Formation of the molten globule-like state of cytochrome c induced by n-alkyl sulfates at low concentrations

The molten globule state of cytochrome c is the major intermediate of protein folding. Elucidation of the thermodynamic mechanism of conformational stability of the molten globule state would enhance our understanding of protein folding. The formation of the molten globule state of cytochrome c was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS, at low concentrations. The refolding states of the protein were monitored by spectroscopic techniques including circular dichroism (CD), visible absorbance and fluorescence. The effect of n-alkyl sulfates on the structure of acid-unfolded horse cytochrome c at pH 2 was utilized to investigate the contribution of hydrophobic interactions to the stability of the molten globule state. The addition of n-alkyl sulfates to the unfolded state of cytochrome c appears to support the stabilized form of the molten globule. The m-values of the refolded state of cytochrome c by SOS, SDeS, SDS, and STS showed substantial variation. The enhancement of m-values as the stability criterion of the molten globule state corresponded with increasing chain length of the cited n-alkyl sulfates. The compaction of the molten globule state induced by SDS, as a prototype for other n-alkyl sulfates, relative to the unfolded state of cytochrome c was confirmed by Stokes radius and thermal transition point (T(m)) measured by microviscometry and differential scanning calorimetry (DSC), respectively. Thus, hydrophobic interactions play an important role in stabilizing the molten globule state.     

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.     

Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dual-functioning protein in the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant-induced unfolding of L-PGDS follows a four-state pathway, including an activity-enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L-PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L-PGDS was a completely reversible process at pH 4.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L-PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two-state unfolding mechanism, the ratio of van't Hoff enthalpy (DeltaH(vH)) to calorimetric enthalpy (DeltaH(cal)) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three-state unfolding process were performed. The heat capacity curves fit well with a three-state process; and the estimated transition temperature (T(m)) and enthalpy change (DeltaH(cal)) of the N<-->I and I<-->U transitions were 48.2 degrees C and 190 kJ.mol(-1), and 60.3 degrees C and 144 kJ.mol(-1), respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290 nm revealed that L-PGDS unfolded through the intermediate state, where its main chain retained the characteristic beta-sheet structure without side-chain interactions.     

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.     

Trifluoroethanol-induced "molten globule" state in stem bromelain

2,2,2-Trifluoroethanol (TFE) denatures proteins but also stabilizes/induces alpha helical conformation in partially/completely unfolded proteins. As reported earlier from this laboratory, stem bromelain is known to exist as a partially folded intermediate (PFI) at pH 2.0. The effect of increasing concentration of TFE on the PFI of bromelain has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino 8-naphthalene sulfonic acid (ANS), and near-UV CD temperature transition. Far-UV CD spectra show considerable accumulation of secondary structure at 70% (v/v) concentration of TFE with spectral features resembling the pH 7.0 preparation. Interestingly the partially folded intermediate regained significant tertiary structure/interactions, with increasing concentration of TFE, and at 60% (v/v) TFE approached almost that of the pseudo native (pH 7.0) state. Further increase to 70% (v/v) TFE, however, resulted in complete loss of tertiary structure/interactions. Studies on tryptophan fluorescence also suggested the induction of some compact structure at 60% (v/v) concentration of TFE. The partially folded intermediate showed enhanced binding of the fluorescent probe (ANS) in the presence of 60% (v/v) TFE. Taken together these observations suggest a "molten globule" state between 60 and 70% (v/v) TFE. Thermal transition studies in the near-UV CD region indicated cooperative transition for PFI in the presence of 60% (v/v) TFE changing to noncooperative transition at 70% (v/v) TFE. This was accompanied by a shift in the midpoint of thermal denaturation (T(m)) from 58 to 51 degrees C. Gradual transition and loss of cooperative thermal unfolding in the 60-70% (v/v) range of TFE also support the existence of the molten globule state.     

