or part of the N-terminal prion domain, were very similar.     

Unfolding/refolding studies of smooth muscle tropomyosin. Evidence for a chain exchange mechanism in the preferential assembly of the native heterodimer

The thermal and the urea-induced unfolding profiles of the coiled-coil alpha-helix of native and refolded tropomyosin from chicken gizzard were studied by circular dichroism. Refolding of tropomyosin at low temperature from alpha + beta subunits, dissociated by guanidinium chloride, urea, or high temperature, predominantly produced alpha alpha + beta beta homodimers in agreement with earlier studies of refolding from guanidinium chloride (Graceffa, P. (1989) Biochemistry 28, 1282-1287). The presence of two unfolding transitions in low salt solutions with about equal helix loss verified the composition with the first unfolding transition of the homodimer mixture originating from alpha alpha. In contrast, refolding by equilibrating at temperatures close to physiological, however, produced the native alpha beta heterodimer, which unfolded in a single transition. The refolding kinetics of dissociated alpha + beta subunits indicated that beta beta homodimers form first, leading to alpha alpha homodimers both of which are relatively stable against chain exchange below approximately 25 degrees C. Equilibrating the homodimer mixture at 37-40 degrees C for long times, however, produced the native alpha beta molecule via chain exchange. The equilibria involved indicate that the free energy of formation from subunits of alpha beta is much less than that of (alpha alpha + beta beta)/2. In vivo folding of alpha beta from the two separate alpha and beta gene products is, therefore, thermodynamically favored over the formation of homodimers and biological factors need not be considered to explain the native preferred alpha beta composition.     

Excimer fluorescence of pyrenyliodoacetamide-labeled tropomyosin: a probe of the state of tropomyosin in reconstituted muscle thin filaments

Rabbit skeletal tropomyosin (Tm) specifically labeled at cysteine groups with N-(1-pyrenyl)-iodoacetamide (PIA) exhibits excimer fluorescence. The excimer fluorescence was sensitive to the local conformation of Tm, to actin binding, and, in reconstituted thin filaments, to the Tm state change induced by binding of myosin subfragment 1 (S1). The properties of PIATm were similar to previously studied pyrenylmaleimide-labeled Tm (PMTm) [Ishii, Y., & Lehrer, S.S. (1985) Biochemistry 24, 6631] except that S1 binding to actin-Tm increased the excimer fluorescence in contrast to the time-dependent decrease seen for PMTm. The fluorescence properties of PIATm are sensitive to the Tm chain-chain interaction via equilibria among pyrene configurations and nonfluorescent dimer as well as the monomer and excimer-forming configurations. The effect of bound troponin (Tn) on the excimer fluorescence of PIATm in the reconstituted systems was dependent on ionic strength with a slight Ca2+ dependence. S1 titrations in the absence and presence of Tn and Ca2+ indicated that the excimer fluorescence probes the state change of Tm from the weak S1 binding state to the strong S1 binding state which is facilitated by Ca2+ [Hill et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186]. Binding of MgADP-S1 and MgAMPPNP-S1 produced the same total excimer fluorescence change as for nucleotide-free S1, showing that the strong S1 binding state of Tm-actin is independent of nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)     

The unfolding pathway of leech carboxypeptidase inhibitor

The unfolding and denaturation curves of leech carboxypeptidase inhibitor (LCI) were elucidated using the technique of disulfide scrambling. In the presence of thiol initiator and denaturant, the native LCI denatures by shuffling its native disulfide bonds and transforms into a mixture of scrambled species. 9 of 104 possible scrambled isomers of LCI, amounting to 90% of total denatured LCI, can be distinguished. The denaturation curve that plots the fraction of native LCI converted into scrambled isomers upon increasing concentrations of denaturant shows that the concentration of guanidine thiocyanate and guanidine hydrochloride required to reach 50% of denaturation is 2.4 and 3.6 m, respectively. In contrast, native LCI is resistant to urea denaturation even at high concentration (8 m). The LCI unfolding pathway was defined based on the evolution of the relative concentration of scrambled isoforms of LCI upon denaturation. Two populations of scrambled species suffer variations along the unfolding pathway. One accumulates as intermediates under strong denaturing conditions and corresponds to open or relaxed structures, among which the beads-form isomer is found. The other population shows an inverse correlation between their relative abundances and the denaturing conditions and should have another kind of non-native structure that is more compact than the unfolded state. The rate constants of unfolding of LCI are low when compared with other disulfide-containing proteins. Overall, the results presented in this study show that LCI, a molecule with potential biotechnological applications, has slow kinetics of unfolding and is highly stable.     

