The location of an engineered inter-subunit disulfide bond in factor for inversion stimulation (FIS) affects the denaturation pathway and cooperativity

Two crossed-linked variants of the homodimeric DNA binding protein factor for inversion stimulation (FIS) were created via engineering of single intermolecular disulfide bonds. The conservative S30C and the nonconservative V58C FIS independent mutations resulted in FIS crossed-linked at the A helix (C30-C30) and at the middle of the B helix (C58-C58). This study sought to investigate how the location of an intermolecular disulfide bond may determine the effect on stability and its propagation through the structure to preserve or alter the denaturation cooperativity of FIS. The oxidized and reduced S30C and V58C FIS exhibited a far-UV CD spectrum and DNA binding affinities that were similar to WT FIS, indicating no significant changes in secondary and tertiary structure. However, the reduced and oxidized forms of the mutants revealed significant differences in the stability and equilibrium denaturation mechanism between the two mutants. In the reduced state, S30C FIS had very little effect on FIS stability, whereas V58C FIS was 2-3 kcal/mol less stable than WT FIS. Interestingly, while both disulfide bonds significantly increased the resistance to urea- and guanidine hydrochloride (GuHCl)-induced denaturation, oxidized V58C FIS exhibited a three-state GuHCl-induced transition. In contrast, oxidized S30C FIS displayed a highly cooperative WT-like transition with both denaturants. The three-state denaturation mechanism of oxidized V58C FIS induced by the GuHCl salt was reproduced by urea denaturation at pH 4, suggesting that disruption of a C-terminus salt-bridge network is responsible for the loss of denaturation cooperativity of V58C FIS in GuHCl or urea, pH 4. A second mutation on V58C FIS created to place a single tryptophan probe (Y95W) at the C-terminus further implies that the denaturation intermediate observed in disulfide crossed-linked V58C FIS results from a decoupling of the stabilities of the C-terminus and the rest of the protein. These results show that, unlike the C30-C30 intermolecular disulfide bond, the C58-C58 disulfide bond did not evenly stabilize the FIS structure, thereby highlighting the importance of the location of an engineered disulfide bond on the propagation of stability and the denaturation cooperativity of a protein.     

The role of the local environment of engineered Tyr to Trp substitutions for probing the denaturation mechanism of FIS

Factor for inversion stimulation (FIS), a 98-residue homodimeric protein, does not contain tryptophan (Trp) residues but has four tyrosine (Tyr) residues located at positions 38, 51, 69, and 95. The equilibrium denaturation of a P61A mutant of FIS appears to occur via a three-state (N₂ ⇆ I₂ ⇆ 2U) process involving a dimeric intermediate (I₂). Although it was suggested that this intermediate had a denatured C-terminus, direct evidence was lacking. Therefore, three FIS double mutants, P61A/Y38W, P61A/Y69W, and P61A/Y95W were made, and their denaturation was monitored by circular dichroism and Trp fluorescence. Surprisingly, the P61A/Y38W mutant best monitored the N₂ ⇆ I₂ transition, even though Trp38 is buried within the dimer removed from the C-terminus. In addition, although Trp69 is located on the protein surface, the P61A/Y69W FIS mutant exhibited clearly biphasic denaturation curves. In contrast, P61A/Y95W FIS was the least effective in decoupling the two transitions, exhibiting a monophasic fluorescence transition with modest concentration-dependence. When considering the local environment of the Trp residues and the effect of each mutation on protein stability, these results not only confirm that P61A FIS denatures via a dimeric intermediate involving a disrupted C-terminus but also suggest the occurrence of conformational changes near Tyr38. Thus, the P61A mutation appears to compromise the denaturation cooperativity of FIS by failing to propagate stability to those regions involved mostly in intramolecular interactions. Furthermore, our results highlight the challenge of anticipating the optimal location to engineer a Trp residue for investigating the denaturation mechanism of even small proteins.     