Volumetric and spectroscopic characterizations of the native and acid-induced denatured states of staphylococcal nuclease

We have characterized the acid-induced denaturation of staphylococcal nuclease (SNase) at different urea concentrations by a combination of ultrasonic velocimetry, high precision densimetry, and CD spectroscopy. Our CD spectroscopic results suggest that, at low salt and acidic pH, the protein is unfolded with disrupted secondary and tertiary structures. Furthermore, as judged by far UV CD spectra, the protein is further unfolded at acidic pH upon the addition of urea up to the concentration of 1.5 M. The midpoint of the transition shifts to more neutral pH values and the cooperativity of the transition decreases as the acid-induced denaturation of SNase occurs at higher urea concentrations. We find that the change in volume, Deltav, accompanying the acid-induced denaturation of SNase increases from -0.013 cm(3) g(-1) (-218 cm(3) mol(-1)) in the absence of urea to 0.011 cm(3) g(-1) (185 cm(3) mol(-1)) at 1.5 M urea. At all urea concentrations, the partial specific adiabatic compressibility, k(o)(s), of the protein decreases upon its unfolding with the values of Deltak(o)(s) equal to -6.3x10(-6) (-0.106 cm(3) mol(-1) bar(-1)), -4.5x10(-6) (-0.076 cm(3) mol(-1) bar(-1)), -4.6x10(-6) (-0.077 cm(3) mol(-1) bar(-1)), and -3.8x10(-6) (-0.064 cm(3) mol(-1) bar(-1)) cm(3) g(-1) bar(-1) at urea concentrations of 0, 0.5, 1.0, and 1.5 M, respectively. In general, our volumetric results suggest that the acid-induced denatured state of SNase is only partially unfolded with the solvent-exposed surface area equal to 70-80 % of that expected for the fully extended conformation.     

Characterization of a partially folded intermediate of stem bromelain at low pH.

Equilibrium studies on the acid included denaturation of stem bromelain (EC 3.4.22.32) were performed by CD spectroscopy, fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino 8-naphthalene sulfonic acid (ANS). At pH 2.0, stem bromelain lacks a well defined tertiary structure as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of some native like secondary structure at pH 2.0. The mean residue ellipticities at 208 nm plotted against pH showed a transition around pH 4.5 with loss of secondary structure leading to the formation of an acid-unfolded state. With further decrease in pH, this unfolded state regains most of its secondary structure. At pH 2.0, stem bromelain exists as a partially folded intermediate containing about 42.2% of the native state secondary structure Enhanced binding of ANS was observed in this state compared to the native folded state at neutral pH or completely unfolded state in the presence of 6 m GdnHCl indicating the exposure of hydrophobic regions on the protein molecule. Acrylamide quenching of the intrinsic tryptophan residues in the protein molecule showed that at pH 2.0 the protein is in an unfolded conformation with more tryptophan residues exposed to the solvent as compared to the native conformation at neutral pH. Interestingly, stem bromelain at pH 0.8 exhibits some characteristics of a molten globule, such as an enhanced ability to bind the fluorescent probe as well as considerable retention of secondary structure. All the above data taken together suggest the existence of a partially folded intermediate state under low pH conditions.     

Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants

The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation <--> X (intermediate) conformation and the second denaturation phase, X conformation <--> D (denatured) conformation are reversible. Conformational characterization of the X state by the far-UV CD, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements led us to conclude that the X state is a molten globule state. Analysis of denaturation transition curves for the stability of different states in terms of Gibbs energy change at pH 6.0 and 25 degrees C led us to conclude that the N state is more stable than the X state by 9.55 +/- 0.32 kcal mol(-1), whereas the X state is more stable than the D state by only 1.40 +/- 0.25 kcal mol(-1). We have also studied the effect of temperature on the equilibria, N conformation <--> X conformation and X conformation <--> D conformation in the presence of different denaturant concentrations using two different optical probes, namely, [theta](222) and Delta epsilon(400). These measurements yielded T(m), (midpoint of denaturation) and Delta H(m) (enthalpy change) at T(m) as a function of denaturant concentration. A plot of Delta H(m) versus corresponding T(m) was used to determine the constant-pressure heat capacity change, Delta C(p) (= ( partial differential Delta H(m)/ partial differential T(m))(p)). Values of Delta C(p) for N conformation <--> X conformation and X conformation <--> D conformation is 0.92 +/- 0.02 kcal mol(-1) K(-1) and 0.41 +/- 0.01 kcal mol(-1) K(-1), respectively. These measurements suggested that about 30% of the hydrophobic groups in the molten globule state are not accessible to the water.     

