Unfolding and refolding of a type kappa immunoglobulin light chain and its variable and constant fragments

By limited proteolysis of a type kappa immunoglobulin light chain (Oku) with clostripain, both the VL and CL fragments were obtained with a high yield. The unfolding and refolding by guanidine hydrochloride of light chain Oku and its VL and CL fragments were studied at pH 7.5 and 25 degrees C with circular dichroism and tryptophyl fluorescence. The concentration of guanidine hydrochloride at the midpoint of the unfolding curve was 1.2 M for the VL fragment and 0.9 M for the CL fragment. As in the case of the CL fragment of light chain Nag (type lambda) [Goto, Y., & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910], the unfolding and refolding kinetics of the CL fragment could be explained in principle on the basis of the three-species mechanism U1 in equilibrium U2 in equilibrium N, where N is native protein and U1 and U2 are the slow-folding and fast-folding species, respectively, of unfolded protein. The unfolding and refolding kinetics of the VL(Oku) fragment were described by a single exponential term. Double-jump experiments, however, showed that two forms of the unfolding molecule exist. The equilibrium and kinetics of unfolding of light chain Oku were explained by the sum of those of the VL and CL fragments. On the other hand, the refolding kinetics of light chain Oku were greatly different from the sum of those of the VL and CL fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ammonium sulfate on the unfolding and refolding of the variable and constant fragments of an immunoglobulin light chain

The equilibria and kinetics of unfolding and refolding by guanidine hydrochloride of the VL and CL fragments of a type kappa immunoglobulin light chain were studied in the presence of ammonium sulfate using circular dichroism and tryptophyl fluorescence at pH 7.5 and 25 degrees C. The unfolding equilibria of the VL and CL fragments were described in terms of the two-state transition. The midpoints of unfolding in the absence of ammonium sulfate were at 0.9 and 1.2 M guanidine hydrochloride for the CL and VL fragments respectively. The transition curves were shifted to higher concentrations of guanidine hydrochloride by 1.4 and 1.6 M for the CL and VL fragments, respectively, per mole of ammonium sulfate. Unfolding reactions of the VL and CL fragments in 3 M guanidine hydrochloride followed first-order kinetics, and the rate constants for the two proteins were both greatly decreased by the presence of ammonium sulfate. The refolding reaction of the CL fragment in 0.3 M guanidine hydrochloride consisted of two phases, and the rate constants were increased a little by the presence of ammonium sulfate. The refolding reaction of the VL fragment in 0.3 M guanidine hydrochloride followed first-order kinetics, and the rate was not affected by the presence of ammonium sulfate. These results showed that ammonium sulfate stabilizes the CL and VL fragments mainly by decreasing the unfolding rate.     

Global fluctuations of the immunoglobulin domains under physiological conditions

Hydrogen-exchange rates of the indole NH proton of a tryptophan residue, buried fully in the interior of each of the constant (CL) and variable (VL) fragments of a type-kappa-immunoglobulin light chain, were studied at various pH values and at 25 degrees C under 1H-nuclear magnetic resonance. The activation energies for the exchange reactions were determined also and compared with those for the unfolding reactions of these fragments induced by guanidine hydrochloride. The pH profiles of the exchange rates of the CL(kappa) and VL(kappa) fragments were very similar to that for a CL (lambda) fragment reported previously. It was found that the CL (kappa) and VL (kappa) fragments as well as the CL (lambda) fragment undergo a global unfolding transition with a conformation very similar to that of the fully unfolded state induced by guanidine hydrochloride even under physiological conditions.     

Unfolding and refolding of the constant fragment of the immunoglobulin light chain containing an intramolecular mercury bridge

The conformation of the constant fragment of the immunoglobulin light chain in which the intrachain disulfide bond is replaced by the bond S-Hg-S (CL-Hg fragment), was as compact as that of the intact CL fragment, but its stability to guanidine hydrochloride was lower than that of the intact CL fragment [Goto, Y. & Hamaguchi, K. (1986) Biochemistry in press]. The kinetics of reversible unfolding and refolding of the CL-Hg fragment by guanidine hydrochloride were studied and compared with those for the intact CL and reduced CL fragments [Goto, Y. & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910, 911-926]. The unfolding kinetics were explained on the basis of a three-species mechanism, U1----U2----F, where U1 and U2 are respectively slow-folding and fast-folding species of unfolded protein, and F is folded protein. However, an additional isomerization, though its contribution to the overall reaction process is small, had to be taken into account to explain the refolding kinetics. The kinetic properties of interconversion between U1 and U2 were similar to those for the intact CL and reduced CL fragments. This suggested that the same prolyl residue is involved in the isomerization reactions in the unfolded states of the intact CL, reduced CL, and CL-Hg fragments. The rate constant for the unfolding process, F to U2, was about 20 times greater than those for the intact CL and reduced CL fragments, while the rate constant for the refolding process, U2 to F, lay between the values for the intact CL and the reduced CL fragment. The free energy profiles of unfolding and refolding of the intact CL, reduced CL, and CL-Hg fragments were compared.     

