Thermodynamic investigations of proteins. II. Calorimetric study of lysozyme denaturation by guanidine hydrochloride.

The thermodynamic parameters of the denaturation of lysozyme are determined at various temperatures (25-60 degrees C) by isothermal calorimetric titrations with guanidine hydrochloride (GuHCl) and by scanning calorimetry in the presence of GuHCl. An approach for the determination of the enthalpy of preferential binding of GuHCl is proposed. It has been shown from GuHCl denaturation experiments that the net enthalpies of denaturation and the denaturational change in the heat capacity of protein can be obtained if preferential binding is taken into consideration. These results are nearly the same as in the case of thermal denaturation in the absence of denaturants. It is concluded that the states of both heat- and GuHCl-denatured lysozyme are thermodynamically indistinguishable.     

Major differences in stability and dimerization properties of two chimeric mutants of human stefins

Stefins A and B are cysteine proteinase inhibitors that have considerable sequence similarity but marked differences in their stability and folding properties. Two chimeric proteins were designed to shed light on these differences. The chimeric mutants have been expressed in Escherichia coli and have been isolated. The first, A37B, consists of 37 residues of stefin A, comprising the N-terminal and the alpha-helix, joined to 61 residues of stefin B; the second, A61B, consists of 61 N-terminal residues of stefin A, followed by 37 residues of stefin B. Spectroscopic properties of the chimeric proteins (absorption, CD, and NMR spectra), together with activity measurements, have confirmed that both have well-defined tertiary structure and are active as cysteine proteinase inhibitors. Characterization consisted of GuHCl denaturation, ANS binding as a function of pH, and monitoring of dimerization under partially denaturing conditions. The c(m) values are 1.3 M GuHCl for A61B as compared with 2.7 M GuHCl for stefin A, and 2.1 M GuHCl for A37B as compared with 1.4 M GuHCl for stefin B (all at pH 7.5, 25 degrees C). However (G degrees (N-U) is lower for both chimeric proteins (18 +/- 3 kJ/mol) than for the parent stefins (28 +/- 3 kJ/mol). In pH denaturation, unlike stefin B, neither chimeric mutant unfolds to I(N) below pH 5.4. At pH 3, where stefin B forms a molten globule and stefin A is native, both A37B and A61B show increased ANS fluorescence and aggregate visibly. Dimers at pre-denaturation conditions are observed in all the proteins under study, but they remain "trapped" only in stefin A.     

Synchrotron SAXS studies on the structural stability of Carcinus aestuarii hemocyanin in solution

The effect of GuHCl and of NaCl on the structural properties of the hemocyanin (Hc) from Carcinus aestuarii has been studied by small angle x-ray scattering (SAXS) using synchrotron radiation. SAXS data collected as a function of perturbant concentration have been used to analyze conformational states of hexameric holo and apoHc as well as the holo and apoforms of the monomeric subunit CaeSS2. In the case of the holoprotein in GuHCl, two concentration domains were identified: at lower concentration, the perturbant induces aggregation of Hc molecules, whereas at higher concentration the aggregates dissociate with concomitant denaturation of the protein. In contrast, with apoHc the denaturation occurs at rather low GuHCl, pointing to an important effect of the active site bound copper for the stabilization of Hc tertiary structure. The effects of NaCl are similar to those of GuHCl as far as CaeSS2 is concerned, namely oligomerization precedes denaturation, whereas in the case of the hexameric form no aggregation occurs. To improve data analysis, on the basis of the current models for Hc monomers and oligomers, the fraction of each aggregation state and/or unfolded protein has been determined by fitting experimental SAXS curves with form factors calculated from Monte Carlo methods. In addition, a global analysis has been carried out on the basis of a thermodynamic model involving an equilibrium between a monomer in a nativelike and denatured form as well as a class of equilibria among the monomer and other aggregates.     