Native-like tertiary structure in the Mucor miehei lipase molten globule state obtained at low pH

Studies on the acid-induced denaturation of Mucor miehei lipase (E.C. 3.1.1.3) were performed by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy and binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS). Acid denaturation of the lipase showed loss of secondary structure and alterations in the tertiary structure in the pH range 4 to 2 and 7 to 2 respectively, suggesting that the lipase exists as an acid-unfolded state approximately pH 2.0. A further decrease in pH (from 2.0 to 1.0) resulted in a second transition, which corresponded to the formation of both secondary and tertiary structures. The acid unfolded state at around pH 2.0 has been characterized by significant loss of secondary structure and a small increase in fluorescence intensity with a blue shift of 2 nm, indicating shift of tryptophan residues to less polar environment. Interestingly, the lipase at pH 1.0 exhibits characteristics of molten globule, such as enhanced binding of hydrophobic dye (ANS), native-like secondary structure and slightly altered tryptophanyl environments. That the molten globule of the lipase at pH 1.0 also possesses native-like tertiary structure is an interesting observation made for this lipase.     

Effect of monomeric and oligomeric sugar osmolytes on ΔGD, the Gibbs energy of stabilization of the protein at different pH values: Is the sum effect of monosaccharide individually additive in a mixture?

Thermal denaturation curves of ribonuclease-A were measured by monitoring changes in the far-UV circular dichroism (CD) spectra in the presence of different concentrations of six sugars (glucose, fructose, galactose, sucrose, raffinose and stachyose) and mixture of monosaccharide constituents of each oligosaccharide at various pH values in the range of 6.0-2.0. These measurements gave values of T(m) (midpoint of denaturation), DeltaH(m) (enthalpy change at T(m)), DeltaC(p) (constant-pressure heat capacity change) under a given solvent condition. Using these values of DeltaH(m), T(m) and DeltaC(p) in appropriate thermodynamic relations, thermodynamic parameters at 25 degrees C, namely, DeltaG(D)(o) (Gibbs energy change), DeltaH(D)(o) (enthalpy change), and DeltaS(D)(o) (entropy change) were determined at a given pH and concentration of each sugar (including its mixture of monosaccharide constituents). Our main conclusions are: (i) each sugar stabilizes the protein in terms of T(m) and DeltaG(D)(o), and this stabilization is under enthalpic control, (ii) the protein stabilization by the oligosaccharide is significantly less than that by the equimolar concentration of the constituent monosaccharides, and (iii) the stabilization by monosaccharides in a mixture is fully additive. Furthermore, measurements of the far- and near-UV CD spectra suggested that secondary and tertiary structures of protein in their native and denatured states are not perturbed on the addition of sugars.     

On the nature of the unfolded intermediate in the in vitro transition of the colicin E1 channel domain from the aqueous to the membrane phase.

The transition of the colicin E1 channel polypeptide from a water-soluble to membrane-bound state occurs in vitro at acid pH values that are associated with an unfolded channel structure whose properties qualitatively resemble those of a "molten globule," or "compact unfolded," intermediate state. The role of such a state for activity was tested by comparing the pH dependence of channel-induced solute efflux and the amplitude of the near-UV CD spectrum. The requirement of a partly unfolded state for activity was shown by the coincidence of the onset of channel activity measured for 4 different lipid compositions with the decrease in near-UV CD amplitude as a function of pH. Tertiary constraints on the 3 tryptophans of the colicin channel, assayed by the amplitude of the near-UV CD spectrum, are retained over the pH range 3-4 where channel activity could be measured and, as well, at pH 2. In addition, the tryptophan fluorescence emission spectrum is virtually unchanged over the pH range 2-6. The temperature independence of the near-UV spectrum at pH 3-6 up to 70 degrees C implies that the colicin E1 channel polypeptide is more stable than that of colicin A. A transition between 53 and 58 degrees C in the amplitude of the near-UV CD is consistent with preservation of part of the hydrophobic core in a destabilized state at pH 2. Thus, the unfolded state associated with colicin activity at acidic pH has the properties of a "compact unfolded" state, having some, but not all of the properties of a "molten globule."(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational states of beta-lactamase: molten-globule states at acidic and alkaline pH with high salt