Reduction of the buried intrachain disulfide bond of the constant fragment of the immunoglobulin light chain: global unfolding under physiological conditions

The constant (CL) fragment of the immunoglobulin light chain contains only one intrachain disulfide bond buried in the interior of the molecule. The kinetics of reduction with dithiothreitol of the disulfide bond were studied at various concentrations of guanidine hydrochloride at pH 8.0 and 25 degrees C. It was found that the disulfide bond is reduced even in the absence of guanidine hydrochloride. The results of the reduction kinetics were compared with those of the unfolding and refolding kinetics of the CL fragment previously reported [Goto, Y., & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910]. It was shown that the reduction of the disulfide bond proceeds through a species with a conformation very similar to that of the fully unfolded one and that the CL fragment undergoes global unfolding transition even in water.     

High stability of a ferredoxin from the hyperthermophilic archaeon A. ambivalens: involvement of electrostatic interactions and cofactors

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric seven-iron protein with a thermal midpoint (T(m)) of 122 degrees C (pH 7). To gain insight into the basis of its thermostability, we have characterized unfolding reactions induced chemically and thermally at various pHs. Thermal unfolding of this ferredoxin, in the presence of various guanidine hydrochloride (GuHCl) concentrations, yields a linear correlation between unfolding enthalpies (DeltaH[T(m)]) and T(m) from which an upper limit for the heat capacity of unfolding (DeltaC(P)) was determined to be 3.15 +/- 0.1 kJ/(mole * K). Only by the use of the stronger denaturant guanidine thiocyanate (GuSCN) is unfolding of A. ambivalens ferredoxin at pH 7 (20 degrees C) observed ([GuSCN](1/2) = 3.1 M; DeltaG(U)[H(2)O] = 79 +/- 8 kJ/mole). The protein is, however, less stable at low pH: At pH 2.5, T(m) is 64 +/- 1 degrees C, and GuHCl-induced unfolding shows a midpoint at 2.3 M (DeltaG(U)[H(2)O] = 20 +/- 1 kJ/mole). These results support that electrostatic interactions contribute significantly to the stability. Analysis of the three-dimensional molecular model of the protein shows that there are several possible ion pairs on the surface. In addition, ferredoxin incorporates two iron-sulfur clusters and a zinc ion that all coordinate deprotonated side chains. The zinc remains bound in the unfolded state whereas the iron-sulfur clusters transiently form linear three-iron species (in pH range 2.5 to 10), which are associated with the unfolded polypeptide, before their complete degradation.     

Release of pyridoxal 5'-phosphate upon unfolding of mitochondrial aspartate aminotransferase

Dimeric mitochondrial aspartate aminotransferase (mAAT) contains a molecule of pyridoxal 5'-phosphate (PLP) tightly attached to each of its two identical active sites. The presence of this natural reporter allows us to study separately local perturbations in the architecture of this critical region of the molecule during unfolding. Upon unfolding of the enzyme with guanidine hydrochloride (GdnHCl), the coenzyme is completely released from the active site. The transition midpoint for the dissociation of PLP is 1.4+/-0.02 M when determined by size-exclusion chromatography (SEC) and 1.6+/-0.02 M when the protein-bound PLP is estimated by electrospray mass spectrometry (ESI-MS). In both cases the transition midpoint is higher than that of inactivation (1.3+/-0.01 M). On the other hand, the midpoint of the unfolding transition obtained by monitoring changes in ellipticity at 356 nm, which reflects the asymmetric environment of the PLP cofactor at the active site, is 1.19+/-0.011 M guanidine. These results indicate that the unfolding of mAAT is a multi-step process which includes an intermediate containing bound PLP but lacking catalytic activity.     

