A Unique Molten Globule State Occurs during Unfolding of Cytochrome c by LiClO 4 Near Physiological pH and Temperature: Structural and Thermodynamic Characterization † (original) (raw)
Related papers
Biochemistry, 2003
Amino acid sequences of seven subfamilies of cytochromes c show that other than heme binding residues there are only four positions which are conserved in all subfamilies: Gly/Ala6, Phe/Tyr10, Leu/Val/Phe94, and Tyr/Trp/Phe97. These residues are 90% conserved in all sequences reported and are also considered to be involved in a common folding nucleus. To determine the importance of conserved interactions offered by the side chain of Leu94, we made an L94G mutant of horse cytochrome c. Characterization of this mutant by the far-UV, near-UV, and Soret circular dichroism, intrinsic and 1-Anilino-8naphthalene sulfonate fluorescence, and dynamic light scattering measurements led to the conclusion that the L94G mutant has all the common structural characteristics of a molten globule at pH 6.0 and 25°C. NaCl induces a cooperative transition between the acid-denatured state and a state of L94G having all the common structural characteristics of a pre-molten-globule state at pH 2 and 25°C. Thermal denaturation studies showed that the midpoint of denaturation of the mutant is 28°C less than that of the wild-type protein. Interestingly, the structure analysis using the coordinates given in the Protein Data Bank (1hrc) also suggested that the L94G mutant would be less stable than the wild-type protein.
PloS one, 2015
While many proteins are recognized to undergo folding via intermediate(s), the heterogeneity of equilibrium folding intermediate(s) along the folding pathway is less understood. In our present study, FTIR spectroscopy, far- and near-UV circular dichroism (CD), ANS and tryptophan fluorescence, near IR absorbance spectroscopy and dynamic light scattering (DLS) were used to study the structural and thermodynamic characteristics of the native (N), denatured (D) and intermediate state (X) of goat cytochorme c (cyt-c) induced by weak salt denaturants (LiBr, LiCl and LiClO4) at pH 6.0 and 25°C. The LiBr-induced denaturation of cyt-c measured by Soret absorption (Δε400) and CD ([θ]409), is a three-step process, N ↔ X ↔ D. It is observed that the X state obtained along the denaturation pathway of cyt-c possesses common structural and thermodynamic characteristics of the molten globule (MG) state. The MG state of cyt-c induced by LiBr is compared for its structural and thermodynamic parameter...
Journal of Biological Inorganic Chemistry - J BIOL INORG CHEM, 2010
It has already been shown that the mutant Leu94Gly of horse cytochrome c exists in a molten globule (MG) state. We have carried out studies of reversible folding and unfolding induced by LiCl of this mutant at pH 6.0 and 25 °C by observing changes in the difference molar absorption coefficient at 402 nm, the mean residue ellipticity at 222 nm, and the difference mean residue ellipticity at 409 nm. This process is a three-state process when measured by these probes. The stable folding intermediate state has been characterized by far- and near-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalenesulfonic acid binding, and dynamic light scattering measurements, which led us to conclude that the intermediate is a premolten globule (PMG). Analysis of the reversible unfolding transition curves for the stability of different states in terms of the Gibbs free energy change at pH 6.0 and 25 °C led us to conclude that the MG state is more stable than the PMG state by 5.4 ± 0.1 kcal mol−1, whereas the PMG state is more stable than the denatured (D) state by only 1.1 ± 0.1 kcal mol−1. A comparison of the conformational and thermodynamic properties of the LiCl-induced PMG state at pH 6.0 with those of the PMG state induced by NaCl at pH 2.0 suggests that a similar PMG state is obtained under both denaturing conditions. Differential scanning calorimetry measurements suggest that heat induces a reversible two-state transition between MG and D states.
Metallomics, 2011
Proteins in cells fold via a number of intermediates. These intermediates are quite important as they guide the protein to attain its unique native conformation. To solve the immensely difficult problem of protein folding, it is necessary to characterize intermediates which will unravel the mystery of the steps involved in the proper folding of proteins. Cytochromes-c (cyts-c) have played an important role in studies of the earliest events and intermediates in protein folding. They have always been considered as model proteins for protein folding studies due to their intrinsic properties that can be measured by multiple probes. A large number of different solvent conditions have been employed to obtain equilibrium intermediates of cyts-c. These intermediates show structural heterogeneity which is mainly due to the different solvent conditions used to induce them. In this review we present results of conformational and thermodynamic characterization of equilibrium intermediates (molten globules and pre-molten globules) of the mammalian cyts-c under different solvent conditions.