A truncated peptide model of the mutant P61A FIS forms a stable dimer

Factor for inversion stimulation (FIS) is a 98-residue homodimeric DNA-binding protein involved in several different cellular processes including DNA inversion and the regulation of multiple genes. FIS contains a flexible and functionally important N-terminus followed by four helices (A-D), the last two of which consist of the DNA-binding region. Helix B, which comprises the main dimerization interface has a 20 degrees kink at its center that was originally thought to be caused by the presence of a proline at position 61. However, it was later shown that the kink remained largely intact and that FIS retained its native-like function when the proline was mutated to an alanine. We previously showed that the P61A mutation increased the stability of FIS, but decreased its equilibrium denaturation cooperativity apparently due to preferential stabilization of the B-helix. Here we studied a peptide of P61A FIS, corresponding to residues 26-71 (26-71(A3) FIS), which encompasses the dimer interface (helices A and B). Circular dichroism (CD) and size-exclusion chromatography/multi-angle light scattering showed that the peptide was alpha-helical and dimeric, respectively, as expected based on the 3D structure of FIS. Urea-induced equilibrium denaturation experiments monitored by far-UV CD revealed a concentration-dependent transition, and data analysis based on a N2<-->2U model yielded a DeltaG of approximately -10 kcal/mol. Our results suggest that 26-71(A3) FIS can form a stable dimeric structure despite lacking the N- and C-terminus of native FIS.     

Differential scanning calorimetry of the thermal denaturation of lactate dehydrogenase

1. Differential scanning calorimetry has been used to study the thermal denaturation of lactate dehydrogenase. At pH 7.0 in 0.1 M potassium phosphate buffer, only one transition was observed. Both the enthalpy of denaturation and the melting temperature are linear function of heating rate. The enthalpy is 430 kcal/mol and the melting temperature 61 degrees C at 0 degrees C/min heating rate. The ratio of the calorimetric heat to the effective enthalpy indicated that the denaturation is highly cooperative. Subunit association does not appear to significantly contribute to the enthalpy of denaturation. 2. Both cofactor and sucrose addition stabilized the protein against thermal denaturation. Pyruvate addition produced no changes. Only a small time-dependent destabilization was observed at low concentrations of urea. Large effects were observed in concentrated NaCl solutions and with sulfhydryl-modified lactate dehydrogenase.     

Biophysical Characterization of the Dimer and Tetramer Interface Interactions of the Human Cytosolic Malic Enzyme

The cytosolic NADP⁺-dependent malic enzyme (c-NADP-ME) has a dimer-dimer quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In this study, the urea-induced unfolding process of the c-NADP-ME interface mutants was monitored using fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation and enzyme activities. Here, we demonstrate the differential protein stability between dimer and tetramer interface interactions of human c-NADP-ME. Our data clearly demonstrate that the protein stability of c-NADP-ME is affected predominantly by disruptions at the dimer interface rather than at the tetramer interface. First, during thermal stability experiments, the melting temperatures of the wild-type and tetramer interface mutants are 8–10°C higher than those of the dimer interface mutants. Second, during urea denaturation experiments, the thermodynamic parameters of the wild-type and tetramer interface mutants are almost identical. However, for the dimer interface mutants, the first transition of the urea unfolding curves shift towards a lower urea concentration, and the unfolding intermediate exist at a lower urea concentration. Third, for tetrameric WT c-NADP-ME, the enzyme is first dissociated from a tetramer to dimers before the 2 M urea treatment, and the dimers then dissociated into monomers before the 2.5 M urea treatment. With a dimeric tetramer interface mutant (H142A/D568A), the dimer completely dissociated into monomers after a 2.5 M urea treatment, while for a dimeric dimer interface mutant (H51A/D90A), the dimer completely dissociated into monomers after a 1.5 M urea treatment, indicating that the interactions of c-NADP-ME at the dimer interface are truly stronger than at the tetramer interface. Thus, this study provides a reasonable explanation for why malic enzymes need to assemble as a dimer of dimers.     

Apolipoprotein A-I(Milano) unfolds via an intermediate state as studied by differential scanning calorimetry and circular dichroism.