Effects of the state of the succinimido-ring on the fluorescence and structural properties of pyrene maleimide-labeled alpha alpha-tropomyosin.

Rabbit skeletal alpha alpha-tropomyosin was labeled at Cys-190 with pyrene maleimide to form (S-[N-(1-pyrene)succinimido])2-tropomyosin (pyreneI-Tm). The product with cleaved succinimido-rings, pyreneII-Tm was also prepared by incubation of pyreneI-Tm at pH greater than 7.5. The pH dependence of the rate of cleavage indicated that hydrolysis rather than aminolysis was the more likely reaction. PyreneI-Tm exhibited a loss in helix content and end-to-end polymerization compared with unlabeled Tm, which increased upon formation of pyreneII-Tm. The cleavage resulted in increased interchain excited state excimer fluorescence originating from pyrene-pyrene interaction between the chains. Thus, increased pyrene-pyrene interaction at Cys 190 leads to an increase in unfolding, the effects of which appear to be transmitted to the ends of tropomyosin. The fluorescence properties of the two types of pyrene-succinimide adducts of dithiothreitol were very similar to the corresponding adducts of pyrene-Tm indicating excimer formation through ground state pyrene-pyrene interaction.     

Conformational stability and domain unfolding of the Von Willebrand factor A domains

Von Willebrand factor (VWF), a multimeric multidomain glycoprotein secreted into the blood from vascular endothelial cells, initiates platelet adhesion at sites of vascular injury. This process requires the binding of platelet glycoprotein Ib-IX-V to the A1 domain of VWF monomeric subunits under fluid shear stress. The A2 domain of VWF monomers contains a proteolytic site specific for a circulating plasma VWF metalloprotease, A Disintegrin and Metalloprotease with Thrombospondin motifs, member #13 of the ADAMTS enzyme family (ADAMTS-13), that functions to reduce adhesiveness of newly released, unusually large (UL), hyperactive forms of VWF. Shear stress assists ADAMTS-13 proteolysis of ULVWF multimers allowing ADAMTS-13 cleavage of an exposed peptide bond in the A2 domain. Shear stress may induce conformational changes in VWF, and even unfold regions of VWF monomeric subunits. We used urea as a surrogate for shear to study denaturation of the individual VWF recombinant A domains, A1, A2, and A3, and the domain triplet, A1-A2-A3. Denaturation was evaluated as a function of the urea concentration, and the intrinsic thermodynamic stability of the domains against unfolding was determined. The A1 domain unfolded in a 3-state manner through a stable intermediate. Domains A2 and A3 unfolded in a 2-state manner from native to denatured. The A1-A2-A3 triple domain unfolded in a 6-state manner through four partially folded intermediates between the native and denatured states. Urea denaturation of A1-A2-A3 was characterized by two major unfolding transitions: the first corresponding to the simultaneous complete unfolding of A2 and partial unfolding of A1 to the intermediate state; and the second corresponding to the complete unfolding of A3 followed by gradual unfolding of the intermediate state of A1 at high urea concentration. The A2 domain containing the cleavage site for ADAMTS-13 was the least stable of the three domains and was the most susceptible to unfolding. The low stability of the A2 domain is likely to be important in regulating the exposure of the A2 domain cleavage site in response to shear stress, ULVWF platelet adherence, and the attachment of ADAMTS-13 to ULVWF.     

Effects of aspartate on rabbit muscle creatine kinase and the salt induced molten globule state

The aspartate (Asp)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, circular dichroism (CD) spectra, native polyacrylamide gel electrophoresis and ultraviolet difference spectra. The results showed that Asp caused inactivation and unfolding of CK, with no aggregation during CK denaturation. The kinetics of CK unfolding followed a one phase process. At higher concentrations of Asp (>2.5mM), the CK dimers were partially dissociated. Inactivation occurred before noticeable conformational change during CK denaturation. Asp denatured CK was mostly reactivated and refolded by dilution. KCl induced the molten globule state with compact structure after CK was denatured with 10mM Asp. These results suggest that the effect of Asp differed from that of other denaturants such as guanidine, HCl or urea during CK unfolding. Asp is a reversible protein denaturant and the molten globule state indicates that intermediates exist during CK folding.     