International Journal of Biochemistry & Cell Biology, 2004
In our earlier communications, we had studied the acid induced unfolding of stem bromelain, glucose oxidase and fetuin [Eur. and effect of salts and alcohols on the acid unfolded state of ␣-chymotrypsinogen and stem bromelain [Biochim. Biophy. Acta 1481 229; Arch. Biochem. Biophys. 413 (2) (2003) 199]. Here, we report the presence of molten globule like equilibrium intermediate state under alkaline, native and acid conditions in the presence of SDS and butanol. A systematic investigation of sodium dodecyl sulphate and butanol induced conformational alterations in alkaline (U 1 ) and acidic (U 2 ) unfolded states of horse heart ferricytochrome c was examined by circular dichroism (CD), tryptophan fluorescence and 1-anilino-8-napthalene sulfonate (ANS) binding. The cytochrome c (cyt c) at pH 9 and 2 shows the loss of approximately 61% and 65% helical secondary structure. Addition of increasing concentrations of butanol (0-7.2 M) and sodium dodecyl sulphate (0-5 mM) led to an increase in ellipticity value at 208 and 222 nm, which is the characteristic of formation of ␣-helical structure. Cyt c is a heme protein in which the tryptophan fluorescence is quenched in the native state by resonance energy transfer to the heme group attached to cystines at positions 14 and 17. At alkaline and acidic pH protein shows enhancement in tryptophan fluorescence and quenched ANS fluorescence. Addition of increasing concentration of butanol and SDS to alkaline or acid unfolded state leads to decrease in tryptophan and increase in ANS fluorescence with a blue shift in λ max , respectively. In the presence of 7.2 M butanol and 5 mM SDS two different intermediate states I 1 and I 2 were obtained at alkaline and acidic pH, respectively. States I 1 and I 2 have native like secondary structure with disordered side chains (loss of tertiary structure) as predicted from tryptophan fluorescence and high ANS binding. These results altogether imply that the butanol and SDS induced intermediate states at alkaline and acid pH lies between the unfolded and native state. At pH 6, in the presence of 7.2 M butanol or 5 mM SDS leads to the loss of CD bands at 208 and 222 nm with the appearance of trough at 228 nm also with increase in tryptophan and ANS fluorescence in contrast to native protein. This partially unfolded intermediate state obtained represents the folding pathway from native to unfolded structure. To summarize; the 7.2 M butanol and 5 mM SDS stabilizes the intermediate state (I 1 and I 2 ) obtained at low and alkaline pH. While the same destabilizes the native structure of protein at pH 6, suggesting a difference in the mechanism of conformational stability.
Formation of a molten-globule-like state of cytochrome c induced by high concentrations of glycerol
Biochimie, 1999
The effect of glycerol on the structure of cytochrome c was investigated by circular dichroism, absorbance and EPR spectroscopy. The results obtained show that an increasing concentration of the organic solvent (70-99.2%, v/v) in aqueouspolyalcohol mixtures converts native cytochrome c into a new, low spin form through a fully reversible, two-state transition. The glycerol-stabilized form (that we call here the G state) retains native-like amounts of α-helix structure while rigid tertiary structure and native Fe(III)-Met(80) axial bond are lost. Analysis of data suggests a molten globule character of the G state; support to this view is afforded by the striking similarities between the spectroscopic (and, thus, structural) properties of the G state with those of the acidic molten globule of the protein (A state). © 1999 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS molten globule / glycerol / A-state / circular dichroism / cytochrome c * Correspondence and reprints Biochimie 81 (1999) 745−751
To understand the role of five extra N-terminal residues, we prepared wild type (WT) yeast iso-1-cytochrome c (y-cyt-c) and its deletants by subsequently deleting these residues. Denaturation of all these proteins induced by LiCl was followed by observing changes in molar absorption coefficient at 405 nm (ε 405), the mean residue ellipticity at 222 nm ([Â] 222), and the difference mean residue ellip-ticity at 409 nm ([Â] 409) near physiological pH and temperature (pH 6.0 and 25 • C). It was observed that in each case LiCl induces biphasic transition, N (native) state ↔ X (intermediate) state ↔ D (dena-tured) state. The intermediate (X) was characterized by the far-UV, near-UV and Soret circular dichroism, ANS (8-anilino-1-naphthalenesulfonic acid) binding and dynamic light scattering measurements. These measurements led us to conclude that X state of each protein has structural characteristics of PMG (pre-molten globule) state. Thermodynamic stability of all proteins was also determined. It was observed that the N-terminal extension stabilizes the native WT protein but it has no effect on the stability of PMG state. Another state was observed for each protein, in the presence of 0.33 M Na 2 SO 4 at pH 2.1, which when characterized showed all structural characteristics of MG (molten globule) state.
