ATP's impact on the conformation and holoenzyme formation in relation to the regulation of brain glutamate decarboxylase

To investigate ATP as a potential factor in the regulation of brain glutamate decarboxylase (GAD), the impact of ATP on the enzyme conformation and holoenzyme formation was investigated. ATP at 100 microM quenches fluorescence emission intensity of the holoenzyme of GAD (holoGAD) by 18% after a correction for the inner filter effect and enhances fluorescence steady-state polarization from 0.158 to 0. 183 when excited at 280 or 295 nm. These findings suggest that ATP moderately changes the microenvironment of one or more tryptophan or tyrosine residues in holoGAD and alters these residues from a more mobile state to a less mobile one. A moderate ATP-induced conformational change in holoGAD is also supported by the observations that ATP increases the thermal denaturation temperature of holoGAD by 2 degrees C, as derived from temperature-dependent fluorescence spectra, and decreases the alpha-helical content of holoGAD by 8-10%, as determined by circular dichroism. Moreover, ATP does not affect the keto-enol tautomerization of holoGAD and has little or no direct effect on its activity, implying that the ATP interacting domain in holoGAD is not at the active site. Kinetics studies, as demonstrated by stopped-flow fluorescence and UV/visible spectroscopy, demonstrate that formation of holoGAD involves two steps: a fast reaction forming an apoGAD-cofactor intermediate complex, followed by a slow reaction involving the conformational change in the intermediate complex. ATP reduces the rate constant of the fast step to one-third and decreases the rate of the slow step and the intermediate complex formation constant to 60% of their original values. The present data suggest that ATP may regulate the interconversion between apoGAD and holoGAD by interacting with apoGAD rather than holoGAD. By slowing down the rate of intermediate complex formation, ATP reduces the amount of holoGAD formed.     

States of tryptophyl residues and stability of recombinant human matrix metalloproteinase 7 (matrilysin) as examined by fluorescence

States of tryptophyl residues and stability of human matrilysin were studied. The activation energy for the thermal inactivation of matrilysin was determined to be 237 kJ/mol, and 50% of the activity was lost upon incubation at 69 degrees C for 10 min. The activity was increased by adding NaCl, and was doubled with 3 M NaCl. Denaturation of matrilysin by guanidine hydrochloride (GdnHCl) and urea was monitored by fluorescence change of tryptophyl residues. Half of the change was observed at 2.2-2.7 M GdnHCl, whereas no change was observed even with 8 M urea. Half of the inactivation was induced at 0.8 M GndHCl and at 2 M urea. The presence of an inactive intermediate with the same fluorescence spectrum as the native enzyme was suggested in the denaturation. Matrilysin contains four tryptophyls, and their states were examined by fluorescence-quenching with iodide and cesium ions and acrylamide. No tryptophyls in the native enzyme were accessible to I(-) and Cs(+), and 2.4 residues were accessible to acrylamide. Based on the crystallographic study, Trp154 is water-accessible, but it should be in a crevice not to contact with I(-) and Cs(+). All tryptophyls in the GdnHCl-denatured enzyme were exposed to the quenchers, while a considerable part was inaccessible in the urea-denatured one.     

Physical-chemical properties of actin in different structural states. New ideas about its folding-unfolding pathways

Results of actin folding-unfolding pathways examination and characterization of intermediate and misfolded states are summarized. Properties of microenvironments and peculiarities of location of tryptophan residues in protein are analysed in detail. This allowed to conclude that the main contribution to the bulk fluorescence of native protein is made by internal tryptophan residues Trp 340 and Trp 356, localized in hydrophobic regions, while tryptophan residues Trp 79 and Trp 86 are quenched. It has been shown that inactivated actin, previously regarded as an intermediate state between native and completely unfolded state of protein is in reality a misfolded aggregated state. The properties of actin in this state were characterized in detail. In particular, it is shown that inactivated actin is a monodisperse associate consisting of 15 monomer unit. Two earlier unknown intermediate states, which precede completely unfolding of protein macromolecule and formation of inactivated actin, were visualized. A new scheme of folding-unfolding processes was proposed. It is shown that the reason of anomalous effects, which are recorded for actin in solutions with small concentrations of GdnHCl, is a specific interaction of actin with a denaturant.     

