Pressure and temperature stability of the main apple allergen Mal d1

High-pressure Fourier-transform infrared (FTIR) spectroscopy was used to determine the pressure and temperature stability of Mal d1. This study was triggered by contradictory results in the literature regarding the success of pressure treatment in the destruction of the allergen. The protein unfolded at 55°C when heated at normal atmospheric pressure. We also studied the effect exerted on pressure stability by environmental factors, which can be important for the stability of the protein in the apple. The pressure unfolding was measured under different pD conditions, and the effect of sugar mixture similar to that of the apple and the effect of ionic strength were also studied. In all cases the allergen unfolded with a transition midpoint in the range of 150–250 MPa. Unfolding was irreversible and was followed by aggregation of the unfolded protein. Lowering the pD destabilized the protein, while addition of sugar mixture and of KCl had stabilizing effect.     

Use of protein unfolding studies to determine the conformational and dimeric stabilities of HIV-1 and SIV proteases.

The free energies of dimer dissociation of the retroviral proteases (PRs) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were determined by measuring the effects of denaturants on the protein fluorescence upon the unfolding of the enzymes. HIV-1 PR was more stable to denaturation by chaotropes and extremes of pH and temperature than SIV PR, indicating that the former enzyme has greater conformational stability. The urea unfolding curves of both proteases were sigmoidal and single phase. The midpoints of the transition curves increased with increasing protein concentrations. These data were best described by and fitted to a two-state model in which folded dimers were in equilibrium with unfolded monomers. This denaturation model conforms to cases in which protein unfolding and dimer dissociation are concomitant processes in which folded monomers do not exist [Bowie, J. U., & Sauer, R. T. (1989) Biochemistry 28, 7140-7143]. Accordingly, the free energies of unfolding reflect the stabilities of the protease dimers, which for HIV-1 PR and SIV PR were, respectively, delta GuH2O = 14 +/- 1 kcal/mol (Ku = 39 pM) and 13 +/- 1 kcal/mol (Ku = 180 pM). The binding of a tight-binding, competitive inhibitor greatly stabilized HIV-1 PR toward urea-induced unfolding (delta GuH2O = 19.3 +/- 0.7 kcal/mol, Ku = 7.0 fM). There were also profound effects caused by adverse pH on the protein conformation for both HIV-1 PR and SIV PR, resulting in unfolding at pH values above and below the respective optimal ranges of 4.0-8.0 and 4.0-7.0     

Dimer dissociation and unfolding mechanism of coagulation factor XI apple 4 domain: spectroscopic and mutational analysis

The blood coagulation protein factor XI (FXI) consists of a pair of disulfide-linked chains each containing four apple domains and a catalytic domain. The apple 4 domain (A4; F272-E362) mediates non-covalent homodimer formation even when the cysteine involved in an intersubunit disulfide is mutated to serine (C321S). To understand the role of non-covalent interactions stabilizing the FXI dimer, equilibrium unfolding of wild-type A4 and its C321S variant was monitored by circular dichroism, intrinsic tyrosine fluorescence and dynamic light scattering measurements as a function of guanidine hydrochloride concentration. Global analysis of the unimolecular unfolding transition of wild-type A4 revealed a partially unfolded equilibrium intermediate at low to moderate denaturant concentrations. The optically detected equilibrium of C321S A4 also fits best to a three-state model in which the native dimer unfolds via a monomeric intermediate state. Dimer dissociation is characterized by a dissociation constant, K(d), of approximately 90 nM (in terms of monomer), which is in agreement with the dissociation constant measured independently using fluorescence anisotropy. The results imply that FXI folding occurs via a monomeric equilibrium intermediate. This observation sheds light on the effect of certain naturally occurring mutations, such as F283L, which lead to intracellular accumulation of non-native forms of FXI. To investigate the structural and energetic consequences of the F283L mutation, which perturbs a cluster of aromatic side-chains within the core of the A4 monomer, it was introduced into the dissociable dimer, C321S A4. NMR chemical shift analysis confirmed that the mutant can assume a native-like dimeric structure. However, equilibrium unfolding measurements show that the mutation causes a fourfold increase in the K(d) value for dissociation of the native dimer and a 1 kcal/mol stabilization of the monomer, resulting in a highly populated intermediate. Since the F283 side-chain does not directly participate in the dimer interface, we propose that the F283L mutation leads to increased dimer dissociation by stabilizing a monomeric state with altered side-chain packing that is unfavorable for homodimer formation.     