Denaturation by urea and renaturation of 20 beta-hydroxysteroid dehydrogenase studied by high-performance size exclusion chromatography

The denaturation by urea and renaturation of 20 beta-hydroxysteroid dehydrogenase, a tetrameric enzyme consisting of four identical subunits, were followed by high-performance size exclusion chromatography to detect intermediates in the processes. During the denaturation process no intermediate form (structured monomers or dimers) between the tetramer and the denatured monomer was observed. During the renaturation process, carried out either with or without NADH, high molecular weight aggregates, native tetramers, and low molecular weight intermediates were evidenced and quantified. The contemporaneous measurement of recovery of activity unambiguously demonstrated that the tetrameric structure is essential for enzymatic activity.     

The peculiar nature of unfolding of the human prion protein

Spontaneous conformational transition of the prion protein from an alpha-helical isoform to a beta-sheet-rich isoform underlies the pathogenesis of sporadic prion diseases. To study the rate-limiting steps of spontaneous conversion, the formation of amyloid fibrils by the recombinant human PrP C-terminal fragment spanning residues 90-231 (recPrP) was monitored in the presence of urea. The kinetics of spontaneous fibril formation displayed sigmoidal behavior involving a lag phase. The shortest lag phase was observed at partially denaturing conditions, close to the concentration of urea corresponding to the middle point of unfolding. This result indicates that unfolding intermediates may be important for the conversion. To test whether unfolding intermediates are formed, we employed size-exclusion chromatography and circular dichroism spectroscopy to monitor urea denaturation of recPrP. Both techniques showed a single sigmoidal transition with very similar thermodynamic parameters of denaturation and that the transition can be described by a simple equilibrium between folded and denatured states. Detailed analyses of data, however, revealed that the dimensions of both the native and denatured species gradually increases with urea. Expansion of the native species is also accompanied by an increase in efficiency of the energy transfer from a single Trp residue to 1-anilinonaphthalene-8-sulfonate dye as measured by fluorescence. These data illustrate that thermodynamic character of the native ensemble changes gradually with environmental conditions. Such behavior is consistent with the thermodynamically variable model, and may be linked to the ability of PrP to adopt distinct abnormal conformations under pathologic conditions.     

Thermodynamic stability of the C-terminal domain of the human inducible heat shock protein 70

The stability of the substrate-binding region of human inducible Hsp70 was studied by a combination of spectroscopic and calorimetric methods. Thermal denaturation of the protein involves four accessible states: the native state, two largely populated intermediates, and the denatured state, with transition temperatures of 52.8, 56.2 and 71.2 degrees C, respectively, at pH 6.5. The intermediate spectroscopic properties resemble those of molten globules but they still retain substantial enthalpy and heat capacity of unfolding. Moreover, the similar heat capacities of the first intermediate and the native state suggests that the hydrophobic core of the intermediate would be highly native-like and that its formation would involve an increased disorder in localized portions of the structure rather than formation of a globally disordered state. The structure of the C-terminal of Hsp70 is destabilized as the pH separates from neutrality. The intermediates become populated under heat shock conditions at acidic and basic pHs. Denaturation by guanidine chloride also indicated that the protein undergoes a sequential unfolding process. The free energy change associated to the loss of secondary structure at 20 degrees C (pH 6.5) is 3.1 kcal.mol(-1) at high salt conditions. These values agree with the free energy changes estimated from differential scanning calorimetry for the transition between the second intermediate and the final denatured state.     

What causes hyperfluorescence: folding intermediates or conformationally flexible native states?