Comparison of inactivation and unfolding of methanol dehydrogenase during denaturation in guanidine hydrochloride and urea

The activity and the conformational changes of methanol dehydrogenase (MDH), a quinoprotein containing pyrrolo-quinoline quinone as its prosthetic group, have been studied during denaturation in guanidine hydrochloride (GdnHCl) and urea. The unfolding of MDH was followed using the steady-state and time resolved fluorescence methods. Increasing the denaturant concentration in the denatured system significantly enhanced the inactivation and unfolding of MDH. The enzyme was completely inactivated at 1 M GdnHCl or 6 M urea. The fluorescence emission maximum of the native enzyme was at 332 nm. With increasing denaturant concentrations, the fluorescence emission maximum red-shifted in magnitude to a maximum value (355 nm) at 5 M GdnHCl or 8 M urea. Comparison of inactivation and conformational changes during denaturation showed that in general accord with the suggestion made previously by Tsou, the active sites of MDH are situated in a region more flexible than the molecule as a whole.     

Steady-state and time-resolved fluorescence studies of the intestinal fatty acid binding protein

The intestinal fatty acid binding protein contains two tryptophan residues (Trp6 and Trp82) both of which have been shown by X-ray and NMR methods to be buried in hydrophobic clusters. By using a combination of steady-state and time-resolved fluorescence experiments, we have deconvoluted the lifetime weighted contribution of each of the tryptophans to the steady-state fluorescence quantum yield. While Trp82 has been implicated in an intermediate that appears at relatively high denaturant concentrations, the variation of the lifetime weighted contribution of Trp6 with urea or guanidium hydrochloride shows formation of an intermediate state at low concentrations of the denaturant before the actual unfolding starts. Trp82 did not show similar behavior. Fluorescence quenching experiments by acrylamide show that while Trp6 in the native protein is less solvent-exposed, its accessibility is increased significantly at low urea concentration indicating that the early intermediate state is partially unfolded. Time-resolved anisotropy experiments indicate that the volume of the partially unfolded intermediates is larger than the native protein and lead to the speculation that the last step of the protein folding might be the removal of solvent molecules from the protein.     

Kinetic differences between the isoforms of glutamate decarboxylase: implications for the regulation of GABA synthesis

Glutamate decarboxylase (GAD) exists as two isoforms, GAD65 and GAD67. GAD activity is regulated by a cycle of activation and inactivation determined by the binding and release of its co-factor, pyridoxal 5'-phosphate. Holoenzyme (GAD with bound co-factor) decarboxylates glutamate to form GABA, but it also catalyzes a slower transamination reaction that produces inactive apoGAD (without bound co-factor). Apoenzyme can reassociate with pyridoxal phosphate to form holoGAD, thus completing the cycle. Within cells, GAD65 is largely apoenzyme (approximately 93%) while GAD67 is mainly holoenzyme (approximately 72%). We found striking kinetic differences between the GAD isoforms that appear to account for this difference in co-factor saturation. The glutamate dependent conversion of holoGAD65 to apoGAD was about 15 times faster than that of holoGAD67 at saturating glutamate. Aspartate and GABA also converted holoGAD65 to apoGAD at higher rates than they did holoGAD67. Nucleoside triphosphates (such as ATP) are known to affect the activation reactions of the cycle. ATP slowed the activation of GAD65 and markedly reduced its steady-state activity, but had little affect on the activation of GAD67 or its steady-state activity. Inorganic phosphate opposed the effect of ATP; it increased the rate of apoGAD65 activation but had little effect on apoGAD67 activation. We conclude that the apo-/holoenzyme cycle of inactivation and reactivation is more important in regulating the activity of GAD65 than of GAD67.     