In this study the thermal and denaturant induced denaturation behaviors of apolipoprotein A-I(Milano) (apo A-IM) have been studied by differential scanning calorimetry and circular dichroism spectroscopy, as well as solution properties by analytical ultracentrifugation. Thermal denaturation is dependent on pH, sodium phosphate concentration and NaCl concentration. The protein is highly self-associated at the protein concentrations used in this study. Denaturation of apo A-IM at pH 7.4 and 8.0 occurs in two steps. The midpoint between the transition is at 37 degrees C. The first step at 31 degrees C involves melting of tertiary structure and rearrangement of protein association complexes, i.e. a transition into an intermediate molten globular-like state. Subsequent melting of this intermediate state into an unfolded state occurs at 52 degrees C. At pH 2.8 the protein lacks all tertiary structure and denaturation occurs over a large temperature interval, indicating the induction of a molten globular-like state at low pH.     

Thermal stability of Phaseolus vulgaris leucoagglutinin: a differential scanning calorimetry study

Phaseolus vulgaris phytohemagglutinin L is a homotetrameric-leucoagglutinating seed lectin. Its three-dimensional structure shows similarity with other members of the legume lectin family. The tetrameric form of this lectin is pH dependent. Gel filtration results showed that the protein exists in its dimeric state at pH 2.5 and as a tetramer at pH 7.2. Contrary to earlier reports on legume lectins that possess canonical dimers, thermal denaturation studies show that the refolding of phytohemagglutinin L at neutral pH is irreversible. Differential scanning calorimetry (DSC) was used to study the denaturation of this lectin as a function of pH that ranged from 2.0 to 3.0. The lectin was found to be extremely thermostable with a transition temperature around 82 degrees C and above 100 degrees C at pH 2.5 and 7.2, respectively. The ratio of calorimetric to vant Hoff enthalpy could not be calculated because of its irreversible-folding behavior. However, from the DSC data, it was discovered that the protein remains in its compact-folded state, even at pH 2.3, with the onset of denaturation occurring at 60 degrees C.     

Unfolding of the trp repressor from Escherichia coli monitored by fluorescence, circular dichroism and nuclear magnetic resonance

The denaturation of the trp repressor from Escherichia coli has been studied by fluorescence, circular dichroism and proton magnetic resonance spectroscopy. The dependences of the fluorescence emission of the two tryptophan residues on the concentration of urea are not identical. The dependence of the quenching of tryptophan fluorescence by iodide as a function of urea concentration also rules out a two-state transition. The circular dichroism at 222 nm decreases in two phases as urea is added. Normalised curves for different residues observed by 1H NMR also do not coincide, and require the presence of at least one stable intermediate. Analysis of the dependence of the denaturation curves on the concentration of protein indicate that the first transition is a partial unfolding of the dimeric repressor, resulting in a loss of about 25% of the helical content. The second transition is the dissociation and unfolding of the partially unfolded dimer. At high concentrations of protein (500 microM) about 73% of the repressor exists as the intermediate in 4 M urea. The apparent dissociation constant is about 10(-4) M; the subunits are probably strongly stabilised by the subunit interaction. The native repressor is stable up to at least 70 degrees C, whereas the intermediate formed at 4 M urea can be denatured reversibly by heating (melting temperature approximately 60 degrees C, delta H approximately 230 kJ/mol).     

Differential scanning calorimetry of the irreversible denaturation of Rapana thomasiana (marine snail, Gastropod) hemocyanin

The thermal denaturation of the hemocyanin from gastropod Rapana thomasiana (RtH) at neutral pH was studied by means of differential scanning calorimetry (DSC). The denaturation was completely irreversible as judged by the absence of any endotherm on rescanning of previously scanned samples. Two transitions, with apparent transition temperatures (T(m)) at 83 and 90 degrees C, were detected by DSC using buffer 20 mM MOPS, containing 0.1 M NaCl, 5 mM CaCl(2) and 5 mM MgCl(2), pH 7.2. Both T(m) were dependent on the scanning rate, suggesting that the thermal denaturation of RtH is a kinetically controlled process. The activation energy (E(A)) of 597+/-20 kJ mol(-1) was determined for the main transition (at 83 degrees C). E(A) for the second transition was 615+/-25 kJ mol(-1). The T(m) and Delta H(cal) values for the thermal denaturation of RtH were found to be independent of the protein concentration, signifying that the dissociation of the protein into monomers does not take place before the rate-determining state of the process of thermal unfolding.     

