Multiple unfolded states of alcohol dehydrogenase I from Kluyveromyces lactis by guanidinium chloride

Inactivation, dissociation, and unfolding of tetrameric alcohol dehydrogenase I from Kluyveromyces lactis (KlADH I) were investigated using guanidinium chloride (GdmCl) as denaturant. Protein transitions were monitored by enzyme activity, intrinsic fluorescence and gel filtration chromatography. At low denaturant concentrations (less than 0.3 M), reversible transformation of enzyme into tetrameric inactive form occurs. At denaturant concentrations between 0.3 and 0.5 M, the enzyme progressively dissociates into structured monomers through an irreversible reaction. At higher denaturant concentrations, the monomers unfold completely. Refolding studies indicate that a total reactivation occurs only with the enzyme denatured between 0 and 0.3 M GdmCl concentrations. The enzyme denatured at GdmCl concentrations higher than 0.3 M refolds only partially. All together, our results indicate that unfolding of the KlADH I is a multistep process, i.e., inactivation of the structured tetramer, dissociation into partially structured monomers, followed by complete unfolding.     

Multiple unfolded states of glutathione transferase bbGSTP1-1 by guanidinium chloride.

Inactivation, dissociation, and unfolding of the homodimeric glutathione transferase (bbGSTP1-1) from Bufo bufo embryos were investigated at equilibrium, using guanidinium chloride (GdmCl) as denaturant. Protein transitions were monitored by enzyme activity, intrinsic fluorescence, far UV circular dichroism, glutaraldehyde cross-linking, and gel-filtration chromatography. At low denaturant concentrations (less than 0.5 M), reversible inactivation of the enzyme occurs. At denaturant concentrations between 0.5 and 1.5 M the enzyme progressively dissociates into structured monomers. At higher denaturant concentrations the monomers unfold completely. Refolding studies indicate that a total reactivation occurs only by starting from the enzyme denatured at concentrations below 0.5 M. The enzyme denatured at GdmCl concentrations higher than 0.5 M only partially refolds. Globally our results indicate that unfolding of the amphibian bbGSTP1-1 is a multistep process, i.e., inactivation of the structured dimer, dissociation into partially structured monomers, followed by complete unfolding.     

Authenticity and reconstitution of immobilized enzymes: characterization and denaturation/renaturation of glucoamylase II.

Glucoamylase II (GA II) immobilized to Eupergit C and CIZ as a porous and nonporous matrix shows enzymatic characteristics indistinguishable from those of the free enzyme, except for reduced specific activity. Since this decrease is equally observed for both matrices, it has to be ascribed to nonproductive fixation of the enzyme or steric hindrance rather that perturbations caused by "inner diffusion" effects. Authenticity refers to the optimum pH for catalytic activity, Michaelis constants for starch and maltoheptaose, as well as identical stability toward temperature, pH, and guanidinium chloride (GdmCl). On the basis of these data, the two-state mechanism observed for the equilibrium transitions of the free enzyme may be assumed to hold also for the immobilized enzyme. Renaturation after preceding denaturation in 6.4 and 7 M GdmCl leads to widely differing yields depending on the conditions. Shifting the denaturant concentration stepwise back to nondenaturing GdmCl concentrations leads to a broad range of "hysteresis" accompanied by aggregation. Rapid dilution of the free and immobilized enzymes at pH greater than 6 and sufficiently low protein concentration leads to reactivation yields of 80 and 45%, respectively. For the free enzyme, reconstitution at lower pH is determined by the kinetic competition of folding and aggregation. In the case of the immobilized enzyme, "entangling" of the matrix with the unfolded polypeptide chain competes with renaturation.     