Cofactor interactions and the regulation of glutamate decarboxylase activity

More than 50% of glutamate decarboxylase (GAD) in brain is present as apoenzyme. Recent work has opened the possibility that apoGAD can be studied in brain by labeling with radioactive cofactor. Such studies would be aided by a compound that inhibits specific binding. One possibility is 4-deoxy-pyridoxine 5-phosphate, a close structural analog of the cofactor pyridoxal 5-phosphate. The effects of deoxypyridoxine-P on the cyclic series of reactions that interconverts apo- and holoGAD was investigated and found to be consistent with simple competitive inhibition of the activation of apoGAD by pyridoxal-P. As expected from the cycle GAD was inactivated when incubated with glutamate and deoxypyridoxine-P even though cofactor was present, but no inactivation was observed with deoxypyridoxine-P in the absence of glutamate. Deoxypyridoxine-P also stabilized apoGAD against heat denaturation. These effects were quantitatively accounted for by a kinetic model of the apo-holoGAD cycle. Deoxypyridoxine-P inhibited the labeling by [³²P]pyridoxal-P of GAD isolated from rat brain. Hippocampal extracts were labeled with [³²P]pyridoxal-P and analyzed by SDS-polyacrylamide gel electrophoresis. Remarkably few bands were strongly labeled. The major labeled band (at 63 kDa) corresponded to one of the forms of GAD. Other strongly-labeled bands were observed at 65 kDa (corresponding to the higher molecular weight form of GAD) and at 69–72 kDa. Labeling of the 63- and 65-kDa bands was inhibited by deoxypyridoxine-P, but the 69–72 kDa bands were unaffected, suggesting that the latter were non-specifically labeled. The results suggest that the 63-kDa form of GAD makes up the majority of apoGAD in hippocampus.Special issue dedicated to Dr. Eugene Roberts.     

Comparison of temperature effect on the guanidine hydrochloride and urea denaturations of lysozyme

Guanidine hydrochloride (GdnHCl) and urea denaturations of lysozyme have been observed at various temperatures by measuring changes in fluorescence. Both transitions appear to be two state, with GdnHCl almost twice as effecitve a denaturant as urea for this protein. By plotting the denaturant concentrations at midpoint of the transition vs. the experimental temperature, it can be demonstrated that urea-denatured lysozyme does not obtain the degree of unfolding found in lysozyme denatured by GdnHCl.     

Conformational change of the dimeric DsbC molecule induced by GdnHCl. A study by intrinsic fluorescence

Unfolding-refolding of Escherichia coli DsbC, a homodimeric molecule, induced by GdnHCl was studied by intrinsic fluorescence. Interpretation of experimental fluorescence data was done together with the analysis of protein 3D structure. It is shown that although Cys 141 is the next neighbor of the single tryptophan residue (Trp 140), the sulfur atoms of the disulfide bond Cys 141-Cys 163 are far apart from the indole ring and cannot quench its fluorescence, while the potential quenchers are Met 136 and His 170. It was revealed that though each subunit of DsbC contains eight tyrosine residues, only three tyrosine residues (Tyr 171, Tyr 38, and Tyr 52) contribute to the bulk fluorescence of the molecule. The character of intrinsic fluorescence intensity changes induced by GdnHCl (equilibrium and kinetic data) and its parametric representation, the existence of an isosbestic point of fluorescence spectra at different GdnHCl concentrations, allowed suggesting a one-step character of DsbC denaturation and its reversibility.     

Determination of the accessibility and localization of tryptophan residues in the influenza virus hemagglutinin molecule

The accessibility and localization of tryptophane residues in the influenza viral hemagglutinin molecule have been determined by measuring specific quenching of tryptophane fluorescence by neutral (acrylamide), anionic (I-) and cationic (Cs+) quenchers. It has been shown that acrylamide quenches 64% of tryptophane fluorescence in H3-hemagglutinin whereas I- and Cs+ quench only 34%. The tryptophanyl residues have been assumed to be located in the hemagglutinin molecule both in the cationic and anionic environments. 64% of tryptophanyls have been shown to be located on the surface of the protein globule.     

Acrylamide quenching of apo- and holo-alpha-lactalbumin in guanidine hydrochloride

We have examined the fluorescence properties and acrylamide quenching of calcium-loaded (holo) and calcium-depleted (apo) alpha-lactalbumin (alpha-LA) as a function of guanidine hydrochloride (GDN/HCl) concentration. The spectral changes accompanying increasing GDN/HCl are consistent with protein unfolding and a release of internal fluorescence quenching, which occurs among the three tryptophan residues located in the region of the so-called "tertiary fold." Values for the intrinsic fluorescence emission, the wavelength maximum of the emission, the Stern/Volmer dynamic quench constant, and the static quench constant are consistent with a significant stabilization effect by calcium against protein unfolding. The dynamic quench constant of apo-alpha-LA increases fourfold to its maximum, in the transition from the native state to protein in 1.5 M GDN/HCl. The dynamic quench constant for holo-alpha-LA remains unchanged until exposed to 2.5 M GDN/HCl, but increases by threefold with addition denaturant to 4 M GDN/HCl. The static quench constant of the apo-protein in the native solvent, approximately 0.2 M(-1), declines to zero in 1 M denaturant, where the molten globule folding intermediate is most populated. A more protracted denaturant-dependent decline in the static quench constant occurs for the holo-protein. Sharp increase in the static quenching occurs for apo-alpha-LA and holo-alpha-LA above 1.5 M GDN/HCl and 3.5 M GDN/HCl, respectively. The results for apo-alpha-LA in dilute GDN/HCl suggest that acrylamide can penetrate the protein molecule (as judged by the collision quenching) but is unable to form a stable complex within the quenching domain for the tryptophans (as judged by the absence of the static quench constant). It seems reasonable to suggest that the protein folding intermediate which occurs in dilute denaturant represents a structure in which the tryptophans are, on average, more accessible to collisional quenching but sufficiently compact to prevent formation of a stable, dark equilibrium complex with acrylamide.     