Free energy changes in denaturation of ribonuclease A by mixed denaturants. Effects of combinations of guanidine hydrochloride and one of the denaturants lithium bromide, lithium chloride, and sodium bromide

The denaturation of ribonuclease A by guanidine hydrochloride, lithium bromide, and lithium chloride and by mixed denaturants consisting of guanidine hydrochloride and one of the denaturants lithium chloride, lithium bromide, and sodium bromide was followed by difference spectral measurements at pH 4.8 and 25 degrees C. Both components of mixed denaturant systems enhance each other's effect in unfolding the protein. The effect of lithium bromide on the midpoint of guanidine hydrochloride denaturation transition is approximately the sum of the effects of the constituent ions. For all the mixed denaturants tested, the dependence of the free energy change on denaturation is linear. The conformational free energy associated with the guanidine hydrochloride denaturation transition in water is 7.5 +/- 0.1 kcal mol-1, and it is unchanged in the presence of low concentrations of lithium bromide, lithium chloride, and sodium bromide which by themselves are not concentrated enough to unfold the protein. The conformational free energy associated with the lithium bromide denaturation transition in water is 11.7 +/- 0.3 kcal mol-1, and it is not affected by the presence of low concentrations of guanidine hydrochloride which by themselves do not disrupt the structure of native ribonuclease A.     

Effect of hydrostatic pressure on unfolding of alpha-lactalbumin: volumetric equivalence of the molten globule and unfolded state

The effect of pressure on the unfolding of bovine alpha-lactalbumin was investigated by ultraviolet absorption methods. The change of molar volume associated with unfolding, deltaV, was measured in the presence or absence of guanidine hydrochloride at pH 7. The deltaV was estimated to be -63 cm3/mol in the absence of a chemical denaturant. While in the presence of guanidine hydrochloride (GuHCl), it was found that deltaV was -66 cm3/mol at 25 degrees C and was independent of the concentration of GuHCl, despite the fact that the molten globule fraction in the total unfolding product decreased with the increase of GuHCl concentration. The results indicate that the volume of alpha-lactalbumin only changes at the transition from a native to a molten globule state, and almost no volume change has been found during the transition from a molten globule to the unfolded state.     

Conformation and disulfide bond formation of the constant fragment of an immunoglobulin light chain: effect of truncation at the C-terminal region

Constant fragments with different carboxyl terminals, CL(109-211), CL(109-207), and CL-(109-200), were prepared by limited carboxypeptidase P or Y proteolysis of the constant fragment, CL-(109-214), of a type lambda immunoglobulin light chain, and their conformations and stabilities, and formation of the disulfide bond from the reduced fragments, were studied. No change in conformation or stability was observed on removal of three residues from the C-terminal end. Removal of seven or more residues from the C-terminal end destabilized the CL fragment. The rate of disulfide bond formation from reduced CL(109-207) was about 7 times faster than that for CL(109-214). These findings suggest that elongation of the polypeptide chain at least beyond the 207th residue is necessary for folding of the CL fragment into a definite conformation.     

Domain unfolding and the stability of thermolysin in guanidine hydrochloride

Equilibrium and kinetic studies of the unfolding and autolysis of the two domain protein thermolysin in guanidine hydrochloride are described. Enzyme activity, circular dichroism, fluorescence, sedimentation, size exclusion chromatography, and viscosity measurements were used to monitor conformational transitions and characterize the native and denatured states. The observation of biphasic transitions for the unfolding of apothermolysin and the spectroscopic changes associated with each phase of the overall unfolding process suggest unfolding of the N-terminal domain at less than 1 M guanidine hydrochloride, followed by the unfolding of the C-terminal domain, with the transition midpoint at 3 M guanidine hydrochloride. The refolding of the C-terminal domain is reversible; however, refolding of the N-terminal domain could not be demonstrated owing to protein aggregation. A quantitative analysis of the two transitions suggest that the unfolding of the two structural domains of thermolysin is not completely independent. Attempts to measure the unfolding of holothermolysin were hampered by autolysis. However, it was possible to show that at least three calcium ions serve to stabilize thermolysin against autolysis or unfolding in guanidine hydrochloride. Similar stabilization was observed for thermolysin with a single terbium ion bound at calcium site S(1). This result is consistent with our earlier findings, which suggest that calcium bound at sites S(1)-S(2) are located at a critical point on the unfolding pathway of thermolysin and serve to act as an interdomain lock.     

Guanidine-induced unfolding of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus

The unfolding induced by guanidine hydrochloride of the small protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus has been investigated by means of circular dichroism and fluorescence measurements. At neutral pH and room temperature the midpoint of the transition occurred at 4M guanidine hydrochloride. Thermodynamic information was obtained by means of both the linear extrapolation model and the denaturant binding model, in the assumption of a two-state N<==>D transition. A comparison with thermodynamic data determined from the thermal unfolding of Sso7d indicated that the denaturant binding model has to be preferred. Finally, it is shown that Sso7d is the most stable against both temperature and guanidine hydrochloride among a set of globular proteins possessing a very similar 3D structure.     