Journal of Biological Inorganic Chemistry, 2009
Amino acid sequences of seven subfamilies of cytochromes c show that other than heme binding residues there are only four positions which are conserved in all subfamilies: Gly/Ala6, Phe/Tyr10, Leu/Val/Phe94, and Tyr/Trp/Phe97. These residues are 90% conserved in all sequences reported and are also considered to be involved in a common folding nucleus. To determine the importance of conserved interactions offered by the side chain of Leu94, we made an L94G mutant of horse cytochrome c. Characterization of this mutant by the far-UV, near-UV, and Soret circular dichroism, intrinsic and 1-Anilino-8-naphthalene sulfonate fluorescence, and dynamic light scattering measurements led to the conclusion that the L94G mutant has all the common structural characteristics of a molten globule at pH 6.0 and 25 °C. NaCl induces a cooperative transition between the acid-denatured state and a state of L94G having all the common structural characteristics of a pre-molten-globule state at pH 2 and 25 °C. Thermal denaturation studies showed that the midpoint of denaturation of the mutant is 28 °C less than that of the wild-type protein. Interestingly, the structure analysis using the coordinates given in the Protein Data Bank (1hrc) also suggested that the L94G mutant would be less stable than the wild-type protein.
International Journal of Biological Macromolecules, 2015
To understand the role of five extra N-terminal residues, we prepared wild type (WT) yeast iso-1cytochrome c (y-cyt-c) and its deletants by subsequently deleting these residues. Denaturation of all these proteins induced by LiCl was followed by observing changes in molar absorption coefficient at 405 nm ( ε 405 ), the mean residue ellipticity at 222 nm ([Â] 222 ), and the difference mean residue ellipticity at 409 nm ( [Â] 409 ) near physiological pH and temperature (pH 6.0 and 25 • C). It was observed that in each case LiCl induces biphasic transition, N (native) state ↔ X (intermediate) state ↔ D (denatured) state. The intermediate (X) was characterized by the far-UV, near-UV and Soret circular dichroism, ANS (8-anilino-1-naphthalenesulfonic acid) binding and dynamic light scattering measurements. These measurements led us to conclude that X state of each protein has structural characteristics of PMG (premolten globule) state. Thermodynamic stability of all proteins was also determined. It was observed that the N-terminal extension stabilizes the native WT protein but it has no effect on the stability of PMG state. Another state was observed for each protein, in the presence of 0.33 M Na 2 SO 4 at pH 2.1, which when characterized showed all structural characteristics of MG (molten globule) state.
Characterization of the polyanion-induced molten globule-like state of cytochromec
Biopolymers, 2007
Cytochrome c (cyt c) undergoes a poly(vinylsulphate) (PVS)-induced transition at slightly acidic pH into a molten globule-like state that resembles the effect that negatively charged membrane surfaces have on this protein. In this work, the thermodynamic properties of the molten globule-like state of cyt c in complex with PVS are studied using differential scanning calorimetry, circular dichroism, fluorescence, and absorbance spectroscopy. The temperature-induced transition of the molten globule-like state of cyt c in the complex with PVS is characterized by a significantly lower calorimetric enthalpy than in the ''typical'' molten globule state of cyt c, i.e. free protein at pH 2.0 in high ionic strength. Moreover, the thermally-denatured state of cyt c in the complex at pH < 6 contains nearly 50% of the native secondary structure. The dependence of the transition temperature on the pH indicates a role for histidine residues in the destabilization of the cyt c structure in the PVS complex and in stabilization of the denatured state with the residual secondary structure. A comparison of the effects of small anions and polyanions demonstrates the importance of cooperativity among the anions in the destabilization of cyt c. Predictably, other hydrophilic flexible polyanions such as heparin, polyglutamate, and polyadenylate also have a destabilizing effect on the structure of cyt c. However, a correlation between the properties of the polyanions and their effect on the protein stability is still unclear.