The pressure effect on the structure and functions of protein disulfide isomerase

Studying on the pressure effects of the structure and functions of the multidomain protein, protein disulfide isomerase (PDI), the intrinsic Trp fluorescence spectra of PDI were measured under high pressure. PDI has 5 Trp residues and the two of all Trp residues are located at the neighborhood of the active site (WCGHC) for isomerase activity. On the basis of the red shift of center of spectral mass (CSM) of the intrinsic Trp fluorescence and the decrease in its fluorescence intensity, the changes in tertiary structure of PDI were observed above 100 MPa. These structural changes were completed at 400 MPa. The CSM of 400 MPa denatured PDI was comparable to that of 6.0 M GdnHCl denatured one. All of the Trp residues included in PDI are completely exposed to aqueous medium at 400 MPa. However, there is the significant difference between the pressure and GdnHCl-denatured PDI. The Trp fluorescence intensity was decreased with increasing pressure, but increased with the increase of the GdnHCl concentration. It is implied that the pressure-denatured state of PDI might remain compact not to be extensively unfolded. In the point of view about the reversibility of pressure-treated PDI, the tertiary structure was completely recovered after released to ambient pressure. The disulfide reduction and chaperone activity of 400 MPa-treated PDI were also recovered to be comparable to those of native one. Despite of a multidomain protein, the excellence in both structural and functional recovery of pressure-denatured PDI is quite remarkable. These unique properties of PDI against high pressure provide the insights into understanding the pressure-induced denaturation of PDI.     

Unfolding and refolding of human glyoxalase II and its single-tryptophan mutants.

Here the structure of human glyoxalase II has been investigated by studying unfolding at equilibrium and refolding. Human glyoxalase II contains two tryptophan residues situated at the N-terminal (Trp57) and C-terminal (Trp199) regions of the molecule. Trp57 is a non-conserved residue located within a "zinc binding motif" (T/SHXHX57DH) which is strictly conserved in all known glyoxalase II sequences as well as in metal-dependent beta-lactamase and arylsulfatase. Site-directed mutagenesis has been used to construct single-tryptophan mutants in order to characterize better the guanidine-induced unfolding intermediates. The denaturation at equilibrium of wild-type glyoxalase II, as followed by activity, intrinsic fluorescence and CD, is multiphasic, suggesting that different regions of varying structural stability characterize the native structure of glyoxalase II. At intermediate denaturant concentration (1.2 M guanidine) a molten globule state is attained. The reactivation of the denatured wild-type enzyme occurs only in the presence of Zn(II) ions. The results show that Zn(II) is essential for the maintenance of the native structure of glyoxalase II and that its binding to the apoenzyme occurs during an essential step of refolding. The comparison of unfolding fluorescence transitions of single-trypthophan mutants with that of wild-type enzyme indicates that the strictly conserved "zinc binding motif" is located in a flexible region of the active site in which Zn(II) participates in catalysis.     

Ca(II)- and Tb(III)-induced stabilization and refolding of anticoagulation factor I from the venom of Agkistrodon acutus

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF I, holo-ACF I, and Tb(3+)-reconstituted ACF I in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism. Metal ions were found to increase the structural stability of ACF I against GdnHCl and thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF I and Tb(3+)-ACF I is a two-state process with no detectable intermediate state(s), whereas the GdnHCl-induced unfolding/refolding of holo-ACF I in the presence of 1 mM Ca(2+) follows a three-step transition, with intermediate state a (Ia) and intermediate state b (Ib). Ca(2+) ions play an important role in the stabilization of the Ia and Ib states. The decalcification of holo-ACF I shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, whereas the reconstitution of apo-ACF I with Tb(3+) ions shifts the initial zone of denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.0 M GdnHCl) at which refolding from the fully denatured state of apo-ACF I to the Ib state of holo-ACF I or to the native state of Tb(3+)-ACF I can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF I, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ions-induced refolding provide evidence that the compact Tb(3+)-binding region forms first, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.     

