Characterization of a molten globule state of bovine carbonic anhydrase III: loss of asymmetrical environment of the aromatic residues has a profound effect on both the near- and far-UV CD spectrum

Bovine muscle carbonic anhydrase (isoenzyme III; BCAIII) exhibited a three-state unfolding process at equilibrium upon denaturation in guanidine hydrochloride (GuHCl). The stable folding intermediate appeared to be of molten globule type. The stability towards GuHCl in terms of mid-point concentrations of denaturation were very similar for BCAIII and human CAII (HCAII). It was further demonstrated that the aromatic amino acid residues contributed significantly to the circular dichroism (CD) spectrum in the far-UV wavelength region during the native-->molten globule state transition. Thus, the ellipiticity change at 218 nm was shown to monitor the loss of tertiary interactions of aromatic side chains at the first unfolding transition as well as the rupture of secondary structure at the second unfolding transition. Similar aromatic contributions to the far-UV CD spectrum, but with varying magnitudes, were also noted for BCAII and HCAII, further emphasizing that interference of aromatic residues should not be neglected at wavelengths that normally are assigned to secondary structural changes.     

Protein folding intermediates of invasin protein IbeA from Escherichia coli

IbeA of Escherichia coli K1 was cloned, expressed and purified as a His(6)-tag fusion protein. The purified fusion protein inhibited E. coli K1 invasion of human brain microvascular endothelial cells and was heat-modifiable. The structural and functional aspects, along with equilibrium unfolding of IbeA, were studied in solution. The far-UV CD spectrum of IbeA at pH 7.0 has a strong negative peak at 215 nm, indicating the existence of beta-sheet-like structure. The acidic unfolding curve of IbeA at pH 2.0 shows the existence of a partially unfolded molecule (molten globule-like structure) with beta-sheet-like structure and displays strong 8-anilino-2-naphthyl sulfonic acid (ANS) binding. The pH dependent intrinsic fluorescence of IbeA was biphasic. At pH 2.0, IbeA exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is in extended beta-sheet conformation and exhibits strong ANS binding. Guanidine hydrochloride denaturation of IbeA in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two domains (possibly) in the molecular structure of IbeA, with differential unfolding stabilities. Furthermore, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. Acid denatured unfolding of IbeA monitored by far-UV CD is non-cooperative with two transitions at pH 3.0-1.5 and 1.5-0.5. At lower pH, IbeA unfolds to the acid-unfolded state, and a further decrease in pH to 2.0 drives the protein to the A state. The presence of 0.5 m KCl in the solvent composition directs the transition to the A state by bypassing the acid-unfolded state. Additional guanidine hydrochloride induced conformational changes in IbeA from the native to the A-state, as monitored by near- and far-UV CD and ANS-fluorescence.     

Local and long-range interactions in the thermal unfolding transition of bovine pancreatic ribonuclease A

This research was undertaken to distinguish between local and global unfolding in the reversible thermal denaturation of bovine pancreatic ribonclease A (RNase A). Local unfolding was monitored by steady-state and time-resolved fluorescence of nine mutants in each of which a single tryptophan was substituted for a wild-type residue. Global unfolding was monitored by far-UV circular dichroism and UV absorbance. All the mutants (except F8W and D38W) exhibited high specific enzymatic activity, and their far-UV CD spectra were very close to that of wild-type RNase A, indicating that the tryptophan substitutions did not affect the structure of any of the mutants (excluding K1W and Y92W) under folding conditions at 20 degrees C. Like wild-type RNase A, the various mutants exhibited reversible cooperative thermal unfolding transitions at pH 5, with transition temperatures 2.5-11 degrees C lower than that of the wild-type transition, as detected by far-UV CD or UV absorbance. Even at 80 degrees C, well above the cooperative transition of all the RNase A mutants, a considerable amount of secondary and tertiary structure was maintained. These studies suggest the following two-stage mechanism for the thermal unfolding transition of RNase A as the temperature is increased. First, at temperatures lower than those of the main cooperative transition, long-range interactions within the major hydrophobic core are weakened, e.g., those involving residues Phe-8 (in the N-terminal helix) and Lys-104 and Tyr-115 (in the C-terminal beta-hairpin motif). The structure of the chain-reversal loop (residues 91-95) relaxes in the same temperature range. Second, the subsequent higher-temperature cooperative unfolding transition is associated with a loss of secondary structure and additional changes in the tertiary contacts of the major hydrophobic core, e.g., those involving residues Tyr-73, Tyr-76, and Asp-38 on the other side of the molecule. The hydrophobic interactions of the C-terminal loop of the protein are enhanced by high temperature, and perhaps are responsible for the preservation of the local structural environment of Trp-124 at temperatures slightly above the major cooperative transition. The results shed new light on the thermal unfolding transitions, generally supporting the thermal unfolding hypothesis of Burgess and Scheraga, as modified by Matheson and Scheraga.     

Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.     

Thermal unfolding of tetrameric melittin: comparison with the molten globule state of cytochrome c.

Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric alpha-helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion- and pH-dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid-denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far-UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2-state transition mechanism between the tetrameric alpha-helical state and the monomeric unfolded state. From the peptide or salt-concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)-1 K-1 at 30 degrees C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)-1 K-1), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins.     

A natively unfolded toxin domain uses its receptor as a folding template

Natively unfolded proteins range from molten globules to disordered coils. They are abundant in eukaryotic genomes and commonly involved in molecular interactions. The essential N-terminal translocation domains of colicin toxins from Escherichia coli are disordered bacterial proteins that bind at least one protein of the Tol or Ton family. The colicin N translocation domain (ColN-(1-90)), which binds to the C-terminal domain of TolA (TolA-(296-421)), shows a disordered far-UV CD spectrum, no near-UV CD signal, and non-cooperative thermal unfolding. As expected, TolA-(296-421) displays both secondary structure in far-UV CD and tertiary structure in near-UV CD. Furthermore it shows a cooperative unfolding transition at 65 degrees C. CD spectra of the 1:1 complex show both increased secondary structure and colicin N-specific near-UV CD signals. A new cooperative thermal transition at 35 degrees C is followed by the unchanged unfolding behavior of TolA-(296-421). Fluorescence and surface plasmon resonance confirm that the new unfolding transition accompanies dissociation of ColN-(1-90). Hence upon binding the disordered structure of ColN-(1-90) converts to a cooperatively folded domain without altering the TolA-(296-421) structure.     

Structural characterization and unfolding mechanism of human 4F2hc ectodomain

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in several functions as amino acid transport, cell fusion, β1-integrin-signaling and transformation. 4F2hc ectodomain has been crystallized and its three-dimensional structure determined. We have carried out a spectroscopical/structural characterization of the recombinant ectodomain in order to obtain information on its dynamic structure in solution and on its ability to form homodimers by itself in the absence of the transmembrane helix and of the potential interactions with the plasma membrane. Analytical ultracentrifugation and crosslinking experiments showed that the ectodomain is monomeric in solution. The secondary structure determined by far-UV circular dichroism (CD) spectroscopy (around 30% α-helix and 20% β-sheets, 12% antiparallel and 8% parallel) reveals a compact and thermally stable structure with a high melting temperature (57-59°C). Tryptophan residues are mainly buried and immobilized in the hydrophobic core of the protein as suggested by near-UV CD spectrum, the position of the Trp maximum fluorescence emission (323nm) and from the acrylamide quenching constant (2.6M(-1)). Urea unfolding equilibrium has been studied by far-UV CD and fluorescence spectroscopy to gain information on the folding/unfolding process of the ectodomain. The analyses suggest the existence of two intermediate states as reported for other TIM barrel-containing proteins rather than an independent unfolding of each domain [A, (βα)(8) barrel; C, antiparallel β(8) sandwich]. Folding seems to be directed by the initial formation of hydrophobic clusters within the first strands of the β-barrel of domain A followed by additional hydrophobic interactions in domain C.     

High- and low-temperature unfolding of human high-density apolipoprotein A-2.