Comparative study of GuHCl denaturation of globular proteins. I. Spectroscopic and chromatographic analysis of the denaturation curves of ribonuclease A,cytochrome c,and pepsinogen

Novel devices for the spectroscopic and chromatographic analysis of the denaturation curves of the protein are described. A multidimensional spectroscopic measuring system makes it possible to carry out simultaneous and continuous acquisition of a set of data of different spectroscopic dimensions at several wavelengths in the course of increasing or decreasing denaturational perturbation. GuHCl-gradient chromatography can provide information about the progressive change of the protein volume in the course of increasing GuHCl concentration. Thus, denaturation curves with a high data-point density can be obtained. The data-storing function by a magnetic disk memory provides enough precision for a rigorous investigation of the correlation among the curves that probe different aspects of denaturation. The GuHCl denaturation of RNase A, cytochrome c, and pepsinogen are studied to demonstrate the high performance of these devices. Three types of transitions are found in these three proteins and the multiphasic nature of the transitions is clearly detected in the last two proteins.     

Reversible denaturation of the gene V protein of bacteriophage f1

The guanidine hydrochloride (GuHCl)-induced denaturation of the gene V protein of bacteriophage f1 has been studied, using the chemical reactivity of a cysteine residue that is buried in the folded protein and the circular dichroism (CD) at 211 and 229 nm as measures of the fraction of polypeptide chains in the folded form. It is found that this dimeric protein unfolds in a single cooperative transition from a folded dimer to two unfolded monomers. A folded, monomeric form of the gene V protein was not detected at equilibrium. The kinetics of unfolding of the gene V protein in 3 M GuHCl and the refolding in 2 M GuHCl are also consistent with a transition between a folded dimer and two unfolded monomers. The GuHCl concentration dependence of the rates of folding and unfolding suggests that the transition state for folding is near the folded conformation.     

Structural characteristics of extra-membrane domains and guanidine hydrochloride-induced denaturation of photosystem 2 core antenna complexes CP43 and CP47

The structural characteristics of the extra-membrane domains and guanidine hydrochloride-induced denaturation of photosystem 2 (PS2) core antenna complexes CP43 and CP47 were investigated using fluorescence emission and circular dichroism (CD) spectra. The extra-membrane domains of CP43 and CP47 possessed a certain degree of secondary and tertiary structure and not a complete random coil conformation. The tertiary structure and the chlorophyll (Chl) a microenvironment of CP47 were more sensitive to guanidine hydrochloride (GuHCl) than that of CP43. Changes in energy transfer from β-carotene to Chl a corresponded well to changes in the tertiary structure while their correlation with changes in the secondary structure was rather poor. Unlike most of water-soluble proteins, both CP43 and CP47 are partly resistant to denaturation induced by guanidine hydrochloride (GuHCl); the denaturation of CP43 or CP47 is not a two-state process. Those features most probably reflect their character as intrinsic membrane proteins.     

Kinetic study of denaturation and subsequent reduction of disulfide bonds of lysozyme by the rapid ultrasonic absorption measurement

A New technique for the rapid measurement of ultrasonic absorption with a sampling interval of 5 msec has been developed and applied to the kinetic study of denaturation and subsequant redution of hen egg-white lysozyme. The lysozyme is denatured by guanidine hydrochloride (GuHCl) orLiBr, and afetr denaturation by GuHCl, its disulfide bonds are reduced by dithiothreitol (DTT). The ultrasonic adsorption coefficient at 9 MHz increases with denaturation but decreases with reduction. The rate constant of denaturation by GuHCl obtained from the rime variance of ultasonic agrees well with that from uv absorption and optical rotation. The time variance if absorption after GuHCl and Dtt have been simultaneously added exhibits two rate constants. Analysis of the constants as functions of regeant concentrations indicates that the intermediates state between native and reduced states is not necessarily the completely denatured state but depends on the concentartions of GuHCl and DTT.     

