Cold denaturation and heat denaturation of Streptomyces subtilisin inhibitor. 1. CD and DSC studies

Cold denaturation and heat denaturation of the protein Streptomyces subtilisin inhibitor (SSI) were studied in the pH range 1.84-3.21 and in the temperature range -3-70 degrees C by circular dichroism and scanning microcalorimetry. The native structure of the protein was apparently most stabilized at about 20 degrees C and was denatured upon heating and cooling from this temperature. Each denaturation was reversible and cooperative, proceeding in two-state transitions, that is, from the native state to the cold-denatured state or from the native state to the heat-denatured state. The two denatured states, however, were not perfect random-coiled structures, and they differed from each other, indicating that there exist three states in this temperature range, i.e., cold denatured, native, and heat denatured. The difference between the cold and heat denaturations was indicated first by circular dichroism. The isodichroic point for the transition from the native state to the cold-denatured state was different from that from the native state to the heat-denatured state in the pH range between 3.21 and 2.45. Moreover, molar ellipticity for the cold-denatured state was different from that of the heat-denatured state, and the transition from the former to the latter was observed at pH values below 2. Values of van't Hoff enthalpies from the native state to the heat-denatured state at pH values between 3.21 and 2.45 were obtained by curve fitting of the CD data, and delta Cp = 1.82 (+/- 0.11) [kcal/(mol.K)] was obtained from the linear plot of the enthalpies against temperature. The parameters obtained from the heat denaturation studies gave curves for delta G zero which were not in agreement with the experimental data in the cold denaturation region when extrapolated to the low temperature. Moreover, the value of the apparent delta Cp for the cold denaturation in the pH range 3.03-2.45 was estimated to be different from that for the heat denaturation, indicating that the mechanism of the cold denaturation of SSI is different from a simple cold denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cold denaturation and heat denaturation of Streptomyces subtilisin inhibitor. 2. 1H NMR studies

Structural transitions of the protein Streptomyces subtilisin inhibitor (SSI) from the native state to the cold-denatured and heat-denatured states were studied by 1H NMR spectroscopy in the temperature range from -10 to 60 degrees C in the acidic pH range. Assignments of some of the 1H NMR signals of SSI in the cold-denatured and heat-denatured states were performed by a combined use of selective deuteration and site-directed mutagenesis. Throughout the pH range from 2.1 to 3.1, both transitions were cooperative and basically only three distinct spectra corresponding to structures in the cold-denatured, native, and heat-denatured states were detected. In the cold-denatured state, the side-chain signals of Met73, His106, at least one Val, and two Leu were observed at distinctly shifted positions from those for a random-coiled structure, suggesting the formation of a tertiary structure, while those of Met70, His43, and Ala2 were observed at positions for a random-coiled structure. This tertiary structure in the cold-denatured state is entirely different from that in the native state, as some amino acid residues exposed to the solvent in the native state (e.g., Met73, His106) are buried while those sequestered in the native state (e.g., His43) are exposed. In the heat-denatured state, however, most 1H NMR signals were observed at random-coiled positions, indicating that there is much less tertiary structure in the heat-denatured state than in the cold-denatured state. At pH values below 2.09, a structural transition was observed from the cold-denatured state to the heat-denatured state without passing through the native state.(ABSTRACT TRUNCATED AT 250 WORDS)     

A native disulfide stabilizes non-native helical structures in partially folded states of equine β-lactoglobulin

Equine β-lactoglobulin (ELG) assumes non-native helices during refolding and in partially folded states. Previously, circular dichroism (CD) combined with site-directed mutagenesis identified helical regions in the acid- and cold-denatured states of ELG. It is also known that a fragment of ELG, CHIBL (residues 88-142), has a structure similar to that of the cold-denatured state. For the study reported herein, the structure of a shorter fragment, CHIBLΔF (residues 97-142), was investigated by CD and nuclear magnetic resonance spectroscopy. The secondary chemical shifts clearly showed that non-native α-helices are present in two different regions, residues 98-107 and 114-135, and are connected by a native disulfide bond. The CD spectra of two peptides that correspond to the helical regions are characterized by weak helical signatures, and the sum of their CD spectra is nearly the same as the spectrum of disulfide-reduced CHIBLΔF. Therefore, the non-native helices are stabilized by the disulfide, and non-native helix formation may occur only during the refolding of the disulfide-intact protein. Supporting this conclusion is the observation that tear lipocalin, a homologue of ELG that lacks the disulfide, does not form non-native helices during folding.     