The effect of carbohydrate on the structure and stability of erythropoietin.

Erythropoietin is a glycoprotein hormone that stimulates the maturation of late erythroid progenitor cells. It has three N-linked and one O-linked carbohydrates which play an important role in the biosynthesis and biological activities of the protein. To determine the role the carbohydrate might have in maintaining the conformational stability of the protein, the protein expressed in mammalian cells (fully glycosylated), the asialo mammalian-expressed protein, and the protein expressed in Escherichia coli (no carbohydrate) were compared for their stability to guanidine HCl, pH, and temperature. Circular dichroism was used to follow protein unfolding. Both the intact and asialo mammalian-expressed proteins unfolded with a cooperative transition in guanidine HCl, with a midpoint at 1.75 M guanidine HCl. The E. coli-expressed material unfolded with a midpoint of 1.2 M guanidine HCl, and a delta G of unfolding which was 1.4 kcal/mol less than that of the two glycosylated molecules. The E. coli-derived protein was also significantly less stable to pH-induced conformational changes, showing a cooperative transition in 35% glycerol with a midpoint at pH 4.4, while both the intact and asialo mammalian-expressed molecules had a transition midpoint of pH 3.75 in the absence of glycerol, and approximately pH 3 in the presence of 35% glycerol. The E. coli-expressed molecule unfolded and precipitated upon heating to 44 degrees C, while the asialo and intact mammalian-expressed proteins remained soluble, with a Tm of 56 degrees C. From these experiments, the carbohydrate appears to play a critical role in stabilizing the erythropoietin molecule to denaturing conditions, and this increased stability does not depend on the presence of sialic acid.     

Unfolding by temperature and guanidine hydrochloride of chicken pancreatic polypeptide

Thermal unfolding of chicken pancreatic polypeptide at two different concentrations was studied at various pH values. The thermal stability was higher at higher protein concentrations. The transition temperatures at two different protein concentrations changed with pH in parallel and decreased by about 30 degrees C on lowering pH from 5 to 2. The results on the thermal unfolding were analyzed by assuming that the dimerization constant is independent of pH, that the thermal unfolding occurs only after the pancreatic polypeptide dimers dissociated into the monomers, and that one ionizable group participates in the acid unfolding of the monomer. The free energy change for the unfolding of the pancreatic polypeptide monomer was estimated to be 1.4 kcal/mol. The unfolding of pancreatic polypeptide by guanidine hydrochloride at pH 6.0 and 25 degrees C was also studied. The stability to guanidine hydrochloride was higher at higher protein concentrations.     

High thermal and chemical stability of Thermus thermophilus seven-iron ferredoxin. Linear clusters form at high pH on polypeptide unfolding.

To probe the stability of the seven-iron ferredoxin from Thermus thermophilus (FdTt), we investigated its chemical and thermal denaturation processes in solution. As predicted from the crystal structure, FdTt is extremely resistant to perturbation. The guanidine hydrochloride-induced unfolding transition shows a midpoint at 6.5 m (pH 7, 20 degrees C), and the thermal midpoint is above boiling, at 114 degrees C. The stability of FdTt is much lower at acidic pH, suggesting that electrostatic interactions are important for the high stability at higher pH. On FdTt unfolding at alkaline pH, new absorption bands at 520 nm and 610 nm appear transiently, resulting from rearrangement of the cubic clusters into linear three-iron species. A range of iron-sulfur proteins has been found to accommodate these novel clusters in vitro, although no biological function has yet been assigned.     

Structure-based stability engineering of the mouse IgG1 Fab fragment by modifying constant domains

A semi-rational approach based on structural data was exploited in a search for CH1 and CL domains with improved intrinsic thermodynamic stabilities. Structural and amino acid level comparisons were carried out against known biophysically well-behaving and thermodynamically beneficial scFv and Fab fragments. A number of mutant Fab fragments were constructed by site-directed mutagenesis of regions in the CH1 and CL domains expected to be most sensitive under physical stress conditions. These mutations were located on three sites in the Fab constant domains; a mobile loop in the CH1 domain, residues surrounding the two largest solvated hydrophobic cavities located in the interface of the CH1 and CL domains and the hydrophobic core regions of both CH1 and CL. Expression levels of functional Fab fragments, denaturant-induced unfolding equilibria and circular dichroism spectroscopy were used to evaluate the relative stabilities of the wild-type and the mutant Fab fragments. The highest thermodynamic stability was reached through the mutation strategy, where the hydrophobicity and the packing density of the solvated hydrophobic cavity in the CH1/CL interface was increased by the replacement of the hydrophilic Thr178 in the CL domain by a more hydrophobic residue, valine or isoleucine. The midpoint of the transition curve from native to unfolded states of the protein, measured by fluorescence emission, occurred at concentrations of guanidine hydrochloride of 2.4 M and 2.6 M for the wild-type Fab and the most stable mutants, respectively. Our results illustrate that point mutations targeted to the CH1/CL interface were advantageous for the overall thermodynamic stability of the Fab fragment.     