Solvent denaturation and stabilization of globular proteins

Statistical thermodynamic theory has recently been developed to account for the stabilities of globular proteins. Here we extend that work to predict the effects of solvents on protein stability. Folding is assumed to be driven by solvophobic interactions and opposed by conformational entropy. The solvent dependence of the solvophobic interactions is taken from transfer experiments of Nozaki and Tanford on amino acids into aqueous solutions of urea or guanidine hydrochloride (GuHCl). On the basis of the assumption of two pathways involving collapse and formation of a core, the theory predicts that increasing denaturant should lead to a two-state denaturation transition (i.e., there is a stable state along each path separated by a free energy barrier). The denaturation midpoint is predicted to occur at higher concentrations of urea than of GuHCl. At neutral pH, the radius of the solvent-denatured state should be much smaller than for a random-flight chain and increase with either denaturant concentration or number of polar residues in the chain. A question of interest is whether free energies of folding should depend linearly on denaturant, as is often assumed. The free energy is predicted to be linear for urea but to have some small curvature for GuHCl. Predicted slopes and exposed areas of the unfolded states are found to be in generally good agreement with experiments. We also discuss stabilizing solvents and compare thermal with solvent denaturation.     

The guanidine hydrochloride-induced denaturation of CP43 and CP47 studied by terahertz time-domain spectroscopy

Terahertz time-domain spectroscopy (THz-TDS) is a new technique in studying the conformational state of a molecule in recent years. In this work, we reported the first use of THz-TDS to examine the denaturation of two photosynthesis membrane proteins: CP43 and CP47. THz-TDS was proven to be useful in discriminating the different conformational states of given proteins with similar structure and in monitoring the denaturation process of proteins. Upon treatment with guanidine hydrochloride (GuHCl), a 1.8 THz peak appeared for CP47 and free chlorophyll a (Chl a). This peak was deemed to originate from the interaction between Chl a and GuHCl molecules. The Chl a molecules in CP47 interacted with GuHCl more easily than those in CP43. Supported by the National Natural Science Foundation of China (Grant No. 39890390)     

The guanidine hydrochloride-induced denaturation of CP43 and CP47 studied by terahertz time-domain spectroscopy

Terahertz time-domain spectroscopy (THz-TDS) is a new technique in studying the conformational state of a molecule in recent years. In this work, we reported the first use of THz-TDS to examine the denaturation of two photosynthesis membrane proteins: CP43 and CP47. THz-TDS was proven to be useful in discriminating the different conformational states of given proteins with similar structure and in monitoring the denaturation process of proteins. Upon treatment with guanidine hydrochloride (GuHCl), a 1.8 THz peak appeared for CP47 and free chlorophyll a (Chl a). This peak was deemed to originate from the interaction between Chl a and GuHCl molecules. The Chl a molecules in CP47 interacted with GuHCl more easily than those in CP43.     

The process of amyloid-like fibril formation by methionine aminopeptidase from a hyperthermophile, Pyrococcus furiosus

Amyloid is associated with serious diseases including Alzheimer's disease and senile-systemic amyloidosis due to misfolded proteins. In the course of study of the denaturation process of methionine aminopeptidase (MAP) from the hyperthermophile P. furiosus, we found that MAP forms amyloid-like fibrils, and we then investigated the mechanism of amyloid fibril formation. The kinetic experiments on denaturation monitored by CD at 222 nm indicated that MAP in the presence of 3.37 M GuHCl at pH 3.31 changed to a conformation containing a considerable content of beta-sheet structure after the destruction of the alpha-helical structure. MAP in this beta-rich conformation was highly associated, and its stability was remarkably high: the midpoint of the GuHCl denaturation curve was 4.82 M at pH 3.0, and a thermal transition was not observed up to 125 degrees C by calorimetry. The amyloid-like fibril formation of MAP was confirmed by Congo red staining with a typical peak at 542 nm in the difference spectrum, showing a cross-beta X-ray diffraction pattern with a clear sharp reflection at 4.7 A and a characteristic unbranched fibrillar appearance with a length of about 1000 A and a diameter of about 70 A in the electron micrographs. Present results indicate that the amyloid-like form of MAP appears just after the protein is almost completely denatured, and even highly stable proteins can also form amyloid-like conformation under conditions where the denatured state of the protein is abundantly populated.     