Human plasma apolipoprotein A-2 (apoA-2) is the second major protein of the high-density lipoproteins that mediate the transport and metabolism of cholesterol. Using CD spectroscopy and differential scanning calorimetry, we demonstrate that the structure of lipid-free apoA-2 in neutral low-salt solutions is most stable at approximately 25 degrees C and unfolds reversibly both upon heating and cooling from 25 degrees C. High-temperature unfolding of apoA-2, monitored by far-UV CD, extends from 25-85 degrees C with midpoint Th = 56 +/- 2 degrees C and vant Hoff's enthalpy delta H(Th) = 17 +/- 2 kcal/mol that is substantially lower than the expected enthalpy of melting of the alpha-helical structure. This suggests low-cooperativity apoA-2 unfolding. The apparent free energy of apoA-2 stabilization inferred from the CD analysis of the thermal unfolding, delta G(app)(25 degrees) = 0.82 +/- 0.15 kcal/mol, agrees with the value determined from chemical denaturation. Enhanced low-temperature stability of apoA-2 observed upon increase in Na2HPO4 concentration from 0.3 mM to 50 mM or addition of 10% glycerol may be linked to reduced water activity. The close proximity of the heat and cold unfolding transitions, that is consistent with low delta G(app)(25 degrees), indicates that lipid-free apoA-2 has a substantial hydrophobic core but is only marginally stable under near-physiological solvent conditions. This suggests that in vivo apoA-2 transfer is unlikely to proceed via the lipid-free state. Low delta H(Th) and low apparent delta Cp approximately 0.52 kcal/mol.K inferred from the far-UV CD analysis of apoA-2 unfolding, and absence of tertiary packing interactions involving Tyr groups suggested by near-UV CD, are consistent with a molten globular-like state of lipid-free apoA-2.     

Role of the C-terminus in the activity, conformation, and stability of interleukin-6.

Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.     

Molten globule-like state of human serum albumin at low pH.

Human serum albumin (HSA), under conditions of low pH, is known to exist in two isomeric forms, the F form at around pH 4.0 and the E form below 3.0. We studied its conformation in the acid-denatured E form using far-UV and near-UV CD, binding of a hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal transition by far-UV and near-UV CD, tryptophan fluorescence, quenching of tryptophan fluorescence using a neutral quencher, acrylamide and viscosity measurements. The results show that HSA at pH 2.0 is characterized by a significant amount of secondary structure, as evident from far-UV CD spectra. The near-UV CD spectra showed a profound loss of tertiary structure. A marked increase in ANS fluorescence signified extensive solvent exposure of non-polar clusters. The temperature-dependence of both near-UV and far-UV CD signals did not exhibit a co-operative thermal transition. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue, Trp214, showed that, in the acid-denatured state, it is buried in the interior in a non-polar environment. Intrinsic viscosity measurements showed that the acid-denatured state is relatively compact compared with that of the denatured state in 7 M guanidine hydrochloride. These results suggest that HSA at pH 2.0 represents the molten globule state, which has been shown previously for a number of proteins under mild denaturing conditions.     

Effect of pH and denaturants on the folding and stability of murine interleukin-6.

The conformation and stability of a recombinant mouse interleukin-6 (mIL-6) has been investigated by analytical ultracentrifugation, fluorescence spectroscopy, urea-gradient gel electrophoresis, and near- and far-ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4.0, the tryptophan fluorescence of mIL-6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence quantum yield was not due to unfolding of the molecule because the conformation of mIL-6, as judged by both urea-gradient gel electrophoresis and CD spectroscopy, was stable over the pH range 2.0-10.0. Sedimentation equilibrium experiments indicated that mIL-6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicochemical studies. Quenching of tryptophan fluorescence (20%) also occurred in the presence of 6 M guanidine hydrochloride upon going from pH 7.4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp-36 and Trp-160, may be partially involved in the quenching of endogenous fluorescence. In this regard, similar results were obtained for a 17-residue synthetic peptide, peptide H1, which corresponds to an N-terminal region of mIL-6 (residues Val-27-Lys-43). The pH-dependent acid quenching of endogenous tryptophan fluorescence of peptide H1 was 30% in the random coil conformation and 60% in the presence of alpha-helix-promoting solvents. Replacement of His-33 with Ala-33 in peptide H1 alleviated a significant portion of the pH-dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His-33 with the indole ring of Trp-36 is a major determinant responsible for the quenching of the endogenous protein fluorescence of mIL-6.     