Fluorescence and conformational stability studies of Staphylococcus nuclease and its mutants, including the less stable nuclease-concanavalin A hybrids

We report steady-state and time-resolved fluorescence studies with the single tryptophan protein, Staphylococcus aureus A, and several of its site-directed mutants. A couple of these mutants, nuclease-conA and nuclease-conA-S28G (which are hybrid proteins containing a six amino acid beta-turn substitute from concanavalin A), are found to have a much lower thermodynamic stability than the wild type. The thermal transition temperatures for nuclease-conA and S28G are 32.8 and 30.5 degrees C, which are about 20 degrees C lower than the Tm for wild-type nuclease A. These mutant proteins also are denatured by a much lower concentration of the denaturants urea and guanidine hydrochloride. We also show that an unfolding transition in the structure of the nuclease-conA hybrids can be induced by relatively low hydrostatic pressure (approximately 700 bar). The free energy for unfolding of nuclease-conA (and nuclease-conA-S28G) is found to be only 1.4 kcal/mol (and 1.2 kcal/mol) by thermal, urea, guanidine hydrochloride, and pressure unfolding. Time-resolved fluorescence intensity and anisotropy measurements with nuclease-conA-S28G show the temperature-, urea-, and pressure-perturbed states each to have a reduced average intensity decay time and to depolarize with a rotational correlation time of approximately 1.0 ns (as compared to a rotational correlation time of 11 ns for the native form of nuclease-conA-S28G at 20 degrees C).     

Equilibrium unfolding of class pi glutathione S-transferase

The equilibrium unfolding transition of class pi glutathione S-transferase, a homodimeric protein, from porcine lung was monitored by spectroscopic methods (fluorescence emission and ultraviolet absorption), and by enzyme activity changes. Solvent (guanidine hydrochloride and urea)-induced denaturation is well described by a two-state model involving significant populations of only the folded dimer and unfolded monomer. Neither a folded, active monomeric form nor stable unfolding intermediates were detected. The conformational stability, delta Gu (H2O), of the native dimer was estimated to be about 25.3 +/- 2 kcal/mol at 20 degrees C and pH6.5.     

Two-domain structure of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase.

NADH-cytochrome b₅ reductase is an amphiphilic protein consisting of a hydrophilic (FAD-containing) moiety and a hydrophobic (membrane-binding) segment and exists in aqueous media as an oligomeric aggregate. Circular dichroism studies have shown that denaturation of the reductase by guanidine hydrochloride in the presence of Emulgen 109P, a nonionic detergent, is a two-stage process as a function of the denaturant concentration. The first transition occurs at about 1 m guanidine hydrochloride and the second one at much higher concentrations. The guanidine hydrochloride concentration causing the second-stage unfolding depends on the concentration of Emulgen 109P. A hydrophilic fragment of the reductase lacking the hydrophobic segment undergoes one-stage denaturation at about 1 m guandine hydrochloride regardless of the presence and absence of Emulgen 109P. Both the reductase as well as the hydrophilic fragment lose their NADH-ferricyanide reductase activity and FAD also at about 1 m guanidine hydrochloride in the presence of the detergent. These findings suggest that the first-stage denaturation of the reductase represents the unfolding of the hydrophilic moiety and the second one that of the hydrophobic segment. Gel chromatography experiments have suggested that in the presence of Emulgen 109P the reductase exists as a mixed micelle with the detergent and this aggregation state persists even after the first-stage denaturation (unfolding of the hydrophilic moiety). The dissociation of the mixed micelle seems to take place concomitant with the second-stage denaturation. It is concluded that the two moieties of the reductase molecule, though linked to each other covalently, exist as independent domains undergoing unfolding separately at least in the presence of Emulgen 109P. This structural feature of the reductase is similar to that of cytochrome b₅ reported by us. The reductase is, therefore, another example of amphiphilic membrane proteins having two structurally independent domains in the molecule.