Characterization of the denatured states distribution of neocarzinostatin by small-angle neutron scattering and differential scanning calorimetry

The denatured states of a small globular protein, apo-neocarzinostatin (NCS), have been characterized using several techniques. Structural properties were investigated by optical spectroscopy techniques and small-angle neutron scattering (SANS), as a function of guanidinium chloride (GdmCl) concentration. SANS experiments show that in heavy water, the protein keeps its native size at GdmCl concentrations below 2.5 M. A sharp transition occurs at about 3.6 M GdmCl, and NCS behaves like an excluded volume chain above 5 M. The same behavior is observed in deuterated buffer by fluorescence and circular dichroism measurements. For the H(2)O buffer, the transition occurs with lower concentration of denaturant, the shift being about 0.6 M. 8-Anilino-1-naphthalenesulfonate (ANS) was used as a hydrophobic fluorescent probe for studying the early stages of protein unfolding. Protein denaturation modifies the fluorescence intensity of ANS, a maximum of intensity being detected close to 2 M GdmCl in hydrogenated buffer, which shows the existence of at least one intermediate state populated at the beginning of the unfolding pathway. Differential scanning calorimetry (DSC) was used to obtain thermodynamic values for NCS denaturation. The melting curves recorded between 20 and 90 degrees C in the presence of various GdmCl concentrations (0-3 M) cannot be explained by a simple two-state model. Altogether, the data presented in this paper suggest that before unfolding the protein explores a distribution of states which is centered around compact states at denaturant concentrations below 2 M in H(2)O, and then shifts to less structured states by increasing denaturant concentrations.     

Modeling and simulation of fed-batch protein refolding process

The simplified kinetic model that assumes competition between first-order folding and third-order aggregation was used to model the fed-batch refolding of denatured-reduced lysozyme. It was found that the model was able to describe the process at limited concentration ranges, i.e., 1-2 and 5-7 mg mL(-)(1), respectively, at extensive guanidinium chloride (GdmCl) concentrations and controlled concentrations of oxidizing and reducing agents. The folding or aggregation rate constant was different at the two protein concentration ranges and strongly dependent on the denaturant concentration. As a result, both rate constants at the two concentration ranges were expressed as functions of GdmCl concentration. The rate constants determined by fed-batch experiments could be employed for the prediction of the fed-batch process but were not able to be extended to a batch refolding by direct dilution. Computer simulations show that the denaturant concentration and fed-batch flow rate are important factors influencing the refolding yield. Prolonged fed-batch time is beneficial to keep the transient intermediate concentration at a low level and to increase the yield of correctly folded protein. This is of importance when the denaturant concentration in refolding buffer solution is low. Thus, at a low denaturant concentration, fed-batch time should be sufficiently long, whereas at an appropriately high GdmCl concentration, a short fed-batch time or a high feed rate of the denatured protein is effective to give a high refolding yield.     

Dissecting contributions to the denaturant sensitivities of proteins

Understanding the molecular basis for protein denaturation by urea and guanidinium chloride (GdmCl) should accommodate the observation that, on a molar basis, GdmCl is generally 2-2.5-fold more effective as a protein denaturant than urea. Previous studies [Smith, J. S., and Scholtz, J. M. (1996) Biochemistry 35, 7292-7297] have suggested that the effects of GdmCl on the stability of alanine-based helical peptides can be separated into denaturant and salt effects, since adding equimolar NaCl to urea enhanced urea-induced unfolding to an extent that was close to that of Gdm. We reinvestigated this observation using an alanine-based helical peptide (alahel) that lacks side chain electrostatic contributions to stability, and compared the relative denaturant sensitivities of this peptide with that of tryptophan zipper peptides (trpzip) whose native conformations are stabilized largely by cross-strand indole ring interactions. In contrast to the observations of Smith and Scholtz, GdmCl was only slightly more powerful as a denaturant of alahel than urea in salt-free buffer (the denaturant m value m(GdmCl)/m(urea) ratio = 1.4), and the denaturation of alahel by urea exhibited only a small dependence on NaCl or KCl. The trpzip peptides were much more sensitive to GdmCl than to urea (m(GdmCl)/m(urea) = 3.5-4). These observations indicate that the m(GdmCl)/m(urea) ratio of 2-2.5 for proteins results from a combination of effects on the multiple contributions to protein stability, for which GdmCl may be only slightly more effective than urea (e.g., hydrogen bonds) or considerably more effective than urea (e.g., indole-indole interactions).     