We present evidence that beta-lactamase is close to fully unfolded (i.e., random coil conformation) at low ionic strength at the extremes of pH and that the presence of salt causes a cooperative transition to a conformation with the properties of a molten globule, namely, a compact state with native-like secondary structure but disordered side chains (tertiary structure). The conformation of beta-lactamase I from Bacillus cereus was examined over the pH 1.5-12.5 region by circular dichroism (CD), tryptophan fluorescence, dynamic light scattering, and 1-anilino-8-naphthalenesulfonate (ANS) binding. Under conditions of low ionic strength (I = 0.05) beta-lactamase was unfolded below pH 2.5 and above pH 11.5, on the basis of the far-UV and near-UV CD and tryptophan fluorescence. However, at high ionic strength and low pH an intermediate conformation (state A) was observed, with a secondary structure content similar to that of the native protein but a largely disordered tertiary structure. The transition from the unfolded state (U) to state A induced by KCl was cooperative and had a midpoint at 0.12 M KCl (I = 0.17 M) at pH 1.6. A similar conformation (state B) was observed at high pH and high ionic strength. The transition from the alkaline U state to state B induced by KCl at pH 12.2 was cooperative and had a midpoint at 0.6 M KCl (I = 0.65 M). Light scattering measurements showed that state B was compact although somewhat expanded compared to the N state. The compactness of state A could not be determined due to its strong propensity to aggregate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characteristics of thermostable immunogenic outer membrane protein from Salmonella enterica serovar Typhi

In this work, we explored the acid-induced unfolding pathway of non-porin outer membrane protein (OMP), an immunogenic protein from Salmonella Typhi, by monitoring the conformational changes over a pH range of 1.0–7.0 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, and dynamic light scattering. The spectroscopic measurements showed that OMP in its native state at pH 7.0 exists in more stable and compact conformation. In contrast, at pH 2.0, OMP retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii, and nearly four-fold increase in ANS fluorescence with respect to the native state, indicating that MG state exists at pH 2.0. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of a partially unfolded state between native and unfolded state. The effect of pH on the conformation and thermostability of OMP points towards its heat resistance at neutral pH (T _m?~?69 °C at pH 7.0, monitored by change in MRE_{222 nm}). Acid unfolded state was also characterized by the lack of a cooperative thermal transition. All these results suggested that acid-induced unfolded state of OMP at pH 2.0 represented the molten globule state. The chemical denaturation studies with GuHCl and urea as denaturants showed dissimilar results. The chemical unfolding experiments showed that in both far-UV CD and fluorescence measurements, GuHCl is more efficient than urea. GuHCl is characterized by low C _m (~1 M), while urea is characterized by high C _m (~3 M). The fully unfolded states were reached at 2 M GuHCl and 4 M urea concentration, respectively. This study adds to several key considerations of importance in the development of therapeutic agents against typhoid fever for clinical purposes.     

Structural studies of N- and C-terminally truncated human apolipoprotein A-I

Apolipoprotein A-I (apoA-I) plays an important structural and functional role in lipid transport and metabolism. This work is focused on the central region of apoA-I (residues 60-183) that is predicted to contain exclusively amphipathic alpha-helices. Six N- and/or C-terminally truncated mutants, delta(1-41), delta(1-59), delta(198-243), delta(209-243), delta(1-41,185-243), and delta(1-59,185-243), were analyzed in their lipid-free state in solution at pH 4.7-7.8 by far- and near-UV CD spectroscopy. At pH 7.8, all mutants show well-defined secondary structures consisting of 40-52% alpha-helix. Comparison of the alpha-helix content in the wild type and mutants suggests that deletion of either the N- or C-terminal region induces helical unfolding elsewhere in the structure, indicating that the terminal regions are important for the integrity of the solution conformation of apoA-I. Near-UV CD spectra indicate significant tertiary and/or quaternary structural changes resulting from deletion of the N-terminal 41 residues. Reduction in pH from 7.8 to 4.7 leads to an increase in the mutant helical content by 5-20% and to a large increase in thermal unfolding cooperativity. Van't Hoff analysis of the mutants at pH 4.7 indicates melting temperatures T(m) ranging from 51 to 59 degrees C and effective enthalpies deltaH(v)(T(m)) = 35 +/- 5 kcal/mol, similar to the values for plasma apoA-I at pH 7.8 (T(m) = 57 degrees C, deltaH(v) = 32 kcal/mol). Our results provide the first report of the pH effects on the secondary, tertiary, and/or quaternary structure of apoA-I variants and indicate the importance of the electrostatic interactions for the solution conformation of apoA-I.     