Acid-induced equilibrium folding intermediate of human platelet profilin

The acid-induced unfolding of human platelet profilin (HPP) can be minimally modeled as a three-state process. Equilibrium unfolding studies have been performed on human platelet profilin1 (HPP) and monitored by far-UV circular dichroism, tryptophan fluorescence, ANS binding, and NMR spectroscopy. Far-UV CD measurements obtained by acid titration demonstrate that HPP unfolds via a three-state mechanism (N --> I --> U), with a highly populated intermediate between pH 4 and 5. Approximately 80% of native helical secondary structural content remains at pH 4, as indicated by monitoring the CD signal at 222 nm. The stability (DeltaGH2O) of the native conformation at pH 7.0 (obtained by monitoring the change in tryptophan signal as a function of urea concentration) is 5.56 +/- 0.51 kcal mol-1; however, the DeltaGH2O for the intermediate species at pH 4 is 2.01 +/- 0.47 kcal mol-1. The calculated m-values for the pH 7.0 and pH 4.0 species were 1.64 +/- 0.15 and 1.34 +/- 0.17 kcal mol-1 M-1, respectively, which is an indication that the native and intermediate species are similarly compact. Additionally, translational diffusion measurements obtained by NMR spectroscopy and ANS binding studies are consistent with a globular and compact conformation at both pH 7.0 and 4.0. The pKa values for the two histidine (His) residues located on helix 4 of HPP were determined to be 5.6 and 5.7 pH units. These pKa values coincide with the midpoint of the far-UV CD acid titration curve and suggest that the protonation of one or both His residues may play a role in the formation of the unfolding intermediate. Stable intermediate species populate the 2D 1H-15N HSQC NMR spectra between pH 4 and 5. A number of backbone and side-chain resonances show significant perturbations relative to the native spectrum; however, considerable nativelike tertiary contacts remain. Interestingly, the residues on HPP that are significantly altered at low pH coincide with segments of the G-actin binding surface and poly-l-proline binding interface. The earlier reports that a decrease in pH below 6.0 induces structural alterations in profilin, favoring dissociation of the profilin-actin complex, corresponds with the structural alterations observed in the partially unfolded species. Our findings suggest that a novel mechanism for pH induced disruption of the profilin-G-actin complex involve a nativelike unfolding intermediate of profilin.     

Characterization of equilibrium intermediates in denaturant-induced unfolding of ferrous and ferric cytochromes c using magnetic circular dichroism, circular dichroism, and optical absorption spectroscopies

Protein unfolding during guanidine HCl denaturant titration of the reduced and oxidized forms of cytochrome c is monitored with magnetic circular dichroism (MCD), natural CD, and absorption of the heme bands and far-UV CD of the amide bands. Direct MCD spectral evidence is presented for bis-histidinyl heme ligation in the unfolded states of both the reduced and oxidized protein. For both redox states, the unfolding midpoints measured with MCD, which is an indicator of tertiary structure, are significantly lower than those measured with far-UV CD, an indicator of secondary structure. The disparate titration curves are interpreted in terms of a compound mechanism for denaturant-induced folding and unfolding involving a molten globulelike intermediate state (MG) with near-native secondary structure and nonnative tertiary structure and heme ligation. A comparison of the dependence of the free energy of formation of the MG intermediate on the redox state with the known contributions from heme ligation and solvation suggests that the heme is significantly more accessible to solvent in the MG intermediate than it is in the native state.     

The isolated C-terminal (F2) fragment of the Escherichia coli tryptophan synthase beta 2-subunit folds into a stable, organized nonnative conformation

Proteolysis of the beta 2-subunit of Escherichia coli tryptophan synthase by the endoproteinase Glu C from Staphylococcus aureus V8 yields a peptide, F2, corresponding to the C-terminal 101 residues of the beta-chain. The conformation and stability of isolated F2 in phosphate buffer at pH 7.8 (where native beta 2 is stable) have been investigated. Circular dichroism spectra in the far-UV showed the presence of large amounts of secondary structure (19% alpha-helices, 34% extended beta-structures). Circular dichroism spectra in the near-UV and sedimentation velocity studies indicated an open globular structure with the aromatic side chains in a symmetric (or disordered) environment. NMR spectra and rates of amide proton exchange showed that F2 fluctuates rapidly between several conformations. The thermal denaturation of F2 observed by the loss of far-UV circular dichroism with increasing temperature appeared noncooperative, and indicates a high thermal stability (Tm = 70 degrees C). Differential scanning microcalorimetry confirmed the absence of cooperativity and indicated a very low value for the calorimetric enthalpy of denaturation (delta H = 17 kJ/mol). All these properties were compatible with a molten globule. However, the low sedimentation coefficient of F2 suggested a very hydrated and/or expanded structure, and the secondary structure content of isolated F2 (see above) differed widely from that reported in the literature for F2 within the context of native beta 2 (49% alpha-helices and 13% extended beta-structures). Thus, neither the secondary nor the tertiary structure of isolated F2 resembled those of native F2. In this respect, isolated F2 is not a "molten globule".(ABSTRACT TRUNCATED AT 250 WORDS)     

