Complete Loss Of Tertiary Structure Research Articles

Stabilization of α-Amylase, the Key Enzyme in Carbohydrates Properties Alterations, at Low pH

The present study correlates the mechanism of α-amylase denaturation under acidic condition and its structural stabilization in presence of selected cosolvents (sorbitol, sucrose, trehalose, and glycerol). The objective of the present study was to minimize the enzyme inactivation at lower pH by stabilizing the enzyme structure. The above cosolvents were found to be an effective stabilizer of α-amylase against denaturation at extreme low pH. The optimum activity of α-amylase was found to be in the pH range of 4.5 to 7. Decreasing the pH of enzyme solution below this range results in a decrease in enzyme activity. The intrinsic and ANS fluorescence indicated the gradual unfolding of protein below pH 4 and resulted in exposure of hydrophobic cluster. The pH-induced unfolding also resulted in protein aggregation. The intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence, acrylamide quenching, near and far UV-CD spectra of α-amylase at lower pH (pH 2.25) indicated the complete loss of tertiary structure and substantial loss of secondary structure. Cosolvents were shown to prevent the acid induced unfolding of the enzyme to a certain extent and help in retaining the secondary structure of enzyme equivalent to native state. Among all the cosolvents used, sorbitol was proven to be the most efficient stabilizer. At lower pH, in presence of cosolvents the enzyme was shown to retain significant amount of secondary structure and poorly defined tertiary structure. This structure resembles the molten globule of the protein, which has substantial amount of secondary structure but poorly defined tertiary structure.

The non-native conformations of cytochrome c in sodium dodecyl sulfate and their modulation by ATP

To understand the interaction of cytochrome c (cyt c) with membranes, a systematic investigation of sodium dodecyl sulfate (SDS)-induced conformational alterations in native horse heart ferricytochrome c (pH 7.0) was carried out using heme absorbance, tryptophan fluorescence and circular dichroism (CD) spectroscopy. ATP interaction with membrane-bound cyt c is known to regulate the process of apoptosis. To understand the effect of nucleotide phosphates on membrane-bound cyt c, we also carried out studies of the interaction of ATP with cyt c in the presence of SDS. Fluorescence and UV-Vis data suggest that SDS induces two different transitions (F to C1, C1 to C2) in cyt c, one in the pre-micellar region and the other in the post-micellar region. The fluorescence data further indicated the increase in distance between Trp 59 and heme in the intermediates in both the regions, suggesting loosening up of cyt c on titration with SDS. The far-UV and near-UV CD data suggest partial loss of secondary and tertiary structure in C1, but complete loss of tertiary structure and no further loss of secondary structure in C2. On titration of C1 and C2 with ATP, the secondary structure is restored. However, the heme ligation pattern and heme exposure change only for C2, but not for C1 on the addition of ATP.

Ionic strength-dependent denaturation of Thermomyces lanuginosus lipase induced by SDS

Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denaturation by sodium dodecyl sulfate (SDS). We have found that at neutral pH, structural integrity is strongly dependent on ionic strength. In 10mM phosphate buffer and SDS, the lipase exhibits a far-UV CD spectrum similar to other proteins denatured in this surfactant while the near-UV CD spectrum shows a complete loss of tertiary structure, observations supported by steady state fluorescence spectroscopy. However, when increasing the ionic strength by the addition of NaCl, the lipase was rendered resistant towards SDS denaturation, as observed by all techniques employed. The effect of salt on the critical micelle concentration (CMC) of SDS was observed to correlate with the effect on the degree of SDS-induced denaturation. This finding is compatible with the notion that the concentration of SDS monomers is a crucial factor for SDS–lipase interactions. The presented results are important for the understanding and improvement of protein stability in surfactant systems.

Spectroscopic study on acid-induced unfolding and refolding of apo-neuroglobin

pH-induced unfolding and refolding of apo-neuroglobin (apo-Ngb) were investigated by UV, fluorescence, circular dichroism (CD) spectra and light scattering measurements. Results revealed that apo-Ngb became partially unfolded at around pH 5.0, with evidences from a red shift in the fluorescence spectra, a decrease in the far-UV CD and a sharp peak in the light scattering intensity. Further lowering of the pH reversed these effects, suggesting that apo-Ngb folds back to a compact state. At pH 2.0, the apo-Ngb forms a folding intermediate known as molten globule (MG), which is possessed of native-like secondary structure and almost complete loss of tertiary structure. Based on these results, the acid-induced denaturation pathway of apo-Ngb can be illustrated from the native state (N), via a partially unfolded state (U(A)) to the molten globule state (MG).

Molten globule-like folding intermediate of asialofetuin at acidic pH

In our earlier communication on acid-induced unfolding of bovine serum fetuin (BSF), we showed the existence of a molten globule (MG)-like state of BSF at pH 1.8. The MG state was characterized by higher content of secondary structure than native and almost complete loss of tertiary structure and more solvent exposed hydrophobic surface [Biochim. Biophys. Acta 1649 (2003) 164]. In this work we have shown the presence of an MG-like partially folded intermediate of asialofetuin at around pH 1.8, which is much different from the MG state observed in BSF in secondary structure contents. The results show that asialofetuin at pH 1.8 retains ∼45% secondary structure, as evident from far-UV CD spectra. The near-UV CD spectra showed almost complete loss of tertiary structure. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue showed that in acid-induced state, it is buried in the interior in a nonpolar environment. The temperature dependence of far-UV CD signal of asialofetuin at pH 1.8 exhibits a weak cooperative thermal transition. A significant increase in ANS fluorescence showed extensive solvent exposure of nonpolar cluster. Size exclusion chromatography (SEC) indicates a slight increase in the hydrodynamic size of acid-induced protein. These results suggest that asialofetuin at pH 1.8 represents the MG-like folding intermediate. Moreover, our results showed that glycosylation might play a role in stabilization of secondary structure during acid and/or thermal denaturation.