Molecular basis for the effect of urea and guanidinium chloride on the dynamics of unfolded polypeptide chains

Chemical denaturants are frequently used to unfold proteins and to characterize mechanisms and transition states of protein folding reactions. The molecular basis of the effect of urea and guanidinium chloride (GdmCl) on polypeptide chains is still not well understood. Models for denaturant--protein interaction include both direct binding and indirect changes in solvent properties. Here we report studies on the effect of urea and GdmCl on the rate constants (k(c)) of end-to-end diffusion in unstructured poly(glycine-serine) chains of different length. Urea and GdmCl both lead to a linear decrease of lnk(c) with denaturant concentration, as observed for the rate constants for protein folding. This suggests that the effect of denaturants on chain dynamics significantly contributes to the denaturant-dependence of folding rate constants for small proteins. We show that this linear dependency is the result of two additive non-linear effects, namely increased solvent viscosity and denaturant binding. The contribution from denaturant binding can be quantitatively described by Schellman's weak binding model with binding constants (K) of 0.62(+/-0.01)M(-1) for GdmCl and 0.26(+/-0.01)M(-1) for urea. In our model peptides the number of binding sites and the effect of a bound denaturant molecule on chain dynamics is identical for urea and GdmCl. The results further identify the polypeptide backbone as the major denaturant binding site and give an upper limit of a few nanoseconds for residence times of denaturant molecules on the polypeptide chain.     

A unique and differential effect of denaturants on cofactor mediated activation of Plasmodium falciparum beta-ketoacyl-ACP reductase

The urea and guanidinium chloride (GdmCl) induced unfolding of FabG, a beta-ketoacyl-ACP reductase of Plasmodium falciparum, was examined in detail using intrinsic fluorescence of FabG, UV-circular dichroism (CD), spectrophotometric enzyme activity measurements, glutaraldehyde cross-linking, and size exclusion chromatography. The equilibrium unfolding of FabG by urea is a multistep process as compared with a two-state process by GdmCl. FabG is fully unfolded at 6.0M urea and 4.0M GdmCl. Approximately 90% of the enzyme activity could be recovered on dialyzing the denaturants, showing that denaturation by both urea and GdmCl is reversible. We found two states in the reversible unfolding process of FabG in presence of NADPH; one is an activity-enhanced state and the other, an inactive state in case of equilibrium unfolding with urea. On the contrary, in presence of NADPH, there is no stabilization of FabG in case of equilibrium unfolding with GdmCl. We hypothesize that the hydrogen-bonding network may be reorganized by the denaturant in the activity-enhanced state formed in presence of 1.0M urea, by interrupting the association between dimer-dimer interface and help in accommodating the larger substrate in the substrate binding tunnel thus, increasing the activity. Furthermore, binding of the active site organizer, NADPH leads to compaction of the FabG in presence of urea, as evident by acrylamide quenching. We have shown here for the first time, the detailed inactivation kinetics of FabG, which have not been evaluated in the past from any of the FabG family of enzymes from any of the other sources. These findings provide impetus for exploring the influences of ligands on the structure-activity relationship of Plasmodium beta-ketoacyl-ACP reductase.     

Evidence for the existence of an unfolding intermediate state for aminoacylase during denaturation in guanidine solutions

The equilibrium unfolding of pig kidney aminoacylase in guanidinium chloride (GdmCl) solutions was studied by following the fluorescence and circular dichroism (CD). At low concentrations of GdmCl, less than 1.0 M, the fluorescence intensity decreased with a slight red shift of the emission maximum (from 335 to 340 nm). An unfolding intermediate was observed in low concentrations of denaturant (between 1.2 and 1.6 M GdmCl). This intermediate was characterized by a decreased fluorescence emission intensity, a red-shifted emission maximum, and increased binding of the fluorescence probe 1-anilino-8-naphthalenesulfonate. No significant changes of the secondary structure were indicated by CD measurement. This conformation state is similar to a molten globule state which may exist in the pathway of protein folding. Further changes in the fluorescence properties occurred at higher concentrations of GdmCl, more than 1.6 M, with a decrease in emission intensity and a significant red shift of the emission maximum from 340 to 354 nm. In this stage, the secondary structure was completely broken. A study of apo-enzyme (Zn2+-free enzyme) produced similar results. However, comparison of the changes of the fluorescence emission spectra of native (Holo-) enzyme with Zn2+-free (Apo-) enzyme at low GdmCl concentrations showed that the structure of the Holo-enzyme was more stable than that of the Apo-enzyme.     