Equilibrium unfolding of kinetically stable serine protease milin: the presence of various active and inactive dimeric intermediates

Kinetically stable homodimeric serine protease milin reveals high conformational stability against temperature, pH and chaotrope [urea, guanidine hydrochloride (GuHCl) and guanidine isothiocynate (GuSCN)] denaturation as probed by circular dichroism, fluorescence, differential scanning calorimetry and activity measurements. GuSCN induces complete unfolding in milin, whereas temperature, urea and GuHCl induce only partial unfolding even at low pH, through several intermediates with distinct characteristics. Some of these intermediates are partially active (viz. in urea and 2 M GuHCl at pH 7.0), and some exhibited strong ANS binding as well. All three tryptophans in the protein seem to be buried in a rigid, compact core as evident from intrinsic fluorescence measurements coupled to equilibrium unfolding experiments. The protein unfolds as a dimer, where the unfolding event precedes dimer dissociation as confirmed by hydrodynamic studies. The solution studies performed here along with previous biochemical characterization indicate that the protein has α-helix and β-sheet rich regions or structural domains that unfold independently, and the monomer association is isologous. The complex unfolding pathway of milin and the intermediates has been characterized. The physical, physiological and probable therapeutic importance of the results has been discussed.     

Dissociation and unfolding of cold-active alkaline phosphatase from atlantic cod in the presence of guanidinium chloride.

Cold-adaptation of enzymes involves improvements in catalytic efficiency. This paper describes studies on the conformational stability of a cold-active alkaline phosphatase (AP) from Atlantic cod, with the aim of understanding more clearly its structural stability in terms of subunit dissociation and unfolding of monomers. AP is a homodimeric enzyme that is only active in the dimeric state. Tryptophan fluorescence, size-exclusion chromatography and enzyme activity were used to monitor alterations in conformational state induced by guanidinium chloride or urea. In cod AP, a clear distinction could be made between dissociation of dimers into monomers and subsequent unfolding of monomers (fits a three-state model). In contrast, dimer dissociation of calf AP coincided with the monophasic unfolding curve observed by tryptophan fluorescence (fits a two-state model). The DeltaG for dimer dissociation of cod AP was 8.3 kcal.mol-1, and the monomer stabilization free energy was 2.2 kcal.mol-1, giving a total of 12.7 kcal.mol-1, whereas the total free energy of calf intestinal AP was 17.3 kcal.mol-1. Thus, dimer formation provided a major contribution to the overall stability of the cod enzyme. Phosphate, the reaction product, had the effect of promoting dimer dissociation and stabilizing the monomers. Cod AP has reduced affinity for inorganic phosphate, the release of which is the rate-limiting step of the reaction mechanism. More flexible links at the interface between the dimer subunits may ease structural rearrangements that facilitate more rapid release of phosphate, and thus catalytic turnover.     

Pressure denaturation of the yeast prion protein Ure2

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

Pressure and denaturants in the unfolding of triosephosphate isomerase: the monomeric intermediates of the enzymes from Saccharomyces cerevisiae and Entamoeba histolytica