Metal ion-induced stabilization and refolding of anticoagulation factor II from the venom of Agkistrodon acutus

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF II, holo-ACF II, and Tb(3+)-reconstituted ACF II in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism (CD). Metal ions were found to increase the structural stability of ACF II against GdnHCl and irreversible thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF II and Tb(3+)-ACF II is a two-state process with no detectable intermediate state, while the GdnHCl-induced unfolding/refolding of holo-ACF II in the presence of 1 mM Ca(2+) follows a three-state transition with an intermediate state. Ca(2+) ions play an important role in the stabilization of both native and I states of holo-ACF II. The decalcification of holo-ACF II shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, while the reconstitution of apo-ACF II with Tb(3+) ions shifts the initial zone of the denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.1 M GdnHCl) at which refolding from the fully denatured state of apo-ACF II to the I state of holo-ACF II or to the native state of Tb(3+)-ACF II can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF II, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ion-induced refolding provide evidence for the fact that the first phase of Tb(3+)-induced refolding should involve the formation of the compact metal-binding site regions, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.     

The structure of toxic protein, the mistletoe lectin, at different pH: the study using intrinsic fluorescence method

The effects of pH on the conformation of mistletoe lectin I and its isolated A- and B-subunits has been investigated by using the methods of intrinsic fluorescence. By the denaturating action of guanidine hydrochloride and the influence of the quenchers (I-, Cs+, acrylamide) the structural stability of the native protein and its isolated subunits was estimated. Treatment of the protein with the denaturant and quenchers revealed its different structure at pH 7.0 and 4.0. At pH 4.0 tryptophan residues become more accessible to quenchers, positive charge of the surrounding area increases and the protein becomes more stable to the action of denaturant. The structure of the isolated A- and B-chains of mistletoe lectin I differs considerably from that of the whole protein: a) its stability to the action of guanidine hydrochloride is lower; b) it depends on the ionic strength of the solvent; c) it is characterized by increased accessibility of tryptophan residues to quenchers (for B-chain). Differences between the conformations of the isolated chains at pH 7.0 and 4.0 are marked more strongly; moreover, at pH 4.5 the B-chain undergoes structural transition. The possible relationship between structural peculiarities of mistletoe lectin I and the mechanism of its transmembrane transfer is discussed.     

Probing folding and fluorescence quenching in human gammaD crystallin Greek key domains using triple tryptophan mutant proteins

Human gammaD crystallin (HgammaD-Crys), a major component of the human eye lens, is a 173-residue, primarily beta-sheet protein, associated with juvenile and mature-onset cataracts. HgammaD-Crys has four tryptophans, with two in each of the homologous Greek key domains, which are conserved throughout the gamma-crystallin family. HgammaD-Crys exhibits native-state fluorescence quenching, despite the absence of ligands or cofactors. The tryptophan absorption and fluorescence quenching may influence the lens response to ultraviolet light or the protection of the retina from ambient ultraviolet damage. To provide fluorescence reporters for each quadrant of the protein, triple mutants, each containing three tryptophan-to-phenylalanine substitutions and one native tryptophan, have been constructed and expressed. Trp 42-only and Trp 130-only exhibited fluorescence quenching between the native and denatured states typical of globular proteins, whereas Trp 68-only and Trp 156-only retained the anomalous quenching pattern of wild-type HgammaD-Crys. The three-dimensional structure of HgammaD-Crys shows Tyr/Tyr/His aromatic cages surrounding Trp 68 and Trp 156 that may be the source of the native-state quenching. During equilibrium refolding/unfolding at 37 degrees C, the tryptophan fluorescence signals indicated that domain I (W42-only and W68-only) unfolded at lower concentrations of GdnHCl than domain II (W130-only and W156-only). Kinetic analysis of both the unfolding and refolding of the triple-mutant tryptophan proteins identified an intermediate along the HgammaD-Crys folding pathway with domain I unfolded and domain II intact. This species is a candidate for the partially folded intermediate in the in vitro aggregation pathway of HgammaD-Crys.     