Biphasic denaturation of human placental alkaline phosphatase in guanidinium chloride

Human placental alkaline phosphatase is a membrane-anchored dimeric protein. Unfolding of the enzyme by guanidinium chloride (GdmCl) caused a decrease of the fluorescence intensity and a large red-shifting of the protein fluorescence maximum wavelength from 332 to 346 nm. The fluorescence changes were completely reversible upon dilution. GdmCl induced a clear biphasic fluorescence spectrum change, suggesting that a three-state unfolding mechanism with an intermediate state was involved in the denaturation process. The half unfolding GdmCl concentrations, [GdmCl]_0.5, corresponding to the two phases were 1.45 M and 2.50 M, respectively. NaCl did not cause the same effect as GdmCl, indicating that the GdmCl-induced biphasic denaturation is not a salt effect. The decrease in fluorescence intensity was monophasic, corresponding to the first phase of the denaturation process with [GdmCl]_0.5 = 1.37 M and reached a minimum at 1.5 M GdmCl, where the enzyme remained completely active. The enzymatic activity lost started at 2.0 M GdmCl and was monophasic but coincided with the second-phase denaturation with [GdmCl]_0.5 = 2.46 M. Inorganic phosphate provides substantial protection of the enzyme against GdmCl inactivation. Determining the molecular weight by sucrose-density gradient ultracentrifugation revealed that the enzyme gradually dissociates in both phases. Complete dissociation occurred at [GdmCl] > 3 M. The dissociated monomers reassociated to dimers after dilution of the GdmCl concentration. Refolding kinetics for the first-phase denaturation is first-order but not second-order. The biphasic phenomenon thereby was a mixed dissociation-denaturation process. A completely folded monomer never existed during the GdmCl denaturation. The biphasic denaturation curve thereby clearly demonstrates an enzymatically fully active intermediate state, which could represent an active-site structure intact and other structure domains partially melted intermediate state. Proteins 33:49–61, 1998. © 1998 Wiley-Liss, Inc.     

The hemoglobin of the aquatic snail, Planorbella duryi (Wetherby)

1. The hemoglobin of the pond snail, Planorbella duryi has a molecular weight of 1.64 x 10(6) to 1.77 x 10(6) as determined by light-scattering at 630 nm and a sedimentation coefficient of 36 S. 2. The analysis of the circular dichroism spectrum obtained in the 190-250 nm region suggests a high degree of helical folding of the polypeptide chains of P. duryi hemoglobin analogous to human hemoglobin and myoglobin, with estimates of alpha-helical folding of about 60-65%, 0-5% beta-structure, and the remaining portion of the chains in unordered form. 3. The dissociated subunits in 6.0 M GdmCl, in the absence and in the presence of reducing reagent (0.1 M dithiothreitol), have a molecular weight of 3.73 +/- 0.23 x 10(5) and 1.93 +/- 0.04 x 10(5), suggesting a di-decameric assembly of the parent hemoglobin organized in the form of five dimers held together by disulfide-linkages. 4. The native hemoglobin is strongly resistant to both pH dissociation and dissociation by urea and such salts as NaCl and NaClO4. Dissociation and denaturation could only be effected in concentrated GdmCl solutions. 5. The influence of the various dissociating agents on the quaternary structure suggest ionic stabilization of the decameric assembly, which is stabilized by salt bridges between the subunits.     

Folding and association of an extremely stable dimeric protein from Sulfolobus islandicus