Triosephosphate isomerase (TIM) is a dimeric enzyme formed by two identical (beta/alpha)8 barrels. In this work, we compare the unfolding and refolding of the TIMs from Entamoeba histolytica (EhTIM) and baker's yeast (yTIM). A monomeric intermediate was detected in the GdnHCl-induced unfolding of EhTIM. The thermodynamic, spectroscopic, catalytic, and hydrodynamic properties of this intermediate were found to be very similar to those previously described for a monomeric intermediate of yTIM observed in GdnHCl. Monomer unfolding was reversible for both TIMs; however, the dissociation step was reversible in yTIM and irreversible in EhTIM. Monomer unfolding induced by high pressure in the presence of GdnHCl was a reversible process. DeltaGUnf, DeltaVUnf, and P1/2 were obtained for the 0.7-1.2 M GdnHCl range. The linear extrapolation of these thermodynamic parameters to the absence of denaturant showed the same values for both intermediates. The DeltaVUnfH2O values calculated for EhTIM and yTIM monomeric intermediates are the same within experimental error (-57 +/- 10 and -76 +/- 14 mL/mol, respectively). These DeltaVUnf H2O values are smaller than those reported for the unfolding of monomeric proteins of similar size, suggesting that TIM intermediates are only partially hydrated. |DeltaVUnf| increased with denaturant concentration; this behavior is probably related to structural changes in the unfolded state induced by GdnHCl and pressure. From the thermodynamic parameters that were obtained, it is predicted that in the absence of denaturants, pressure would induce monomer unfolding (P1/2 approximately 140 MPa) prior to dimer dissociation (P1/2 approximately 580 MPa). Therefore, dimerization prevents the pressure unfolding of the monomer.     

High pressure reveals that the stability of interdimeric contacts in the R- and T-state of HbA is influenced by allosteric effectors: Insights from computational simulations

The molecular details of the mechanism of action of allosteric effectors on hemoglobin oxygen affinity are not clearly understood. The global allostery model proposed by Yonetani et al. suggests that the binding of allosteric effectors can take place both in the R and T states and that they influence oxygen affinity through inducing global tertiary changes in the subunits. Recently published high pressure studies yielded dissociation constants at atmospheric pressure that showed a stabilizing effect of heterotropic allosteric effectors on the dimer interface in the R state, and a more pronounced destabilizing effect in a T state model. In the present work, we report on computational modeling used to interpret the high pressure experimental data. We show structural changes in the hemoglobin interdimeric interfaces, indicative of a global tertiary structural change induced by the binding of allosteric effectors. We also show that the number of water molecules bound at the interface is significantly influenced by binding effectors in the T state in accordance with the experimental data. Our results suggest that the binding of effectors at definite sites leads to tertiary changes that propagate to the interfaces and results in overall structural re-organizations.     

Subunit exchange by CheA histidine kinases from the mesophile Escherichia coli and the thermophile Thermotoga maritima

Dimerization of the chemotaxis histidine kinase CheA is required for intersubunit autophosphorylation [Swanson, R. V., Bourret, R. B., and Simon, M. I. (1993) Mol. Microbiol. 8, 435-441]. Here we show that CheA dimers exchange subunits by the rate-limiting dissociation of a central four-helix bundle association domain (P3), despite the high stability of P3 versus unfolding. P3 alone determines the stability and exchange properties of the CheA dimer. For CheA proteins from the mesophile Escherichia coli and the thermophile Thermotoga maritima, subunit dissociation activates at temperatures where the respective organisms live (37 and 80 degrees C). Under destabilizing conditions, P3 dimer dissociation is cooperative with unfolding. Chemical denaturation is reversible for both EP3 and TP3. Aggregation accompanies thermal unfolding for both proteins under most conditions, but thermal unfolding is reversible and two-state for EP3 at low protein concentrations. Residue differences within interhelical loops may account for the contrasted thermodynamic properties of structurally similar EP3 and TP3 (41% sequence identity). Under stabilizing conditions, greater correlation between activation energy for dimer dissociation and P3 stability suggests more unfolding in the dissociation of EP3 than TP3. Furthermore, destabilization of extended conformations by glycerol slows relative dissociation rates more for EP3 than for TP3. Nevertheless, at physiological temperatures, neither protein likely unfolds completely during subunit exchange. EP3 and TP3 will not exchange subunits with each other. The receptor coupling protein CheW reduces the subunit dissociation rate of the T. maritima CheA dimer by interacting with the regulatory domain P5.     