Unfolding rates of barstar determined in native and low denaturant conditions indicate the presence of intermediates

The folding and unfolding rates of the small protein, barstar, have been monitored using stopped-flow measurements of intrinsic tryptophan fluorescence at 25 degrees C, pH 8.5, and have been compared over a wide range of urea and guanidine hydrochloride (GdnHCl) concentrations. When the logarithms of the rates of folding from urea and from GdnHCl unfolded forms are extrapolated linearly with denaturant concentration, the same rate is obtained for folding in zero denaturant. Similar linear extrapolations of rates of unfolding in urea and GdnHCl yield, however, different unfolding rates in zero denaturant, indicating that such linear extrapolations are not valid. It has been difficult, for any protein, to determine unfolding rates under nativelike conditions in direct kinetic experiments. Using a novel strategy of coupling the reactivity of a buried cysteine residue with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to the unfolding reaction of barstar, the global unfolding and refolding rates have now been determined in low denaturant concentrations. The logarithms of unfolding rates obtained at low urea and GdnHCl concentrations show a markedly nonlinear dependence on denaturant concentration and converge to the same unfolding rate in the absence of denaturant. It is shown that the native protein can sample the fully unfolded conformation even in the absence of denaturant. The observed nonlinear dependences of the logarithms of the refolding and unfolding rates observed for both denaturants are shown to be due to the presence of (un)folding intermediates and not due to movements in the position of the transition state with a change in denaturant concentration.     

Conformational changes of disulfide isomerase C induced by guanidine hydrochloride

Unfolding--refolding of Escherichia coli disulfide isomerase C (DsbC) induced by GdnHCl was studied by intrinsic fluorescence. Interpretation of experimental fluorescence data was done together with the analysis of protein 3D structure. It is shown that although Cys 141 is the next neighbour of a single tryptophan residue Trp 140, sulfur atoms of the disulfide bond Cys 141--Cys 163 are far apart from the indole ring and cannot quench its fluorescence, while the potential quenchers are Met 136 and His 170. It has been revealed that, though each subunit of DsbC contains eight tyrosine residues, only three tyrosine residues (Tyr 171, Tyr 38 and Tyr 52) contribute to the bulk fluorescence of the molecule. The character of intrinsic fluorescence intensity changes induced by GdnHCl (equilibrium and kinetic data), the character of parametric dependencies between fluorescence intensity recorded at 320 and 365 nm, and the existence of an isosbestic point of protein fluorescence spectra in solutions with different GdnHCl concentrations, allowed suggesting a one-step character of DsbC denaturation. The reversibility of this process is also shown.     

Comparison of Guanidine Hydrochloride (GdnHCl) and Urea Denaturation on Inactivation and Unfolding of Human Placental Cystatin (HPC)

The activity and conformational change of human placental cystatin (HPC), a low molecular weight thiol proteinase inhibitor (12,500) has been investigated in presence of guanidine hydrochloride (GdnHCl) and urea. The denaturation of HPC was followed by activity measurements, fluorescence spectroscopy and Circular Dichroism (CD) studies. Increasing the denaturant concentration significantly enhanced the inactivation and unfolding of HPC. The enzyme was 50% inactivated at 1.5 M GdnHCl or 3 M urea. Up to 1.5 M GdnHCl concentration there was quenching of fluorescence intensity compared to native form however at 2 M concentration intensity increased and emission maxima had 5 nm red shift with complete unfolding in 4–6 M range. The mid point of transition was in the region of 1.5–2 M. In case of urea denaturation, the fluorescence intensity increased gradually with increase in the concentration of denaturant. The protein unfolded completely in 6–8 M concentration of urea with a mid-point of transition at 3 M. CD spectroscopy shows that the ellipticity of HPC has increased compared to that of native up to 1.5 M GdnHCl and then there is gradual decrease in ellipticity from 2 to 5 M concentration. At 6 M GdnHCl the protein had random coil conformation. For urea the ellipticity decreases with increase in concentration showing a sigmoidal shaped transition curve with little change up to 1 M urea. The protein greatly loses its structure at 6 M urea and at 8 M it is a random coil. The urea induced denaturation follows two-state rule in which Native→Denatured state transition occurs in a single step whereas in case of GdnHCl, intermediates or non-native states are observed at lower concentrations of denaturant. These intermediate states are possibly due to stabilizing properties of guanidine cation (Gdn⁺) at lower concentrations, whereas at higher concentrations it acts as a classical denaturant.