ORF56 is a plasmid-encoded protein from Sulfolobus islandicus, which probably controls the copy number of the pRN1 plasmid by binding to its own promotor. The protein showed an extremely high stability in denaturant, heat, and pH-induced unfolding transitions, which can be well described by a two-state reaction between native dimers and unfolded monomers. The homodimeric character of native ORF56 was confirmed by analytical ultracentrifugation. Far-UV circular dichroism and fluorescence spectroscopy gave superimposable denaturant-induced unfolding transitions and the midpoints of both heat as well as denaturant-induced unfolding depend on the protein concentration supporting the two-state model. This model was confirmed by GdmSCN-induced unfolding monitored by heteronuclear 2D NMR spectroscopy. Chemical denaturation was accomplished by GdmCl and GdmSCN, revealing a Gibbs free energy of stabilization of -85.1 kJ/mol at 25 degrees C. Thermal unfolding was possible only above 1 M GdmCl, which shifted the melting temperature (t(m)) below the boiling point of water. Linear extrapolation of t(m) to 0 M GdmCl yielded a t(m) of 107.5 degrees C (5 microM monomer concentration). Additionally, ORF56 remains natively structured over a remarkable pH range from pH 2 to pH 12. Folding kinetics were followed by far-UV CD and fluorescence after either stopped-flow or manual mixing. All kinetic traces showed only a single phase and the two probes revealed coincident folding rates (k(f), k(u)), indicating the absence of intermediates. Apparent first-order refolding rates depend linearly on the protein concentration, whereas the unfolding rates do not. Both lnk(f) and lnk(u) depend linearly on the GdmCl concentration. Together, folding and association of homodimeric ORF56 are concurrent events. In the absence of denaturant ORF56 refolds fast (7.0 x 10(7)M(-1)s(-1)) and unfolds extremely slowly (5.7 year(-1)). Therefore, high stability is coupled to a slow unfolding rate, which is often observed for proteins of extremophilic organisms.     

Low concentrations of guanidinium chloride expose apolar surfaces and cause differential perturbation in catalytic intermediates of rhodanese

The conformations of sulfur-free and sulfur-containing rhodanese were followed with and without the detergent lauryl maltoside after guanidinium chloride (GdmCl) addition to 5 M to study the apparent irreversibility of denaturation. Without lauryl maltoside, sulfur-containing rhodanese denatured in a transition giving, at approximately 2.3 M GdmCl, 50% of the total denaturation induced change observed by activity, CD, or intrinsic fluorescence. Sulfur-free rhodanese gave more complex behavior by intrinsic fluorescence and CD. CD showed loss of secondary structure in a broad, complex, and apparently biphasic transition extending from 0.5 to 3 M GdmCl. The interpretation of the transition was complicated by time-dependent aggregation due to noncovalent interactions. Results with the apolar fluorescence probe 2-anilinonaphthalene-8-sulfonic acid, implicated apolar exposure in aggregation. Sulfhydryl reactivity indicated that low GdmCl concentrations induced intermediates affecting the active site conformation. Lauryl maltoside prevented aggregation with no effect on activity or any conformational parameter of native enzyme. Transitions induced by GdmCl were still observed and consistent with several phases. Even in lauryl maltoside, an increase in apolar exposure was detected by 2-anilinonaphthalene-8-sulfonic acid, and by protein adsorption to octyl-Sepharose well below the major unfolding transitions. These results are interpreted with a model in which apolar interdomain interactions are disrupted, thereby increasing active site accessibility, before the intradomain interactions.     

The unfolding intermediate state of calf intestinal alkaline phosphatase during denaturation in guanidine solutions

The equilibrium unfolding of calf intestinal alkaline phosphatase in guanidinium chloride (GdmCl) solutions was studied by following the fluorescence and ultraviolet difference spectra. At low concentrations of GdmCl (< 1.6 M), the fluorescence intensity decreased with a slight red shift of the emission maximum from 332 nm to 344 nm. An unfolding intermediate state was observed at a broad concentration range of GdmCl as a denaturant (between 1.6 and 2.6 M). This intermediate was characterized by increased fluorescence emission intensity, ultraviolet difference absorption at 236 nm and 260 nm, as well as increased binding to the protein and red shift of the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid.     

Denaturation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by guanidine hydrochloride; identification of inactive, partially unfolded, dimeric intermediates.