Does beta-lactoglobulin denaturation occur via an intermediate state?

The denaturation of beta-lactoglobulin (BLG) in the presence of urea and GuHCl has been investigated at different pH values with various spectroscopic techniques. The equilibrium denaturation free energy values, obtained by linearly extrapolating the data to vanishing denaturant (DeltaG(D)(H2O)), are compared and discussed. The fit of the spectroscopic data monitoring the denaturation of BLG has been approached, at first, with a two-state model that describes the protein transition from the folded state (at each pH and in the absence of denaturant) to the denatured state, but in particular, along the GuHCl denaturation pathway some evidence is found of the presence of an intermediate state. Time-resolved fluorescence experiments performed on the BLG-ANS (1-anilino-8-naphthalenesulfonate) complex help to understand the results. Fluorescence polarization anisotropy (FPA) measurements accompanying the denaturation process show the presence of a fast rotational diffusion of the ANS probe, and the data are interpreted in terms of local fluctuations of a still structured tract of the denatured protein where the probe is bound.     

Accumulation of partly folded states in the equilibrium unfolding of ervatamin A: spectroscopic description of the native, intermediate, and unfolded states

Ervatamin A, a cysteine proteases from Ervatamia coronaria, has been used as model system to examine structure-function relationship by equilibrium unfolding methods. Ervatamin A belongs to alpha+beta class of proteins and exhibit stability towards temperature and chemical denaturants. Acid induced unfolding of ervatamin A was incomplete with respect to the structural content of the enzyme. Between pH 0.5 and 2.0, the enzyme is predominantly in beta-sheet conformation and shows a strong ANS binding suggesting the existence of a partially unfolded intermediate state (I(A) state). Surprisingly, high concentrations of GuHCl required to unfold this state and the transition mid points GuHCl induced unfolding curves are significantly higher. GuHCl induced unfolding of ervatamin A at pH 3.0 as well as at pH 4.0 is complex and cannot be satisfactorily fit to a two-state model for unfolding. Besides, a strong ANS binding to the protein is observed at low concentration of GuHCl, indicating the presence of intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8M) the enzyme retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to urea unfolding at pH 3.0 and below. Urea induced unfolding of ervatamin A at pH 3.0 is cooperative and the transitions curves obtained by different probes are and non-coincidental. Temperature denaturation of ervatamin A in I(A) state is non-cooperative, contrary to the cooperativity seen with native protein, suggesting the presence of two parts in the molecular structure of ervatamin A may be domains, with different stability that unfolds in steps. Careful inspection of biophysical properties of intermediate states populated in urea and GuHCl (I(UG) state) induced unfolding suggests all these three intermediates are identical and populated in different conditions. However, the properties of the intermediate (I(A) state) identified at pH approximately 1.5 are different from those of the I(UG) state.     

Reversible thermal denaturation of human FGF-1 induced by low concentrations of guanidine hydrochloride.

Human acidic fibroblast growth factor (FGF-1) is a powerful mitogen and angiogenic factor with an apparent melting temperature (Tm) in the physiological range. FGF-1 is an example of a protein that is regulated, in part, by stability-based mechanisms. For example, the low Tm of FGF-1 has been postulated to play an important role in the unusual endoplasmic reticulum-independent secretion of this growth factor. Despite the close relationship between function and stability, accurate thermodynamic parameters of unfolding for FGF-1 have been unavailable, presumably due to effects of irreversible thermal denaturation. Here we report the determination of thermodynamic parameters of unfolding (DeltaH, DeltaG, and DeltaCp) for FGF-1 using differential scanning calorimetry (DSC). The thermal denaturation is demonstrated to be two-state and reversible upon the addition of low concentrations of added guanidine hydrochloride (GuHCl). DeltaG values from the DSC studies are in excellent agreement with values from isothermal GuHCl denaturation monitored by fluorescence and circular dichroism (CD) spectroscopy. Furthermore, the results indicate that irreversible denaturation is closely associated with the formation of an unfolding intermediate. GuHCl appears to promote reversible two-state denaturation by initially preventing aggregation of this unfolding intermediate, and at subsequently higher concentrations, by preventing formation of the intermediate.     