Urea-induced conformational changes in cold- and heat-denatured states of a protein, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI) is known to exist in at least two distinct denatured states, cold-denatured (D') and heat-denatured (D) under acidic conditions. In the present work, we investigated the manner how increasing urea concentration from 0 to 8 M changes the polypeptide chain conformation of SSI that exists initially in the D' and D states as well as in the native state (N), in terms of the secondary structure, the tertiary structure, and the chain form, based on the results of the experiments using circular dichroism (CD), small-angle X-ray scattering (SAXS) and 1H-NMR spectroscopy. Our results indicate that the urea-induced conformational transitions of SSI under typical conditions of D' (pH 1.8, 3 degrees C) occur at least in two steps. In the urea concentration range of 0-2 M (step 1), a cooperative destruction of the tertiary structure occurs, resulting in a mildly denatured state (DU), which may still contain a little amount of secondary structures. In the concentration range of 2-4 M urea (step 2), the DU state gradually loses its residual secondary structure, and increases the radius of gyration nearly to a maximum value. At 4 M urea, the polypeptide chain is highly disordered with highly mobile side chains. Increasing the urea concentration up to 8 M probably results in the more highly denatured or alternatively the stiffer chain conformations. The conformational transition starting from the N state proceeds essentially the same way as in the above scheme in which D' is replaced with N. The conformational transition starting from the D state lacks step 1 because the D state contains no tertiary structures and is similar to the DU state. The fact that similar conformations are reached at urea concentrations above 2 M from different conformations of D', D, and N indicates that the effect of urea dominates in determining the polypeptide conformation of SSI in the denatured states rather than the pH and temperature.     

A 13C nuclear magnetic resonance and circular dichroism study of the collagen-gelatin transformation in enzyme solubilized collagen.

Natural abundance Fourier transform 13C nuclear magnetic resonance (13C NMR) were obtained for enzyme solubilized collagen at 1 degrees intervals through the transition region. The transition of collagen molecules from the rigid triple helical state to single-stranded, random-coil state is accompanied by a change from broadened carbon resonances unobservable under high-resolution conditions to narrow line spectra. Thus distinction can be made between helical and random-coil states of individual residues. The transition is monophasic, as determined by examination of 14 different carbon resonances, and the entire structure is found to melt cooperatively over a temperature interval of 5 +/- 1 degrees. All the residues seem to be involved in the unfolding process concurrently. The transition was also studied by examining the changes in the circular dichroism spectrum brought about by heating. The experiments corroborated the observation that the transition proceeded cooperatively over a temperature interval of 4 degrees. Enzyme soluble collagen is seen to melt less cooperatively than native collagen. The enthalpy change was determined by assuming an equilibrium between three random coil gelatin chains and tropocollogen molecules. From the enthalpy, the average length of the tripeptide sequences (70-85) involved in the transition can be estimated. The shortening of the cooperative unit could arise as a result of some alteration of the native conformation through proctase treatment.     

Hairpin folding dynamics: the cold-denatured state is predisposed for rapid refolding

Cold denaturation is a general phenomenon in globular proteins, and the associated cold-denatured states of proteins have important fundamental and practical significance. Here, we have characterized the cold-denatured state of a beta-hairpin forming peptide, MrH3a, in 8% hexafluoro-2-propanol (HFIP) and the dynamics of its refolding following a laser-induced T-jump. Beta-hairpins constitute an important class of protein structural elements, yet their folding mechanisms are not fully understood. Characterization of MrH3a using NMR, CD, and IR spectroscopies reveals residual structure in the cold-denatured state, in contrast with the highly disordered heat-denatured state. The residual structure in the cold-denatured state comprises relatively compact and solvent protected conformations. Furthermore, we find a substantial acceleration in the rate of folding from the cold-denatured state compared to that of the heat-denatured state. In addition, the cold-denatured state is not populated in 20% HFIP; folding occurs only from the fully unfolded state and is significantly slower. We interpret the acceleration of the folding rate of MrH3a in 8% HFIP as a direct consequence of the collapsed conformations of the cold-denatured state. Finally, there may be some reduction of the loop search cost when starting from the cold-denatured state, since this state may have some of the stabilizing cross-strand interactions already formed.     