Urea-dependent unfolding of murine adenosine deaminase: sequential destabilization as measured by 19F NMR

Murine adenosine deaminase (mADA) is a 40 kDa (beta/alpha)(8)-barrel protein consisting of eight central beta-strands and eight peripheral alpha-helices containing four tryptophan residues. In this study, we investigated the urea-dependent behavior of the protein labeled with 6-fluorotryptophan (6-(19)F-Trp). The (19)F NMR spectrum of 6-(19)F-Trp-labeled mADA reveals four distinct resonances in the native state and three partly overlapped resonances in the unfolded state. The resonances were assigned unambiguously by site-directed mutagenesis. Equilibrium unfolding of 6-(19)F-Trp-labeled mADA was monitored using (19)F NMR based on these assignments. The changes in intensity of folded and unfolded resonances as a function of urea concentration show transition midpoints consistent with data observed by far-UV CD and fluorescence spectroscopy, indicating that conformational changes in mADA during urea unfolding can be followed by (19)F NMR. Chemical shifts of the (19)F resonances exhibited different changes between 1.0 and 6.0 M urea, indicating that local structures around 6-(19)F-Trp residues change differently. The urea-induced changes in local structure around four 6-(19)F-Trp residues of mADA were analyzed on the basis of the tertiary structure and chemical shifts of folded resonances. The results reveal that different local regions in mADA have different urea-dependent behavior, and that local regions of mADA change sequentially from native to intermediate topologies on the unfolding pathway.     

Structurally homologous all beta-barrel proteins adopt different mechanisms of folding

Acidic fibroblast growth factors from human (hFGF-1) and newt (nFGF-1) (Notopthalamus viridescens) are 16-kDa, all beta-sheet proteins with nearly identical three-dimensional structures. Guanidine hydrochloride (GdnHCl)-induced unfolding of hFGF-1 and nFGF-1 monitored by fluorescence and far-UV circular dichroism (CD) shows that the FGF-1 isoforms differ significantly in their thermodynamic stabilities. GdnHCl-induced unfolding of nFGF-1 follows a two-state (Native state to Denatured state(s)) mechanism without detectable intermediate(s). By contrast, unfolding of hFGF-1 monitored by fluorescence, far-UV circular dichroism, size-exclusion chromatography, and NMR spectroscopy shows that the unfolding process is noncooperative and proceeds with the accumulation of stable intermediate(s) at 0.96 M GdnHCl. The intermediate (in hFGF-1) populated maximally at 0.96 M GdnHCl has molten globule-like properties and shows strong binding affinity to the hydrophobic dye, 1-Anilino-8-naphthalene sulfonate (ANS). Refolding kinetics of hFGF-1 and nFGF-1 monitored by stopped-flow fluorescence reveal that hFGF-1 and nFGF-1 adopts different folding mechanisms. The observed differences in the folding/unfolding mechanisms of nFGF-1 and hFGF-1 are proposed to be either due to differential stabilizing effects of the charged denaturant (Gdn(+) Cl(-)) on the intermediate state(s) and/or due to differences in the structural interactions stabilizing the native conformation(s) of the FGF-1 isoforms.     