Denaturation and proteolytic digestion of porcine low-density lipoprotein in aqueous guanidine hydrochloride solutions.

The denaturation of porcine low-density lipoprotein (LDL) in aqueous guanidine hydrochloride (GuHCl) was studied by flotation velocity experiments, optical rotatory dispersion and fluorescence spectroscopy. The denaturation of LDL occurred between 2 and 4M GuGCl, where small sigmoidal changes in iptical rotation and fluorescence intensity were noted. The hydrated density of the native LDL was 1.036g/cm-3 and this remained constant upon denaturation in 4M GuHCl. The slope of the flotation coefficient-solvent density curve was 35 per cent less for denatured LDL than for the native LDL. Since there is no indication of splitting of LDL in 4M GuHCl, it is natural to interpret the result in terms of an increase of the translational frictional coefficient by 50 per cent. The observed changes in optical rotation, fluorescence intensity and flotation coefficient in 4M GuHCl were readily reversed and native LDL was recovered after removal of GuHCl by dialysis. Proteolytic treatment of denatured LDL produced digested LDL which had a hydrated density of 1.021g/cm-3 corresponding to the loss of 30 per cent of apo-LDL. The digested LDL behaved like a compact, globular particle in aqueous NaCl solution and in 4M GuHCl. These results can best be interpreted by a model of the LDL particle in which approximately 30 per cent of apo-LDL is exposed to the solvent, such that it can be reversibly denatured by GuHCl and at the same time is easily avalable to proteolytic enzymes, whereas the rest of apo-LDL is tightly associated with lipids and possibly buried inside the lipid moiety. SDS-polyacrylamide gel electrophoresis of the digested LDL revealed four major peptide fragments with sizes ranging from 70,000 to 100,000 daltons. We believe that the method and results described in this paper will have meaningful applications in the study of membrane proteins.     

Polyacrylamide gel electrophoresis of visna virus polypeptides isolated by agarose gel chromatography.

The proteins of visna are separated into nine major peaks by agarose gel chromatography in 6 M guanidine hydrochloride (GuHCl). The polypeptides in eack peak were isolated by acid precipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns of SDS-PAGE show that the excluded material from the GuHCl column contains an aggregate of 10 non-glycosylated polypeptides. It is shown that this aggregate represents virus substructures that are not completely solubilized by GuHCl. Two glycoproteins, gp175 and gp115, were isolated from the column eluate. The major glycoprotein gp115 was coeluted with P90, P68, and P61 in GuHCl 4. Each of the four major peaks (GuHCl 5 to 8) contains more than one nonglycosylated polypeptide. However, a small polypeptide, P12, can be isolated in a homogeneous form in the last peak, GuHCl 9. Analysis of the virus proteins (100 microgram) by SDS-PAGE shows that 20 radioactive bands can be recognized. During fractionation of the protein on agarose gel columns followed by analysis with SDS-PAGE, a number of minor polypeptides that were not detected before became clearly recognizable. Thus, the combined use of column chromatography and SDS-PAGE shows that visna virus is composed of 25 proteins.     