Enthalpic and entropic contributions to actin stability: calorimetry, circular dichroism, and fluorescence study and effects of calcium

The delta H associated with the thermal unfolding of G-actin has been determined by differential scanning calorimetry (DSC) to be 142 +/- 5 kcal/mol, with the Tm (melting temperature) at 57.2 +/- 0.5 degrees C, at pH 8.0 (heating rate 0.5 K/min). The transition is broad and cannot be treated as a single transition that mimics a two-state process, suggesting the existence of domains. Deconvolution is done to fit it into two quasi-independent two-state transitions. For F-actin, the transition is more cooperative, with a cooperative ratio (the ratio of van't Hoff enthalpy and calorimetric enthalpy) of 1.4, indicating intermonomer interaction. The delta H of the thermal unfolding of F-actin is 162 +/- 10 kcal/mol with a Tm at 67.0 +/- 0.5 degrees C. A state of G-actin similar to that of the heat-denatured form, designated D-actin, is obtained by removing tightly bound Ca2+ with EGTA. The DSC-detectable cooperative transition is completely lost when the free calcium concentration of the medium is 1 x 10(-11) M or lower, using a Ca2+/EGTA buffer system. However, circular dichroism (CD) shows that the helix content of actin, 32% in the G-form, is only partially reduced to 19% in this apo form. The CD spectrum and the helix content of the calcium-depleted actin are almost identical with those of the heat-denatured D form. This loss of 40% of the native helical content is irreversible in both cases. The remaining 60% of the native helical content cannot be further eliminated by heating to 95 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature control for kinetic refolding of heat-denatured ovalbumin.

The folding of heat-denatured ovalbumin, a non-inhibitory serpin with a molecular size of 45 kDa, was examined. Ovalbumin was heat-denatured at 80 degrees C under nonreducing conditions at pH 7.5 and then cooled either slowly or rapidly. Slow cooling allowed the heat-denatured ovalbumin to refold to its native structure with subsequent resistance to digestion by trypsin. Upon rapid cooling, by contrast, the heat-denatured molecules assumed the metastable non-native conformations that were susceptible to trypsin. The non-native species were marginally stable for several days at a low temperature, but the molecules were transformed slowly into the native conformation. Considering data from size-exclusion chromatography and from analyses of CD, intrinsic tryptophan fluorescence, and adsorption of the dye 1-anilinonaphthalene-8-sulfonate, we postulated that the non-native species that accumulated upon rapid cooling were compact but structureless globules with disordered side chains collectively as a folding intermediate. Temperature-jumped CD experiments revealed biphasic kinetics for the refolding process of heat-denatured ovalbumin, with the features of increasing and subsequently decreasing amplitude of the rapid and the slow phases, respectively, with the decrease in folding temperature. The temperature dependence of the refolding kinetics indicated that the yield of renaturation was maximal at about 55 degrees C. These findings suggested the kinetic partitioning of heat-denatured ovalbumin between alternative fates, slow renaturation to the native state and rapid collapse to the metastable intermediate state. Analysis of disulfide pairing revealed the formation of a scrambled form with non-native disulfide interactions in both the heat-denatured state and the intermediate state that accumulated upon rapid cooling, suggesting that non-native disulfide pairing is responsible for the kinetic barriers that retard the correct folding of ovalbumin.     

Helix-rich transient and equilibrium intermediates of equine beta-lactoglobulin in alkaline buffer

Acidic buffer conditions are known to stabilize helix-rich states of even those proteins with a predominantly beta-sheet native secondary structure. Here we investigated whether such states also exist under alkaline buffer conditions. The guanidine hydrochloride (GuHCl)-induced unfolding transition and kinetic refolding of equine beta-lactoglobulin (ELG) by GuHCl-jump were investigated at pH 8.7 by far-ultraviolet circular dichroism. We found that an equilibrium intermediate appeared in 45% ethylene glycol (EGOH) buffer with 1.5 M GuHCl. The intermediate is rich in non-native alpha-helix, which is similar to the helix-rich state of ELG at pH 4.0. A kinetic study was done on the folding rate of ELG and compared with bovine beta-lactoglobulin (BLG). Transient intermediates, which were observed as the burst phase of the refolding reaction, were also rich in alpha-helix. The activation enthalpy of ELG was calculated to be c.a. 80 kJ/mol, whereas that of BLG was c.a. 70 kJ/mol in the presence of 45% EGOH. The ellipticities of the transient intermediate of ELG show temperature dependence in the presence of 45% EGOH, whereas that of BLG did not show significant dependence. This study therefore extends the existence of helix-rich equilibrium and transient intermediates of predominantly beta-sheet proteins to alkaline buffer conditions.     

Native-like beta-hairpin retained in the cold-denatured state of bovine beta-lactoglobulin.