Effect of Ligands on HP-Induced Unfolding and Oligomerization of β-Lactoglobulin

To probe intermediate states during unfolding and oligomerization of proteins remains a major challenge. High pressure (HP) is a powerful tool for studying these problems, revealing subtle structural changes in proteins not accessible by other means of denaturation. Bovine β-lactoglobulin (BLG), the main whey protein, has a strong propensity to bind various bioactive molecules such as retinol and resveratrol, two ligands with different affinity and binding sites. By combining in situ HP-small-angle neutron scattering (SANS) and HP-ultraviolet/visible absorption spectroscopy, we report the specific effects of these ligands on three-dimensional conformational and local changes in BLG induced by HP. Depending on BLG concentration, two different unfolding mechanisms are observed in situ under pressures up to ∼300 MPa: either a complete protein unfolding, from native dimers to Gaussian chains, or a partial unfolding with oligomerization in tetramers mediated by disulfide bridges. Retinol, which has a high affinity for the BLG hydrophobic cavity, significantly stabilizes BLG both in three-dimensional and local environments by shifting the onset of protein unfolding by ∼100 MPa. Increasing temperature from 30 to 37°C enhances the hydrophobic stabilization effects of retinol. In contrast, resveratrol, which has a low binding affinity for site(s) on the surface of the BLG, does not induce any significant effect on the structural changes of BLG due to pressure. HP treatment back and forth up to ∼300 MPa causes irreversible covalent oligomerization of BLG. Ab initio modeling of SANS shows that the oligomers formed from the BLG-retinol complex are smaller and more elongated compared to BLG without ligand or in the presence of resveratrol. By combining HP-SANS and HP-ultraviolet/visible absorption spectroscopy, our strategy highlights the crucial role of BLG hydrophobic cavity and opens up new possibilities for the structural determination of HP-induced protein folding intermediates and irreversible oligomerization.     

Thermal,Chemical and pH Induced Unfolding of Turmeric Root Lectin: Modes of Denaturation

Curcuma longa rhizome lectin, of non-seed origin having antifungal, antibacterial and α-glucosidase inhibitory activities, forms a homodimer with high thermal stability as well as acid tolerance. Size exclusion chromatography and dynamic light scattering show it to be a dimer at pH 7, but it converts to a monomer near pH 2. Circular dichroism spectra and fluorescence emission maxima are virtually indistinguishable from pH 7 to 2, indicating secondary and tertiary structures remain the same in dimer and monomer within experimental error. The tryptophan environment as probed by acrylamide quenching data yielded very similar data at pH 2 and pH 7, implying very similar folding for monomer and dimer. Differential scanning calorimetry shows a transition at 350.3 K for dimer and at 327.0 K for monomer. Thermal unfolding and chemical unfolding induced by guanidinium chloride for dimer are both reversible and can be described by two-state models. The temperatures and the denaturant concentrations at which one-half of the protein molecules are unfolded, are protein concentration-dependent for dimer but protein concentration-independent for monomer. The free energy of unfolding at 298 K was found to be 5.23 Kcal mol⁻¹ and 14.90 Kcal mol⁻¹ for the monomer and dimer respectively. The value of change in excess heat capacity upon protein denaturation (ΔC_p) is 3.42 Kcal mol⁻¹ K⁻¹ for dimer. The small ΔC_p for unfolding of CLA reflects a buried hydrophobic core in the folded dimeric protein. These unfolding experiments, temperature dependent circular dichroism and dynamic light scattering for the dimer at pH 7 indicate its higher stability than for the monomer at pH 2. This difference in stability of dimeric and monomeric forms highlights the contribution of inter-subunit interactions in the former.     

