Conformational and Thermodynamic Characterization of the Molten Globule State Occurring during Unfolding of Cytochromes-c by Weak Salt Denaturants (original) (raw)
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JBIC Journal of Biological Inorganic Chemistry, 2010
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.
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.
Biomolecular NMR Assignments, 2019
Proteins fold via a number of intermediates that help them to attain their unique native 3D structure. These intermediates can be trapped under extreme conditions of pH, temperature and chemical denaturants. Similar states can also be achieved by other processes like chemical modification, site directed mutagenesis (or point mutation) and cleavage of covalent bonds of natural proteins under physiological conditions usually taken as dilute buffer (near neutral pH) and 25 °C. Structural characterization of molten globules is hampered due to (i) their transient nature, (ii) very low population at equilibrium, and (iii) prone to aggregation at high concentration. Furthermore, the dynamic nature of these folding intermediates makes them unsuitable for X-ray diffraction. Hence, understanding their structures at the atomic level is often a challenge. However, characterization of these intermediates at the atomic level is possible by NMR, which could possibly unravel new details of the protein folding process. We have previously shown that the L94G mutant of horse cytochrome-c displays characteristics of the molten globule (MG) state at pH 6.0 and 25 °C. As a first step towards characterizing this MG state at the atomic level by NMR, we report its complete backbone, side chain and heme chemical shift assignments.
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.
Stability of uniformly labeled (13C and 15N) cytochrome c and its L94G mutant
Scientific Reports
Cytochrome c (cyt c) is widely used as a model protein to study (i) folding and stability aspects of the protein folding problem and (ii) structure–function relationship from the evolutionary point of view. Databases of cyts c now contain 285 cyt c sequences from different organisms. A sequence alignment of all these proteins with respect to horse cyt c led to several important conclusions. One of them is that Leu94 is always conserved in all 30 mammalian cyts c. It is known that mutation L94G of the wild type (WT) horse cyt c is destabilizing and mutant exists as molten globule under the native condition (buffer pH 6 and 25 °C). We have expressed and purified uniformly labeled (13C and 15N) and unlabeled WT horse cyt c and its L94G mutant. We report that labeling does not affect the thermodynamic stability of proteins. To support this conclusion, the secondary and tertiary structure of each protein in labeled and unlabeled forms was determined by conventional techniques (UV–Vis abs...
Biochemistry, 2006
We have carried out denaturation studies of bovine cytochrome c (cyt c) by LiClO 4 at pH 6.0 and 25°C by observing changes in difference molar absorbance at 400 nm (∆ 400 ), mean residue ellipticities at 222 nm ([θ] 222 ) and difference mean residue ellipticity at 409 nm (∆[θ] 409 ). The denaturation is a three-step process when measured by ∆ 400 and ∆[θ] 409 , and it is a two-step process when monitored by [θ] 222 . The stable folding intermediate state has been characterized by near-and far-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements. A comparison of the conformational and thermodynamic properties of the LiClO 4 -induced molten globule (MG) state with those induced by other solvent conditions (e.g., low pH, LiCl, and CaCl 2 ) suggests that LiClO 4 induces a unique MG state, i.e., (i) the core in the LiClO 4 -induced state retains less secondary and tertiary structure than that in the MG states obtained in other solvent conditions, and (ii) the thermodynamic stability associated with the LiClO 4 -induced process, native state T MG state, is the same as that observed for each transition between native and MG states induced by other solvent conditions.
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 Molecular Biology, 1996
One unique aspect of cytochrome c folding concerns the involvement of the 1 Department of Biology covalently attached heme group and its axial ligands. To elucidate the role Faculty of Science of the ligands in stabilizing the native and molten globule states, we studied Osaka University, Toyonaka the conformational and thermodynamic features of the iron-free derivative Osaka 560, Japan of horse cyctochrome c (porphyrin-cytochrome c). At neutral pH, far-UV 2 Protein Engineering Research circular dichroism suggested that porphyrin-cytochrome c has native-like Institute, Furuedai, Suita a-helices, whereas near-UV CD suggested that the side-chains are flexible. Osaka 565, Japan Its stability against heat or denaturants was much less than that of the intact protein, and similar to that of the acidic molten globule state of the 3 Department of Earth and holoprotein. These results indicate that, at neutral pH, the ligation of His18 Space Science, Faculty of to the iron is important for the maintenance of the native structure whereas Science, Osaka University the Met80 ligation is not essential, and that porphyrin-cytochrome c Toyonaka, Osaka 560, Japan assumes a molten globule-like state. Porphyrin-cytochrome c was largely 4 Institute for Protein unfolded at pH 2.0 in the absence of salt, but assumed another molten Research, Osaka University globule-like structure in the presence of anions. The salt-induced Suita, Osaka 565, Japan stabilization of the molten globule-like state was the same as that of apocytochrome c, requiring a much higher salt concentration than 5 Institute for Enzyme holocytochrome c. These results indicate that, at acidic pH, the His18 Research, University of ligation is important, although not essential, for stabilizing the molten Tokushima, Tokushima, 770 globule state. Taken together, both specific (i.e. the His18 axial ligand, as Japan observed at acidic pH) and nonspecific interactions (the hydrophobic effects of the heme, as observed at neutral pH) contribute to stabilizing the molten globule state.
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.
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.