Bovine beta-lactoglobulin is denatured by increased temperature (heat denaturation) and by decreased temperature (cold-denaturation) in the presence of 4 M urea at pH 2.5. We characterized the structure of the cold-denatured state of beta-lactoglobulin using circular dichroism (CD), small-angle X-ray scattering (SAXS) and heteronuclear nuclear magnetic resonance (NMR). CD and SAXS indicated that the cold-denatured state, in comparison with the highly denatured state induced by urea, is rather compact, retaining some secondary structure, but no tertiary structure. The location of the residual structures in the cold-denatured state and their stability were characterized by 1H/2H exchange combined with heteronuclear NMR. The results indicated that the residues adjacent to the disulfide bond (C106-C119) connecting beta-strands G and H had markedly high protection factors, suggesting the presence of a native-like beta-hairpin stabilized by the disulfide bond. Since this beta-hairpin is conserved between different conformational states, including the kinetic refolding intermediate, it should be of paramount importance for the folding and stability of beta-lactoglobulin. On the other hand, the non-native alpha-helix suggested for the folding intermediate was not detected in the cold-denatured state. The 1H/2H exchange experiments showed that the protection factors of a mixture of the native and cold-denatured states is strongly biased by that of the labile cold-denatured state, consistent with a two-process model of the exchange.     

Influence of Ca2+ binding on the structure and stability of bovine alpha-lactalbumin studied by circular dichroism and nuclear magnetic resonance spectra

Both the Ca2+-bound and Ca2+-free forms of alpha-lactalbumin can assume essentially the same folded conformation as evidenced by similarity in their CD and proton n.m.r. spectra. Thermal unfolding followed by the aromatic CD has shown that the stability of the folded state is markedly enhanced by Ca2+ and that the stabilization is almost entirely entropic; addition of 0.1 mM Ca2+ shifts the transition temperature from 40 degrees to 62 degrees in 0.1M Na+ at pH 7.0. The enthalpy change of the unfolding, coincident between the two forms, is, however, significantly smaller than that known for lysozyme. The n.m.r. spectrum under the condition that both the forms of the protein are in the folded state reflects minor environmental changes of certain protons upon Ca2+ binding, and these changes are shown to afford useful probes for assessment of the location of the binding site. From the pH dependence and temperature dependence of the spectrum and also by using spin decoupling in the aromatic region (6.4-8.7 p.p.m.), it is shown that none of histidyl residues are affected and that at least two tryptophanyl ring protons experience environmental changes upon Ca2+ binding to the folded apo-protein. Effect of free excess Ca2+ on the spectrum has also shown that in native alpha-lactalbumin there is only one Ca2+-binding site that is detectable by the present method.     

Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased beta-sheet and reduced alpha-helix content relative to the native state. Urea-induced unfolding at 25 degrees C reveals a two-step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15 degrees to 60 degrees C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near-ultraviolet CD spectra at 25 degrees C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.     

Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.     

Conformation and stability of thiol-modified bovine beta-lactoglobulin

Bovine beta-lactoglobulin A assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. Beta-lactoglobulin A has a free thiol at Cys121, which is buried between the beta-barrel and the C-terminal major alpha-helix. This thiol group was specifically reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 1.0 M Gdn-HCI at pH 7.5, producing a modified beta-lactoglobulin (TNB-bIg) containing a mixed disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). The conformation and stability of TNB-bIg were studied by circular dichroism (CD), tryptophan fluorescence, analytical ultracentrifugation, and one-dimensional 1H-NMR. The CD spectra of TNB-bIg indicated disordering of the native secondary structure at pH 7.5, whereas a slight increase in the alpha-helical content was observed at pH 2.0. The tryptophan fluorescence of TNB-bIg was significantly quenched compared with that of the intact protein, probably by the energy transfer to TNB. Sedimentation equilibrium analysis indicated that, at neutral pH, TNB-bIg is monomeric while the intact protein is dimeric. In contrast, at pH 2.0, both the intact beta-lactoglobulin and TNB-bIg were monomeric. The unfolding transition of TNB-bIg induced by Gdn-HCl was cooperative in both pH regions, although the degree of cooperativity was less than that of the intact protein. The 1H-NMR spectrum for TNB-bIg at pH 3.0 was native-like, whereas the spectrum at pH 7.5 was similar to that of the unfolded proteins. These results suggest that modification of the buried thiol group destabilizes the rigid hydrophobic core and the dimer interface, producing a monomeric state that is native-like at pH 2.0 but is molten globule-like at pH 7.5. Upon reducing the mixed disulfide of TNB-bIg with dithiothreitol, the intact beta-lactoglobulin was regenerated. TNB-bIg will become a useful model to analyze the conformation and stability of the intermediate of protein folding.     

Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase

The effect of trehalose (0.5 M) on the thermal stability of cutinase in the alkaline pH range was studied. The thermal unfolding induced by increasing temperature was analyzed in the absence and in the presence of trehalose according to a two-state model (which assumes that only the folded and unfolded states of cutinase were present). Trehalose delays the reversible unfolding. The midpoint temperature of the unfolding transition (Tm) increases by 4.0 degrees C and 2. 6 degrees C at pH 9.2 and 10.5, respectively, in the presence of trehalose. At pH 9.2 the thermal unfolding occurs at higher temperatures (Tm is 52.6 degrees C compared to 42.0 degrees C at pH 10.5) and a refolding yield of around 80% was obtained upon cooling. This pH value was chosen to study the irreversible inactivation (long-term stability) of cutinase. Temperatures in the transition range from folded to unfolded state were selected and the rate constants of irreversible inactivation determined. Inactivation followed first-order kinetics and trehalose reduced the observed rate constants of inactivation, pointing to a stabilizing effect on the irreversible inactivation step of thermal denaturation. However, if the contribution of reversible unfolding on the irreversible inactivation of cutinase was taken into account, i.e., considering the fraction of cutinase molecules in the reversible unfolded conformation, the intrinsic rate constants can be calculated. Based on the intrinsic rate constants it was concluded that trehalose does not delay the irreversible inactivation. This conclusion was further supported by comparing the activation energy of the irreversible inactivation in the absence and in the presence of trehalose. The apparent activation energy in the absence and in the presence of trehalose were 67 and 99 Kcal/mol, respectively. The activation energy calculated from intrinsic rate constants was higher in the absence (30 Kcal/mol) than in the presence of trehalose (16 Kcal/mol), showing that kinetics of the irreversible inactivation step increased in the presence of trehalose. In fact, trehalose stabilized only the reversible step of thermal denaturation of cutinase.     

Conformational intermediates in the folding of a coiled-coil model peptide of the N-terminus of tropomyosin and alpha alpha-tropomyosin.

Circular dichroism was used to study the folding of alpha alpha-tropomyosin and AcTM43, a 43-residue peptide designed to serve as a model for the N-terminal domain of tropomyosin. The sequence of the peptide is AcMDAIKKKMQMLKLDVENLLDRLEQLEADLKALEDRYKQLEGGC. The peptide appeared to form a coiled coil at low temperatures (< 25 degrees C) in buffers with physiological ionic strength and pH. The folding and unfolding of the peptide, however, were noncooperative. When CD spectra were examined as a function of temperature, the apparent degree of folding differed when the ellipticity was followed at 222, 208, and 280 nm. Deconvolution of the spectra suggested that at least three component curves contributed to the CD in the far UV. One component curve was similar to the CD spectrum of the coiled-coil alpha-helix of native alpha alpha-tropomyosin. The second curve resembled the spectrum of single-stranded short alpha-helical segments found in globular proteins. The third was similar to that of polypeptides in the random coil conformation. These results suggested that as the peptide folded, the alpha-helical content increased before most of the coiled coil was formed. When the CD spectrum of striated muscle alpha alpha-tropomyosin was examined as a function of temperature, the unfolding was also not totally cooperative. As the temperature was raised from 0 to 25 degrees C, there was a decrease in the coiled coil and an increase in the conventional alpha-helix type spectrum without formation of random coil. The major transition, occurring at 40 degrees C, was a cooperative transition characterized by the loss of all of the remaining coiled coil and a concomitant increase in random coil.     

Investigation of apomyoglobin stability depending on urea and temperature at two different pH values

Equilibrium unfolding of apomyoglobin by urea was investigated in the temperature range from 5 to 25 degrees C at two pH values. The thermodynamic parameters of the apomyoglobin native-unfolded state transition were determined. Conformational changes in the protein structure were monitored by tryptophan fluorescence and far UV circular dichroism. Apomyoglobin preserves its native conformation at pH 5.7 and 6.2 in the temperature range used. It was shown that the apomyoglobin stability and its unfolding cooperativity are substantially lower at 5 degrees C than at other temperatures. This fact should be taken in account at the investigation of apomyoglobin.     

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.     

Conformational stability of apoflavodoxin.

Flavodoxins are alpha/beta proteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin from Anaboena PCC 7119 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 11 and pH 6, the apoprotein is folded properly as judged from near-ultraviolet (UV) circular dichroism (CD) and high-field 1H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two-state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5-2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far-UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two-state process at acidic pH. Apoflavodoxin constitutes a promising system for the analysis of the stability and folding of alpha/beta proteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors.     

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.