Protein unfolded states populated at high and ambient pressure are similarly compact

The relationship between the dimensions of pressure-unfolded states of proteins compared with those at ambient pressure is controversial; resolving this issue is related directly to the mechanisms of pressure denaturation. Moreover, a significant pressure dependence of the compactness of unfolded states would complicate the interpretation of folding parameters from pressure perturbation and make comparison to those obtained using alternative perturbation approaches difficult. Here, we determined the compactness of the pressure-unfolded state of a small, cooperatively folding model protein, CTL9-I98A, as a function of temperature. This protein undergoes both thermal unfolding and cold denaturation, and the temperature dependence of the compactness at atmospheric pressure is known. High-pressure small angle x-ray scattering studies, yielding the radius of gyration and high-pressure diffusion ordered spectroscopy NMR experiments, yielding the hydrodynamic radius were carried out as a function of temperature at 250 MPa, a pressure at which the protein is unfolded. The radius of gyration values obtained at any given temperature at 250 MPa were similar to those reported previously at ambient pressure, and the trends with temperature are similar as well, although the pressure-unfolded state appears to undergo more pronounced expansion at high temperature than the unfolded state at atmospheric pressure. At 250 MPa, the compaction of the unfolded chain was maximal between 25 and 30°C, and the chain expanded upon both cooling and heating. These results reveal that the pressure-unfolded state of this protein is very similar to that observed at ambient pressure, demonstrating that pressure perturbation represents a powerful approach for observing the unfolded states of proteins under otherwise near-native conditions.     

Strength of hydrogen bond network takes crucial roles in the dissociation process of inhibitors from the HIV-1 protease binding pocket

To understand the underlying mechanisms of significant differences in dissociation rate constant among different inhibitors for HIV-1 protease, we performed steered molecular dynamics (SMD) simulations to analyze the entire dissociation processes of inhibitors from the binding pocket of protease at atomistic details. We found that the strength of hydrogen bond network between inhibitor and the protease takes crucial roles in the dissociation process. We showed that the hydrogen bond network in the cyclic urea inhibitors AHA001/XK263 is less stable than that of the approved inhibitor ABT538 because of their large differences in the structures of the networks. In the cyclic urea inhibitor bound complex, the hydrogen bonds often distribute at the flap tips and the active site. In contrast, there are additional accessorial hydrogen bonds formed at the lateral sides of the flaps and the active site in the ABT538 bound complex, which take crucial roles in stabilizing the hydrogen bond network. In addition, the water molecule W301 also plays important roles in stabilizing the hydrogen bond network through its flexible movement by acting as a collision buffer and helping the rebinding of hydrogen bonds at the flap tips. Because of its high stability, the hydrogen bond network of ABT538 complex can work together with the hydrophobic clusters to resist the dissociation, resulting in much lower dissociation rate constant than those of cyclic urea inhibitor complexes. This study may provide useful guidelines for design of novel potent inhibitors with optimized interactions.     

Folding of Escherichia coli DsbC: characterization of a monomeric folding intermediate

The homodimeric protein DsbC is a disulfide isomerase and a chaperone located in the periplasm of Escherichia coli. We have studied the guanidine hydrochloride (GdnHCl)-induced unfolding and refolding of DsbC using mutagenesis, intrinsic fluorescence, circular dichroism spectra, size-exclusion chromatography, and sedimentation velocity analysis. The equilibrium refolding and unfolding of DsbC was thermodynamically reversible. The equilibrium folding profile measured by fluorescence excited at 280 nm exhibited a three-state transition profile with a stable folding intermediate formed at 0-2.0 M GdnHCl followed by a second transition at higher GdnHCl concentrations. Sedimentation velocity data revealed dissociation of the dimer to the monomer over the concentration range of the first transition (0-2.0 M). In contrast, fluorescence emission data for DsbC excited at 295 nm showed a single two-state transition. Fluorescence emission data for the equilibrium unfolding of the monomeric G49R mutant, excited at either 295 or 280 nm, indicated a single two-state transition. Data obtained for the dimeric Y52W mutant indicated a strong protein concentration dependence of the first transition but no dependence of the second transition in equilibrium unfolding. This suggests that the fluorescence of Y52W sensitively reports conformational changes caused by dissociation of the dimer. Thus, the folding of DsbC follows a three-state transition model with a monomeric folding intermediate formed in 0-2.0 M GdnHCl. The folding of DsbC in the presence of DTT indicates an important role for the non-active site disulfide bond in stabilizing the conformation of the molecule. Dimerization ensures the performance of chaperone and isomerase functions of DsbC.     