Hyperfluorescent intensity maxima during protein unfolding titrations are often taken as a sign for a thermodynamic folding intermediate. Here we explore another possibility: that hyperfluorescence could be the signature of a "pretransition" conformationally loosened native state. To model such native states, we study mutants of a fluorescent ubiquitin variant, placing cavities at various distances from the tryptophan fluorophore. We examine the correlation between protein flexibility and enhanced fluorescence intensity by using circular dichroism, fluorescence intensity unfolding titrations, fluorescence anisotropy measurements, and molecular dynamics. Based on experiment and simulation, we propose a simple model for hyperfluorescence in terms of static and dynamic conformational properties of the native state during unfolding. Apomyoglobin denaturant unfolding and phosphoglycerate kinase cold denaturation are discussed as examples. Our results do not preclude the existence of thermodynamic intermediates but do raise caution that by itself, hyperfluorescence during unfolding titrations is not conclusive proof of thermodynamic folding intermediates.     

Unfolding kinetics of glutathione reductase from cyanobacterium Spirulina maxima

The kinetics of the irreversible unfolding of glutathione reductase (NAD[P]H:GSSG oxidoreductase, EC 1.6.4.2.) from cyanobacterium Spirulina maxima was studied at pH 7.0 and room temperature. Denaturation was induced by guanidinium chloride and the changes in enzyme activity, aggregation state, and tertiary structure were monitored. No full reactivation of enzyme was obtained, even after very short incubation times in the presence of denaturant. Reactivation plots were complex, showing biphasic kinetics. A very fast early event in the denaturation pathway was the dissociation of tetrameric protein into reactivatable native-like dimers, followed by its conversion into a nonreactivatable intermediary, also dimeric. In the final step of the unfolding pathway the latter was dissociated into denatured monomers. Fluorescence measurements revealed that denaturation of S. maxima glutathione reductase is a slow process. Release of the prostethic group FAD was previous to the unfolding of the enzyme. No aggregated species were detected in the unfolding pathway, dismissing the aggregation of denatured polypeptide chains as the origin of irreversibility. Instead, the transition between the two dimeric intermediates is proposed as the cause of irreversibility in the denaturation of S. maxima glutathione reductase. A value of 106.6 +/- 3 kJ mol(-1) was obtained for the activation free energy of unfolding in the absence of denaturant. No evidence for the native monomer in the unfolding pathway was obtained which suggests that the dimeric nature of glutathione reductase is essential for the maintenance of the native subunit conformation.     

Conformational stability and thermodynamic characterization of the lipoic acid bearing domain of human mitochondrial branched chain alpha-ketoacid dehydrogenase

The lipoic acid bearing domain (hbLBD) of human mitochondrial branched chain alpha-ketoacid dehydrogenase (BCKD) plays important role of substrate channeling in oxidative decarboxylation of the branched chain alpha-ketoacids. Recently hbLBD has been found to follow two-step folding mechanism without detectable presence of stable or kinetic intermediates. The present study describes the conformational stability underlying the folding of this small beta-barrel domain. Thermal denaturation in presence of urea and isothermal urea denaturation titrations are used to evaluate various thermodynamic parameters defining the equilibrium unfolding. The linear extrapolation model successfully describes the two-step; native state <-->denatured state unfolding transition of hbLBD. The average temperature of maximum stability of hbLBD is estimated as 295.6 +/- 0.9 K. Cold denaturation of hbLBD is also predicted and discussed.     

Thermodynamics of the unfolding of the cold-shock protein from Thermotoga maritima.

Proteins from (hyper-)thermophiles are known to exhibit high intrinsic stabilities. Commonly, their thermodynamic characterization is impeded by irreversible side reactions of the thermal analysis or calorimetrical problems. Small single-domain proteins are suitable candidates to overcome these obstacles. Here, the thermodynamics of the thermal denaturation of the recombinant cold-shock protein (Csp) from the hyperthermophilic bacterium Thermotoga maritima (Tm) was studied by differential scanning calorimetry. The unfolding transition can be described over a broad pH range (3.5-8.5) by a reversible two-state process. Maximum stability (DeltaG (25 degrees C)=6.5 kcal/mol) was observed at pH 5-6 where Tm Csp unfolds with a melting temperature at 95 degrees C. The heat capacity difference between the native and the denatured states is 1.1(+/-0.1) kcal/(mol K). At pH 7, thermal denaturation occurs at 82 degrees C. The corresponding free energy profile has its maximum at 30 degrees C with DeltaGN-->U=4.8(+/-0.5) kcal/mol. At the optimal growth temperature of T. maritima (80 degrees C), Tm Csp in the absence of ligands is only marginally stable, with a free energy of stabilization not far beyond the thermal energy. With the known stabilizing effect of nucleic acids in mind, this suggests a highly dynamical interaction of Tm Csp with its target molecules.     