Purification and characterization of a cobalt-activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus

A novel metallocarboxypeptidase (PfuCP) has been purified to homogeneity from the hyperthermophilic archaeon, Pyrococcus furiosus, with its intended use in C-terminal ladder sequencing of proteins and peptides at elevated temperatures. PfuCP was purified in its inactive state by the addition of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) to purification buffers, and the activity was restored by the addition of divalent cobalt (K, = 24 +/- 4 microM at 80 degrees C). The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) had no effect on the activity. The molecular mass of monomeric PfuCP is 59 kDa as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 58 kDa by SDS-PAGE analysis. In solution, PfuCP exists as a homodimer of approximately 128 kDa as determined by gel filtration chromatography. The activity of PfuCP exhibits a temperature optimum exceeding 90 degrees C under ambient pressure, and a narrow pH optimum of 6.2-6.6. Addition of Co2+ to the apoPfuCP at room temperature does not alter its far-UV circular dichroism (CD) or its intrinsic fluorescence spectrum. Even when the CoPfuCP is heated to 80 degrees C, its far-UV CD shows a minimal change in the global conformation and the intrinsic fluorescence of aromatic residues shows only a partial quenching. Changes in the intrinsic fluorescence appear essentially reversible with temperature. Finally, the far-UV CD and intrinsic fluorescence data suggest that the overall structure of the holoenzyme is extremely thermostable. However, the activities of both the apo and holo enzyme exhibit a similar second-order decay over time, with 50% activity remaining after approximately 40 min at 80 degrees C. The N-blocked synthetic dipeptide, N-carbobenzoxy-Ala-Arg (ZAR), was used in the purification assay. The kinetic parameters at 80 degrees C with 0.4 mM CoCl2 were: Km, 0.9 +/- 0.1 mM; Vmax, 2,300 +/- 70 U mg(-1); and turn over number, 600 +/- 20 s(-1). Activity against other ZAX substrates (X = V, L, I, M, W, Y, F, N, A, S, H, K) revealed a broad specificity for neutral, aromatic, polar, and basic C-terminal residues. This broad specificity was confirmed by the C-terminal ladder sequencing of several synthetic and natural peptides, including porcine N-acetyl-renin substrate, for which we have observed (by MALDI-TOF MS) stepwise hydrolysis by PfuCP of up to seven residues from the C-terminus: Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser.     

Structural intermediates of acid unfolded Con-A in different co-solvents: fluoroalcohols and polyethylene glycols

A molten globule-like intermediate of Con-A was obtained when subjected to acid unfolding. At pH 2 the intermediate was found to have native-like secondary structure, somewhat denatured tertiary structure and maximum ANS binding. Further the stability of this intermediate was studied in presence of fluoroalcohols (TFE and HFIP) and polyethylene glycols (PEG-400, 4000 and 20,000). Secondary structural changes were monitored by far-UV CD while alterations in the tertiary structure of the acid unfolded intermediate were probed by near-UV CD. To study the environment and position of the tryptophan residues present intrinsic fluorescence studies were performed. ANS binding studies were also made to know the extent of exposure of the hydrophobic patches. Using the above-mentioned techniques it was found that in presence of fluoroalcohols the pH 2 intermediate was transformed to a state with predominant alpha-helical secondary and denatured tertiary structures. In the pathway of these transformations MG-like intermediates were formed at 10% TFE and 6% HFIP. The folding intermediate of Con-A obtained at pH 2 underwent a series of conformational changes when exposed to different molecular weight PEGs. Secondary structure was induced by low molecular weight PEG-400 and low concentrations of PEG-4000 and PEG-20,000 while at higher concentrations transition in structure was observed. Tertiary structure was stabilized only at low concentrations of PEG-400. PEG-4000 and PEG-20,000 in the whole concentration range resulted in the loss of tertiary structure.     

Multiparameter kinetic study on the unfolding and refolding of bovine carbonic anhydrase B

The kinetics of unfolding and refolding of bovine carbonic anhydrase B by guanidinium chloride have been studied by simultaneously monitoring several spectroscopic parameters, each of which reflects certain unique conformational features of the protein molecule. In the present report, far-UV circular dichroism (CD) was used to follow the secondary structural change, UV difference absorption was used to follow the exposure or burying of aromatic amino acid residues, and near-UV CD was used to follow tertiary structural changes during unfolding and refolding. The unfolding is described by two unimolecular rate processes, and refolding is described by three unimolecular rate processes. The minimum number of conformational species involved in the mechanism is five. The refolding of the protein followed by the above three parameters indicates that the process consists of an initial rapid phase in which the random-coiled protein is converted to an intermediate state(s) having secondary structure comparable to that of the native protein. This is followed by the burying of the aromatic amino acid residues to form the interior of the protein molecule. Subsequently, the protein molecule acquires its tertiary structure and folds into a unique conformation with the formation of aromatic clusters.