Equilibrium titrations of acid-induced unfolding-refolding and salt-induced molten globule of cytochrome c by FT-IR spectroscopy

Despite extensive investigations on the acid-unfolded and acid/salt-induced molten globule(-like) states of cytochrome c using variety of techniques, structural features of the acid-unfolded state in terms of residual secondary structures and the structural transition between the acid-unfolded and acid/salt-refolded states have not been fully characterized beyond the circular dichroism (CD) spectroscopy. It is unusual that secondary structure(s) of the unfolded state leading to the molten globule state, an important protein folding intermediate, as determined by CD was not fully corroborated by independent experimental method(s). In this study, we carried out an equilibrium titration of acid-induced unfolding and subsequent acid- and salt-induced refolding of cytochrome c using Fourier transform infrared spectroscopy. The spectral profiles of the equilibrium titration reveal new structural details about the acid-unfolded state and the structural transition associated with the acid/salt-refolded molten globule(-like) states of cytochrome c.     

Alcohol-induced versus anion-induced states of alpha-chymotrypsinogen A at low pH

Characterization of conformational transition and folding intermediates is central to the study of protein folding. We studied the effect of various alcohols (trifluoroethanol (TFE), butanol, propanol, ethanol and methanol) and salts (K(3)FeCN(6), Na(2)SO(4), KClO(4) and KCl) on the acid-induced state of alpha-chymotrypsinogen A, a predominantly beta-sheet protein, at pH 2.0 by near-UV circular dichroism (CD), far-UV CD and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence measurements. Addition of alcohols led to an increase in ellipticity value at 222 nm indicating the formation of alpha-helical structure. The order of effectiveness of alcohols was shown to be TFE>butanol>propanol>ethanol>methanol. ANS fluorescence data showed a decrease in fluorescence intensity on alcohol addition, suggesting burial of hydrophobic patches. The near-UV CD spectra showed disruption of tertiary structure on alcohol addition. No change in ellipticity was observed on addition of salts at pH 2.0, whereas in the presence of 2 M urea, salts were found to induce a molten globule-like state as evident from the increases in ellipticity at 222 nm and ANS fluorescence indicating exposure of hydrophobic regions of the protein. The effectiveness in inducing the molten globule-like state, i.e. both increase in ellipticity at 222 nm and increase in ANS fluorescence, followed the order K(3)FeCN(6)>Na(2)SO(4)>KClO(4)>KCl. The loss of signal in the near-UV CD spectrum on addition of alcohols indicating disordering of tertiary structure results suggested that the decrease in ANS fluorescence intensity may be attributed to the unfolding of the ANS binding sites. The results imply that the alcohol-induced state had characteristics of an unfolded structure and lies between the molten globule and the unfolded state. Characterization of such partially folded states has important implications for protein folding.     

Alcohol-induced conformational transitions in ervatamin C. An alpha-helix to beta-sheet switchover

Alcohol-induced conformational transitions of erv C, a highly stable cysteine protease, were followed by CD, fluorescence, and activity. At acidic pH, the addition of different alcohols caused two types of conformational transitions. Increasing the concentration of nonfluorinated alkyl alcohols induced a conformational switch from -helix to -sheet. Under these conditions, the protein lost its proteolytic activity and tertiary structure. The switch was a sudden one, observed in 50% methanol, 45% ethanol, and 40% propanol. Under similar conditions of pH and concentration, however, glycerol and TFE enhanced the -helicity of the protein. Methanol-induced denaturation was observed to occur in two stages; the first is the -sheet state stabilized at low alcohol concentrations, and the other is the -sheet state with enhanced ellipticity stabilized at high alcohol concentrations. This -sheet conformation can be attained from the native as well as 6 M GuHCl-denatured state by addition of methanol and exhibits properties different from the native or unfolded state. This state shows loss of tertiary structure and activity, enhanced nonnative secondary structure, noncooperative temperature unfolding, and higher stability toward denaturants as compared to the native state, which are characteristic of the molten globule-like state or O-state, and thus this state may be functioning as an intermediate in the folding pathway of erv C.     