Drug resistance mutations can effect dimer stability of HIV-1 protease at neutral pH.

The monomer-dimer equilibrium for the human immunodeficiency virus type 1 (HIV-1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in beta-sheet structure and a lower degree of ANS binding. An autolysis-resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild-type enzyme is typically unstable in the absence of bound inhibitor. The dimer dissociation constant (KD) of the triple mutant was 5.8 microM at pH 7.0 and was below the limit of measurement (approximately 100 nM) at pH 4.5. Similar studies using the catalytically inactive D25N mutant yielded a KD value of 1.0 microM at pH 7.0. These values differ significantly from a previously reported value of 23 nM obtained indirectly from inhibitor binding measurements (Darke et al., 1994). We show that the discrepancy may result from the thermodynamic linkage between the monomer-dimer and inhibitor binding equilibria. Under conditions where a significant degree of monomer is present, both substrates and competitive inhibitors will shift the equilibrium toward the dimer, resulting in apparent increases in dimer stability and decreases in ligand binding affinity. Sedimentation equilibrium studies were also carried out on several drug-resistant HIV-1 protease mutants: V82F, V82F/I84V, V82T/I84V, and L90M. All four mutants exhibited reduced dimer stability relative to the autolysis-resistant mutant at pH 7.0. Our results indicate that reductions in drug affinity may be due to the combined effects of mutations on both dimer stability and inhibitor binding.     

High-pressure FTIR study of the stability of horseradish peroxidase. Effect of heme substitution,ligand binding,Ca++ removal,and reduction of the disulfide bonds

The pressure stability of horseradish peroxidase isoenzyme C and the identification of possible stabilizing factors are presented. The effect of heme substitution, removal of Ca(2+), binding of a small substrate molecule (benzohydroxamic acid), and reduction of the disulfide bonds on the pressure stability were investigated by FTIR spectroscopy. HRP was found to be extremely stable under high pressure with an unfolding midpoint of 12.0 +/- 0.1 kbar. While substitution of the heme for metal-free mesoporphyrin did not change the unfolding pressure, Ca(2+) removal and substrate binding reduced the midpoint of the unfolding by 2.0 and 1.2 kbar, respectively. The apoprotein showed a transition as high as 10.4 kbar. However, the amount of folded structure present at the atmospheric pressure was considerably lower than that in all the other forms of HRP. Reduction of the disulfide bonds led to the least pressure stable form, with an unfolding midpoint at 9.5 kbar. This, however, is still well above the average pressure stability of proteins. The high-pressure stability and the analysis of the pressure-induced spectral changes indicate that the protein has a rigid core, which is responsible for the high stability, while there are regions with less stability and more conformational mobility.     

Solution structure of the mature HIV-1 protease monomer: insight into the tertiary fold and stability of a precursor

We present the first solution structure of the HIV-1 protease monomer spanning the region Phe1-Ala95 (PR1-95). Except for the terminal regions (residues 1-10 and 91-95) that are disordered, the tertiary fold of the remainder of the protease is essentially identical to that of the individual subunit of the dimer. In the monomer, the side chains of buried residues stabilizing the active site interface in the dimer, such as Asp25, Asp29, and Arg87, are now exposed to solvent. The flap dynamics in the monomer are similar to that of the free protease dimer. We also show that the protease domain of an optimized precursor flanked by 56 amino acids of the N-terminal transframe region is predominantly monomeric, exhibiting a tertiary fold that is quite similar to that of PR1-95 structure. This explains the very low catalytic activity observed for the protease prior to its maturation at its N terminus as compared with the mature protease, which is an active stable dimer under identical conditions. Adding as few as 2 amino acids to the N terminus of the mature protease significantly increases its dissociation into monomers. Knowledge of the protease monomer structure and critical features of its dimerization may aid in the screening and design of compounds that target the protease prior to its maturation from the Gag-Pol precursor.     