Multiple unfolding states of glutathione transferase from Physa acuta (Gastropoda [correction of Gastropada]: Physidae)

The equilibrium unfolding of the major Physa acuta glutathione transferase isoenzyme (P. acuta GST(3)) has been performed using guanidinium chloride (GdmCl), urea, and acid denaturation to investigate the unfolding intermediates. Protein transitions were monitored by intrinsic fluorescence. The results indicate that unfolding of P. acuta GST(3) using GdmCl (0-3.0M) is a multistep process, i.e., three intermediates coexist in equilibrium. The first intermediate, a partially dissociated dimer, exists at low GdmCl concentration (approximately at 0.7M). At 1.2M GdmCl, a dimeric intermediate with a compact structure was observed. This intermediate undergoes dissociation into structural monomers at 1.75M of GdmCl. The monomeric intermediate started to be completely unfolding at higher GdmCl concentrations (>1.8M). Unfolding using urea (0-7.0M) and acid-induced structures as well as the fluorescence of 8-anilino-1-naphthalenesulfonate in the presence of different GdmCl concentrations confirmed that the unfolding is a multistep process. At concentrations of GdmCl or urea less than the midpoints or at the midpoint pH (pH 4.2-4.6), the unfolding transition is protein concentration independent and involved a change in the subunit tertiary structure yielding a partially active dimeric intermediate. The binding of glutathione to the enzyme active site stabilizes the native dimeric state.     

Folding mechanism of FIS, the intertwined, dimeric factor for inversion stimulation

FIS, the factor for inversion stimulation, from Escherichia coli and other enteric bacteria, is an interwined alpha-helical homodimer. Size exclusion chromatography and static light scattering measurements demonstrated that FIS is predominately a stable dimer at the concentrations (1-10 microM monomer) and buffer conditions employed in this study. The folding and unfolding of FIS were studied with both equilibrium and kinetic methods by circular dichroism using urea and guanidinium chloride (GdmCl) as the perturbants. The equilibrium folding is reversible and well-described by a two-state folding model, with stabilities at 10 degrees C of 15.2 kcal mol(-1) in urea and 13.5 kcal mol(-1) in GdmCl. The kinetic data are consistent with a two-step folding reaction where the two unfolded monomers associate to a dimeric intermediate within the mixing time for the stopped-flow instrument (<5 ms), and a slower, subsequent folding of the dimeric intermediate to the native dimer. Fits of the burst phase amplitudes as a function of denaturant showed that the free energy for the formation of the dimeric intermediate constitutes the majority of the stability of the folding (9.6 kcal mol(-1) in urea and 10.5 kcal mol(-1) in GdmCl). Folding-to-unfolding double jump kinetic experiments were also performed to monitor the formation of native dimer as a function of folding delay times. The data here demonstrate that the dimeric intermediate is obligatory and on-pathway. The folding mechanism of FIS, when compared to other intertwined, alpha-helical, homodimers, suggests that a transient kinetic dimeric intermediate may be a common feature of the folding of intertwined, segment-swapped, alpha-helical dimers.     

Structural and conformational changes of beta-lactoglobulin B: an infrared spectroscopic study of the effect of pH and temperature

Infrared spectra of 2.5 mM solutions of beta-lactoglobulin B were recorded as a function of pH (from pH 2 to pH 13) and as a function of temperature (from -100 degrees C to +90 degrees C). An analysis of the pH- and temperature-induced changes in the secondary structure was performed based on changes in the conformation-sensitive amide I bands of beta-lactoglobulin. Whereas the total amount of beta-structure remains constant (56-59%) between pH 2 and pH 10, the proportions of the various beta-components do change. In particular, the dimerization of the monomeric protein, induced by raising the pH from 2 to 3 , leads to an increase in the intensity of the 1636 cm-1 band (associated with antiparallel beta-sheet), at the expense of the 1626 cm-1 band (associated with exposed beta-strands). Both the thermal and alkaline denaturation of beta-lactoglobulin occur in two distinct stages. Although the spectra (i.e., the structures) after complete thermal or alkaline denaturation are clearly different, the spectrum of the protein after the first stage of thermal denaturation (at about 60 degrees C) is the same as that after the first stage of alkaline denaturation (at pH 11), suggesting a common denaturation intermediate, which probably represents a crossover point in a complex potential hypersurface.     