A recombinant transductor-effector system: in vitro study of G inhibitory protein (G-alpha-i1) direct activators

Most protease prosegments are co-synthesized at the N-termini of cysteine proteases and are involved in folding assistance, inhibition, and activation of their mature enzymes. By using circular dichroism, UV-difference and fluorescence spectroscopies, we studied the thermal unfolding of papain prosegment. The transition seems to be two-state and reversible, with an unfolded state prone to aggregation. Unfolding thermodynamic parameters obtained show low values both for deltaH(Tm) and deltaCp(U), indicative of a loosely packed three-dimensional conformation for the prosegment at near-neutral pH conditions. In spite of these results, fluorescence experiments demonstrate that papain prosegment is able to recognize and inhibit its cognate protease. An acid medium induces a molten globule-like state without intermediates, which in turn undergoes an irreversible thermal unfolding. Our results suggest that papain prosegment has a high degree of conformational flexibility, with the ability to form not only a molten globule-like structure in activating conditions, but also requiring an induced fit in order to be functional as inhibitor.     

Chitosan-soyprotein interaction as determined by thermal unfolding experiments

Chitosan interaction with soybean beta-conglycinin beta(3) was investigated by thermal unfolding experiments using CD spectroscopy. The negative ellipticity of the protein was enhanced with rising solution temperature. The transition temperature of thermal unfolding of the protein (T(m)) was 63.4 degrees C at pH 3.0 (0.15 M KCl). When chitosan was added to the protein solution, the T(m) value was elevated by 7.7 degrees C, whereas the T(m) elevation upon addition of chitosan hexamer (GlcN)(6) was 2.2 degrees C. These carbohydrates appear to interact with the protein stabilizing the protein structure, and the interaction ability could be evaluated from the T(m) elevation. Similar experiments were conducted at various pHs from 2.0 to 3.5, and the T(m) elevation was found to be enhanced in the higher pH region. We conclude that chitosan interacts with beta-conglycinin through electrostatic interactions between the positive charges of the chitosan polysaccharide and the negative charges of the protein surface.     

Compressibility changes accompanying conformational transitions of apomyoglobin

We used high-precision density and ultrasonic velocity measurements to characterize the native (N), molten globule (MG), and unfolded (U) conformations of apomyoglobin. The molten globule states that were studied in this work include the MG(pH4)(NaCl) state observed at pH 4 and 20 mM NaCl, the MG(pH4)(NaTCA) state observed at pH 4 and 20 mM sodium trichloracetate (NaTCA), the MG(pH2)(NaCl) state observed at pH 2 and 200 mM NaCl, and the MG(pH2)(NaTCA) state observed at pH 2 and 20 mM NaTCA. We used our densimetric and acoustic data to evaluate changes in adiabatic compressibility associated with the acid- or salt-induced N-to-MG, MG-to-U, MG-to-MG, and U-to-MG transitions of the protein. The N-to-MG(pH4)(NaCl) and N-to-MG(pH4)(NaTCA) transitions are accompanied by decreases in compressibility of -(3.0 +/- 0.6) x 10(-6) and -(2.0 +/- 0.6) x 10(-6) cm3 g(-1)bar(-1), respectively. The N-to-MG(pH2)(NaCl) and N-to-MG(pH2)(NaTCA) transitions are associated with compressibility changes of -(4.9 +/- 1.1) x 10(-6) and (0.7 +/- 0.9) x 10(-6) cm3 g(-1) bar(-1), respectively. We interpret these data in terms of the degree of unfolding of the various molten globule forms of apomyoglobin. In general, our compressibility data reveal significant disparities between the various equilibrium molten globule states of apomyoglobin while also quantitatively characterizing each of these states. Volumetric insights provided by our data facilitate gaining a better understanding of the folding pathways, intermediates, and kinetics of apomyoglobin folding.