The unfolding pathway and conformational stability of potato carboxypeptidase inhibitor

The unfolding and denaturation curves of potato carboxypeptidase inhibitor (PCI) were investigated using the technique of disulfide scrambling. In the presence of denaturant and thiol initiator, the native PCI denatures by shuffling its native disulfide bonds and converts to form a mixture of scrambled PCI that consists of 9 out of a possible 14 isomers. The denaturation curve is determined by the fraction of native PCI converted to scrambled isomers under increasing concentrations of denaturant. The concentration of guanidine thiocyanate, guanidine hydrochloride, and urea required to denature 50% of the native PCI was found to be 0.7, 1.45, and 8 m, respectively. The PCI unfolding curve was constructed through the analysis of structures of scrambled isomers that were denatured under increasing concentrations of denaturant. These results reveal the existence of structurally defined unfolding intermediates and a progressive expansion of the polypeptide chain. The yield of the beads-form isomer (Cys(8)-Cys(12), Cys(18)-Cys(24), and Cys(27)-Cys(34)) as a fraction of total denatured PCI was shown to be directly proportional to the strength of the denaturing condition. Furthermore, the PCI sequence was unable to fold quantitatively into a single native structure. Under physiological conditions, the scrambled isomers of PCI that constitute about 4% of the protein were in equilibrium with native PCI.     

A theoretical DFT investigation of the lysozyme mechanism: computational evidence for a covalent intermediate pathway

To test the occurrence of local particularities during the unfolding of Ca2+-loaded goat alpha-lactalbumin (GLA) we replaced Trp60 and -118, either one or both, by Phe. In contrast with alternative studies, our recombinant alpha-lactalbumins are expressed in Pichia pastoris and do not contain the extra N-terminal methionine. The substitution of Trp60 leads to a reduction of the global stability. The effect of the Trp118Phe substitution on the conformation and stability of the mutant, however, is negligible. Comparison of the fluorescence spectra of these mutants makes clear that Trp60 and -118 are strongly quenched in the native state. They both contribute to the quenching of Trp26 and -104 emission. By the interplay of these quenching effects, the fluorescence intensity changes upon thermal unfolding of the mutants behave very differently. This is the reason for a discrepancy of the apparent transition temperatures derived from the shift of the emission maxima (Tm,Fl lambda) and those derived from DSC (Tm,DSC). However, the transition temperatures derived from fluorescence intensity (Tm,Fl int) and from DSC (Tm,DSC), respectively, are quite similar, and thus, no local rearrangements are observed upon heat-induced unfolding. At room temperature, the occurrence of specific local rearrangements upon GdnHCl-induced denaturation of the different mutants is deduced from the apparent free energies of their transition state obtained from stopped-flow fluorescence measurements. By phi-value analysis it appears that, while the surroundings of Trp118 are exposed in the kinetic transition state, the surroundings of Trp60 remain native.     

Urea-induced denaturation of human serum albumin labeled with acrylodan

We induced the denaturation of unlabeled human serum albumin (HSA) and of similar albumin labeled with acrylodan (6-acryloyl-2-dimethylamino naphthalene) with urea and studied the transition profiles using circular dichroism and fluorescence spectroscopy. The circular dichroism spectra for both albumin preparations resulted in the same curves, thus indicating that labeling with acrylodan does not perturb the conformation of HSA. Our results indicate that the denaturation of both albumin preparations takes place at a single, two-state transition with midpoint at about 6 M urea, due to the unfolding of its domain II. It is important to point out that even at 8 M urea, some residual structure remains in the HSA. Great changes in the fluorescence of the dye bound to the protein were observed by addition of solid guanidine hydrochloride to the protein labeled with acrylodan dissolved in 8 M urea, indicating that domain I of this protein was not denatured by urea.