A 2D-IR study of heat- and [(13)C]urea-induced denaturation of sarcoplasmic reticulum Ca(2+)-ATPase

Two-dimensional infrared correlation spectroscopy (2D-IR) was applied to the study of urea- and heat-induced unfolding denaturation of sarcoplasmic reticulum Ca(2+)-ATPase (SR ATPase). Urea at 2-3 M causes reversible loss of SR ATPase activity, while higher concentrations induce irreversible denaturation. Heat-induced denaturation is a non-two-state process, with an "intermediate state" (at t approximately 45 degrees C) characterized by the presence of protein monomers, instead of the native oligomers. 2D-IR reveals that urea denaturation causes loss of the structural transition to the "intermediate state". Whenever the urea effect can be reversed, the transition to the "intermediate state" is re-established.     

Equilibrium and transient intermediates in folding of human macrophage migration inhibitory factor.

Acid, guanidinium-Cl and urea denaturations of recombinant human macrophage migration inhibitory factor (MIF) were measured using CD and fluorimetry. The acid-induced denaturation was followed by CD at 200, 222, and 278 nm and by tryptophan fluorescence. All four probes revealed an acid-denatured state below pH 3 which resembled a typical molten globule. The pH transition is not two-state as the CD data at 222 nm deviated from all other probes. Urea and guanidinium-Cl denaturations (pH 7, 25 degrees C) both gave an apparent DeltaGU app H2O of 31 +/- 3 kJ.mol-1 when extrapolated to zero denaturant concentration. However, denaturation transitions recorded by fluorescence (at the same protein concentration) occurred at lower urea or guanidinium-Cl concentrations, consistent with an intermediate in the course of MIF denaturation. CD at 222 nm was not very sensitive to protein concentration (in 10-fold range) even though size-exclusion chromatogryphy (SEC) revealed a dimer-monomer dissociation prior to MIF unfolding. Refolding experiments were performed starting from acid, guanidinium-Cl and urea-denatured states. The kinetics were multiphasic with at least two folding intermediates. The intrinsic rate constant of the main folding phase was 5.0 +/- 0.5 s-1 (36.6 degrees C, pH 7) and its energy of activation 155 +/- 12 kJ.mol-1.     

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.     

Comparison of thermal properties of bovine spectrin and fodrin

Thermal properties of bovine brain fodrin have been studied by circular dichroism and electron spin resonance and compared to those of bovine erythrocyte spectrin. Protein unfolding was induced either by urea or by a combination of heat and urea. The denaturation profiles of the two proteins, as measured by the changes in ellipticity at 222 nm as a function of temperature, were very similar but fodrin denaturation occurred at both higher temperatures and higher urea concentrations. In the absence of urea the thermal transition of spectrin was centered at 51 degrees C and that of fodrin at 54.5 degrees C. Proteins were also labeled with a maleimide analog spin probe. Spin-labeled fodrin showed a thermal transition similar to that of spectrin but centered at 46 degrees C instead of 42 degrees C. These findings indicated a close structural similarity of the two proteins but a slightly higher conformational stability of fodrin to both heat and urea.     

Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

Reversibility of thermal transitions in proteins: Racemization and aggregation as factors in the reversible denaturation of a soluble keratin derivative (SCMKA)

The reversibility of the thermal denaturation of a low-sulfur fraction of solubilized wool keratin (SCMKA) has been studied under a variety of conditions of time, protein concentration, and pH. Two types of irreversibility for the transition have been encountered. One of these is associated with an aggregation of the protein on denaturation to give a product which may contain elements of a β conformation. This type of irreversibility is favored by high protein concentration, and the original conformation may in fact be regained if the aggregated structure is broken down by a solvent such as 8M urea and the urea subsequently removed by dialysis. The other type of irreversibility appears to be due to racemization of the protein. It does not seem to be dependent on protein concentration and is apparent only at temperatures beyond the actual transition range (～40–65°C) at pH values below 11, At pH 12, however, racemization appears to proceed slowly even at 4°C. The thermal transition at pH 9 and pH 10 has been shown to be multistage in nature. Over the pH range 9–12 there is a progressive decrease in thermal stability with increase of pH. Addition of NaCl at pH 10 leads to an increase in thermal stability of the molecule.