The denaturation of the dimeric enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by guanidine hydrochloride has been studied using enzymatic activity, intrinsic fluorescence, circular dichroism, and light scattering measurements. Equilibrium experiments at 25 degrees C revealed that between 0.9 and 1.2 M denaturant the enzyme underwent a conformational change, exposing tryptophan residues to solvent, with some loss of secondary structure and a complete loss of enzymatic activity but without dimer dissociation to subunits. This inactive, partially unfolded, dimeric intermediate was susceptible to slow aggregation, perhaps due to exposure of 'sticky' hydrophobic stretches of the polypeptide chain. A second equilibrium transition, reflecting extensive unfolding and dimer dissociation, occurred only at denaturant concentrations above 1.4 M. Kinetics experiments demonstrated that in the denaturant concentration range of 1.7-1.9 M the fluorescence change occurred in two distinct steps. The first step involved a large, very rapid drop in fluorescence whose rate was strongly dependent on the denaturant concentration. This was followed by a small, relatively slow rise in the emission intensity, the rate of which was independent of denaturant concentration. Enzymatic activity was lost with a denaturant-concentration-dependent rate, which was approx. 3-times slower than the rate of the first step in fluorescence change. A denaturation mechanism incorporating several unfolding intermediates and which accounts for all the above results is presented and discussed. While the fully unfolded enzyme regained up to 55% of its original activity upon dilution of denaturant to a concentration that would be expected to support native enzyme, denaturation intermediates were able to reactivate only minimally and in fact were found to aggregate and precipitate out of solution.     

Enzyme activation by denaturants in organic solvent systems with a low water content.

The effect of urea and guanidine hydrochloride (GdmCl) on the activity of heart lactate dehydrogenase, glycerol-3-phosphate dehydrogenase, hexokinase, inorganic pyrophosphatase, and glyceraldehyde-3-phosphate dehydrogenase was studied in low-water systems. Most of the experiments were made in a system formed with toluene, phospholipids, Triton X-100, and water in a range that varied over 1.0-6.5% (by vol.) [Garza-Ramos, G., Darszon, A., Tuena de Gómez-Puyou, M. & Gómez-Puyou, A. (1990) Biochemistry 29, 751-757]. In such conditions at saturating substrate concentrations, the activity of the enzymes was more than 10 times lower than in all-water media. However the activity of the first four aforementioned enzymes was increased between 4 and 20 times by the denaturants. The most marked activating effect was found with lactate dehydrogenase; with 3.8% (by vol.) water maximal activation was observed with 1.5 M GdmCl (about 20-fold); 4 M urea activated, but to a lower extent. Activation by guanidine thiocyanate was lower than with GdmCl. The activating and inactivating effects of GdmCl on lactate dehydrogenase depended on the amount of water; as the amount of water was increased from 2.0% to 6.0% (by vol.), activation and inactivation took place with progressively lower GdmCl concentrations. When activity was measured as a function of the volume of 1.5 M GdmCl solution, a bell-shaped activation curve was observed. In a low-water system formed with n-octane, hexanol, cetyltrimethylammonium bromide and 3.0% water, a similar activation of lactate dehydrogenase by GdmCl and urea was observed. The water solubility diagrams were modified by GdmCl and urea, and this could reflect on enzyme activity. However, from a comparison of denaturant concentrations on the activity of the enzymes studied, it would seem that, independently of their effect on the characteristics of the low-water systems, denaturants bring about activation through their known mechanism of action on the protein. It is suggested that the effect of denaturants is due to the release of constraints in enzyme catalysis imposed by a low-water environment.     

Anion shielding of electrostatic repulsions in transthyretin modulates stability and amyloidosis: insight into the chaotrope unfolding dichotomy

The balance between stabilizing forces and the localized electrostatic repulsions destabilizing the transthyretin (TTR) tetramer is tunable via anion shielding. The two symmetrical anion interaction sites in TTR are comprised of residues Lys15 and Lys15' from opposing subunits on the periphery of the two thyroxine binding sites. These epsilon-ammonium groups repel one another and destabilize the tetramer, unless an appropriate anion is present, which stabilizes the tetramer. Chaotrope denaturation of TTR exhibits unusual behavior in that urea appears to be a stronger denaturant than GdmCl (guanidinium chloride), even though GdmCl is typically twice as powerful as a denaturant. The shift in the midpoint of the urea denaturation curve to higher concentrations as well as the increase in the mole fraction of tetramer that is highly resistant to denaturation with increasing KCl concentration provides strong evidence that anion shielding stabilizes the TTR tetramer. A consequence of tetramer stabilization is folding hysteresis, because the high GdmCl concentrations required to denature the anion-stabilized tetramer do not allow refolding of the unfolded monomers. The formation of amyloid fibrils by TTR requires that its normal tetrameric structure dissociate to alternatively folded monomers, a process mediated by acidification (pH 5-4). This process is inhibited by Cl(-) ions in a concentration-dependent fashion. Chloride ion may not be the relevant physiological TTR stability modulator, but it is the main focus of these studies explaining the hysteresis observed in the denaturation and refolding studies with GdmCl.     