Urea-dependent unfolding of HIV-1 protease studied by circular dichroism and small-angle X-ray scattering

HIV-1 protease is responsible for the maturation of infective virions, and is one of the targets of drugs against AIDS. It is an aspartic protease with a 99-resiude polypeptide dimerized. Previous study with fluorescence and sedimentation measurements revealed that the protein was unfolded with concomitant dissociation of the subunits. In the present study, we investigated urea-dependent unfolding of HIV-1 protease with CD and SAXS in order to monitor the secondary structure and the global size and shape of the molecule, respectively. The unfolding parameters estimated by both methods were almost the same, indicating that the dissociation of the subunits accompanied the disruption of their internal structures. This is in line with the previous results, and moreover some residual structures were suggested to be present in the unfolded state. The distinct difference, as compared with the unfolding of pepsin, was interpreted from the point of their molecular architectures.     

Mutational and structural studies aimed at characterizing the monomer of HIV-1 protease and its precursor

An experimental protocol for folding the mature human immunodeficiency virus-1 (HIV-1) protease is presented that facilitates NMR studies at a low protein concentration of approximately 20 micoM. Under these conditions, NMR spectra show that the mature protease lacking its terminal beta-sheet residues 1-4 and 96-99 (PR(5-95)) exhibits a stable monomer fold spanning the region 10-90 that is similar to that of the single subunit of the wild-type dimer and the dimer bearing a D25N mutation (PR(D25N)). Urea-induced unfolding monitored both by changes in (1)H-(15)N heteronuclear single quantum correlation spectra and by protein fluorescence indicates that although PR(5-95) monomer displays a transition profile similar to that of the PR(D25N) dimer (50% unfolded (U(50)) = approximately 1.9 M), extending the protease with 4 residues (SFNF) of its N-terminally flanking sequence in the Gag-Pol precursor ((SFNF)PR(D25N)) decreases the stability of the fold (U(50) = approximately 1.5 M). Assigned backbone chemical shifts were used to elucidate differences in the stability of the PR(T26A) (U(50) = 2.5 M) and (SFNF)PR(D25N) monomers and compared with PR(D25N/T26A) monomer. Discernible differences in the backbone chemical shifts were observed for N-terminal protease residues 3-6 of (SFNF)PR(D25N) that may relate to the increase in the equilibrium dissociation constant (K(d)) and the very low catalytic activity of the protease prior to its autoprocessing at its N terminus from the Gag-Pol precursor.     

Activity and dimerization of human immunodeficiency virus protease as a function of solvent composition and enzyme concentration.

The activity of human immunodeficiency virus 1 (HIV-1) protease has been examined as a function of solvent composition, incubation time, and enzyme concentration at 37 degrees C in the pH 4.5-5.5 range. Glycerol and dimethyl sulfoxide inhibit the enzyme, while polyethylene glycol and bovine serum albumin activate the enzyme. When incubated at a concentration of 50-200 nM, the activity of the protease decreases irreversibly with an apparent first-order rate constant of 4-9 x 10(-3) min-1. The presence of 0.1% (w/v) polyethylene glycol or bovine serum albumin in the reaction buffer dramatically stabilizes enzyme activity. In the absence of prolonged incubation of the enzyme at submicromolar concentration, the specific activity of HIV-1 protease in buffers of either high or low ionic strength is constant over the enzyme concentration range of 0.25-5 nM, indicating that dissociation of the dimeric protease, if occurring, can only be governed by a picomolar dissociation constant. Similarly, the variation of the specific activity of HIV-2 protease over the enzyme concentration of 4-85 nM is consistent only with a dimer dissociation constant of less than 10 nM. We conclude that: 1) the assumption of a nondissociating HIV-1 protease is a valid one for kinetic studies of tight-binding inhibitors where nanomolar concentrations of the enzymes are employed; 2) stock protease solutions of submicromolar concentration in the absence of activity-stabilizing compounds may lead to erroneous kinetic data and complicate mechanistic interpretations.