The Sequential Mechanism of Guanidine Hydrochloride-Induced Denaturation of cAMP Receptor Protein from Escherichia coli. A Fluorescent Study Using 8-Anilino-1-Naphthalenesulfonic Acid

cAMP receptor protein (CRP) regulates expression of a number of genes in Escherichia coli. The protein is a homodimer and each monomer is folded into two structural domains. The biological activation of CRP upon cAMP binding may involve the subunit realignment as well as reorientation between the domains within each subunit. In order to study the interactions between the subunits or domains, we performed stopped-flow measurements of the guanidine hydrochloride (GuHCl)-induced denaturation of CRP. The changes in CRP structure induced by GuHCl were monitored using both intrinsic Trp fluorescence as well as the fluorescence of an extrinsic probe, 8-anilino-1-Naphthalenesulfonic acid (ANS). Results of CRP denaturation using Trp fluorescence detection are consistent with a two-step model [Malecki, and Wasylewski, (1997), Eur. J. Biochem. 243, 660], where the dissociation of dimer into subunits is followed by the monomer unfolding. The denaturation of CRP monitored by ANS fluorescence reveals the existence of two additional processes. One occurs before the dissociation of CRP into subunits, whereas the second takes place after the dissociation, but prior to proper subunit unfolding. These additional processes suggest that CRP denaturation is described by a more complicated mechanism than a simple three-state equilibrium and may involve additional changes in both inter- and intrasubunit interactions. We also report the effect of cAMP on the kinetics of CRP subunit unfolding and refolding.     

A tetrahedral zinc(II)-binding site introduced into a designed protein

The ultimate goal of protein engineering is to create novel proteins which will adopt predetermined structures, bind specified ligands, and catalyze new reactions. Here we describe the successful introduction of metal-binding activity into a model four helix bundle protein. The designed binding site is tetrahedral and is formed by two Cys and two His ligands on adjacent helices. We have introduced this site into the protein and characterized the binding activity. Using 65Zn(II), we have shown that the protein binds Zn(II), that the sulfhydryls are essential for binding, and that binding occurs to the protein monomer. The designed protein binds metals with high affinity: we estimate the dissociation constants as 2.5 X 10(-8) M for Zn(II) and 1.6 X 10(-5) M for Co(II). The characteristic absorption spectrum of the Co(II)-substituted protein fully supports the model of a tetrahedral binding site comprised of two Cys and two His ligands. Circular dichroism studies indicate that no significant changes in secondary structure occur between the metal-bound and metal-free forms of the protein. However, the metal-bound form is substantially stabilized toward denaturation by GuHCl compared to the metal-free form.     

Guanidinium chloride-induced spectral perturbations of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid confound interpretation of data on molten globule states

We describe limitations in the use of 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) to examine unfolding intermediates associated with guanidinium chloride (GuHCl)-induced protein denaturation. Several studies have used alterations in fluorescence emission of bis-ANS to quantify the population of “molten globule” states. Our findings indicate that the observed changes in bis-ANS spectroscopic properties could originate from the interactions of bis-ANS and GuHCl and the aggregation of the dye at higher GuHCl concentrations. We posit that in the absence of additional complementary structural or spectroscopic measurements, the use of bis-ANS emission alone to monitor protein conformations can be misleading.     

Kietics of thermal unfolding and refolding of thermostable phosphoglycerate kinase.

The kinetics of denaturation by guanidine hydrochloride (GuHCl) of a thermostable phosphoglycerate kinase (PGK) extracted from Thermus thermophilus and of yeast PGK at neutral pH were studied by circular dichroism. Denaturation by GuHCl proceeded as a first-order reaction. The activation free energy of the denaturation reactions (delta Gf not identical to ) in the absence of GuHCl was estimated to be 32.7 kcal/mol for T. thermophilus PGK and 27.9 kcal/mol for yeast PGK (at 25 degrees C). Measurements of the rate constants at various temperatures indicated that delta Gf not identical to has maximum values at 29 degrees C for T. thermophilus PGK and at 20 degrees C for yeast PGK, and that the temperature dependences of delta Gf not identical to, delta Hf not identical to, and delta Sf not identical to for T. thermophilus PGK are smaller than those of yeast PGK. Values of delta Sf not identical to for thermal denaturation for both PGK's are approximately 200 e.u.