Effects of denaturants on amide proton exchange rates: a test for structure in protein fragments and folding intermediates

A method for detecting structure in marginally stable forms of a protein is described. The principle is to measure amide proton exchange rates in the absence and presence of varying concentrations of a denaturant. Unfolding of structure by the denaturant is reflected by an acceleration of amide proton exchange rates, after correction for the effects of the denaturant on the intrinsic rate of exchange. This exchange-rate test for structure makes no assumptions about the rate of exchange in the unfolded state. The effects of 0-8 M urea and 0-6 M guanidinium chloride (GdmCl) on acid- and base-catalyzed exchange from model compounds have been calibrated. GdmCl does not appear to be well-suited for use in the exchange-rate test; model compound studies show that the effects of GdmCl on intrinsic exchange rates are complicated. In contrast, the effects of urea are a more uniform function of denaturant concentration. Urea increases acid-catalyzed, and decreases base-catalyzed, rates in model compounds. The exchange-rate test is used here to study structure formation in the S-protein (residues 21-124 of ribonuclease A). In conditions where an equilibrium folding intermediate of S-protein (I3) is known to be populated (pH 1.7, 0 degree C), the exchange-rate test for structure is positive. At higher temperatures (greater than 32 degrees C) I3 is unfolded, but circular dichroism data suggest that residual structure remains [Labhardt, A. M. (1982) J. Mol. Biol. 157, 357-371].(ABSTRACT TRUNCATED AT 250 WORDS)     

Aggregation as the basis for complex behaviour of cutinase in different denaturants

We have previously described the complexity of the folding of the lipolytic enzyme cutinase from F. solani pisi in guanidinium chloride. Here we extend the refolding analysis by refolding from the pH-denatured state and analyze the folding behaviour in the presence of the weaker denaturant urea and the stronger denaturant guanidinium thiocyanate. In urea there is excellent consistency between equilibrium and kinetic data, and the intermediate accumulating at low denaturant concentrations is off-pathway. However, in GdmCl, refolding rates, and consequently the stability of the native state, vary significantly depending on whether refolding takes place from the pH- or GdmCl-denatured state, possibly due to transient formation of aggregates during folding from the GdmCl-denatured state. In GdmSCN, stability is reduced by several kcal/mol with significant aggregation in the unfolding transition region. The basis for the large variation in folding behaviour may be the denaturants' differential ability to support formation of exposed hydrophobic regions and consequent changes in aggregative properties during refolding.     

Rapid subunit exchange in dimeric lipoprotein lipase and properties of the inactive monomer

Lipoprotein lipase (LPL), a key enzyme in the metabolism of triglyceride-rich plasma lipoproteins, is a homodimer. Dissociation to monomers leads to loss of activity. Evidence that LPL dimers rapidly exchange subunits was demonstrated by fluorescence resonance energy transfer between lipase subunits labeled with Oregon Green and tetrametylrhodamine, respectively, and also by formation of heterodimers composed of radiolabeled and biotinylated lipase subunits captured on streptavidine-agarose. Compartmental modeling of the inactivation kinetics confirmed that rapid subunit exchange must occur. Studies of activity loss indicated the existence of a monomer that can form catalytically active dimers, but this intermediate state has not been possible to isolate and remains hypothetical. Differences in solution properties and conformation between the stable but catalytically inactive monomeric form of LPL and the active dimers were studied by static light scattering, intrinsic fluorescence, and probing with 4,4'-dianilino-1,1'-binaphtyl-5,5'-disulfonic acid and acrylamide. The catalytically inactive monomer appeared to have a more flexible and exposed structure than the dimers and to be more prone to aggregation. By limited proteolysis the conformational changes accompanying dissociation of the dimers to inactive monomers were localized mainly to the central part of the subunit, probably corresponding to the region for subunit interaction.