Molten globule-like folding intermediate of asialofetuin at acidic pH

In our earlier communication on acid-induced unfolding of bovine serum fetuin (BSF), we showed the existence of a molten globule (MG)-like state of BSF at pH 1.8. The MG state was characterized by higher content of secondary structure than native and almost complete loss of tertiary structure and more solvent exposed hydrophobic surface [Biochim. Biophys. Acta 1649 (2003) 164]. In this work we have shown the presence of an MG-like partially folded intermediate of asialofetuin at around pH 1.8, which is much different from the MG state observed in BSF in secondary structure contents. The results show that asialofetuin at pH 1.8 retains ∼45% secondary structure, as evident from far-UV CD spectra. The near-UV CD spectra showed almost complete loss of tertiary structure. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue showed that in acid-induced state, it is buried in the interior in a nonpolar environment. The temperature dependence of far-UV CD signal of asialofetuin at pH 1.8 exhibits a weak cooperative thermal transition. A significant increase in ANS fluorescence showed extensive solvent exposure of nonpolar cluster. Size exclusion chromatography (SEC) indicates a slight increase in the hydrodynamic size of acid-induced protein. These results suggest that asialofetuin at pH 1.8 represents the MG-like folding intermediate. Moreover, our results showed that glycosylation might play a role in stabilization of secondary structure during acid and/or thermal denaturation.

Functional analysis of chemically synthesized derivatives of the human CC chemokine CCL15/HCC‐2, a high affinity CCR1 ligand

The CCL15 is a human CC chemokine that activates the receptors, CCR1 and CCR3. Unlike other chemokines, it contains an unusually long N-terminal domain of 31 amino acids preceding the first cysteine residue and a third disulfide bond. To elucidate the functional role of distinct structural determinants, a series of sequential amino-terminal truncated and point-mutated CCL15 derivatives as well as mutants lacking the third disulfide bond and the carboxy-terminal alpha-helix were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. We demonstrate that a truncation of 24 amino acid residues (delta24-CCL15) converts the slightly active 92-residue delta0-CCL15 into a potent agonist of CC chemokine receptor 1 (CCR1) and a weak agonist of CCR3 in cell-based assays. The biological activity decreases from delta24-CCL15 to delta29-CCL15, and re-increases from delta29-CCL15 to delta30-CCL15. Thus, an exocyclic N-terminal region of only one amino acid residue is sufficient for efficient CCR1 activation. As none of the peptides investigated except for delta24-CCL15 activates CCR3, we suggest that CCR1 is the major receptor for CCL15 in vivo. Further we demonstrate that the third disulfide bond of CCL15 and an exchange of tyrosine in position 70 by a leucine residue, which is conserved in CXC chemokines, do not alter the interaction with CCR1. In contrast, a CCL15 derivative lacking the carboxy-terminal alpha-helix exhibits a complete loss of tertiary structure and hence loss of CCR1 agonistic and binding activity. This study demonstrates that specific protein residues in chemokines, which contribute to receptor-ligand interaction, vary significantly between chemokines and cannot be extrapolated using data from functionally related chemokines.

Alcohol and temperature induced conformational transitions in ervatamin B: sequential unfolding of domains.

The structural aspects of ervatamin B have been studied in different types of alcohol. This alcohol did not affect the structure or activity of ervatamin B under neutral conditions. At a low pH (3.0), different kinds of alcohol have different effects. Interestingly, at a certain concentration of non-fluorinated, aliphatic, monohydric alcohol, a conformational switch from the predominantly alpha-helical to beta-sheeted state is observed with a complete loss of tertiary structure and proteolytic activity. This is contrary to the observation that alcohol induces mostly the alpha-helical structure in proteins. The O-state of ervatamin B in 50% methanol at pH 3.0 has enhanced the stability towards GuHCl denaturation and shows a biphasic transition. This suggests the presence of two structural parts with different stabilities that unfold in steps. The thermal unfolding of ervatamin B in the O-state is also biphasic, which confirms the presence of two domains in the enzyme structure that unfold sequentially. The differential stabilization of the structural parts may also be a reflection of the differential stabilization of local conformations in methanol. Thermal unfolding of ervatamin B in the absence of alcohol is cooperative, both at neutral and low pH, and can be fitted to a two state model. However, at pH 2.0 the calorimetric profiles show two peaks, which indicates the presence of two structural domains in the enzyme with different thermal stabilities that are denatured more or less independently. With an increase in pH to 3.0 and 4.0, the shape of the DSC profiles change, and the two peaks converge to a predominant single peak. However, the ratio of van't Hoff enthalpy to calorimetric enthalpy is approximated to 2.0, indicating non-cooperativity in thermal unfolding.

Acid- and base-induced conformational transitions of equinatoxin II

We have investigated the acid- and base-induced conformational transitions of equinatoxin II (EqTxII), a pore-forming protein, by a combination of CD-spectroscopy, ultrasonic velocimetry, high precision densimetry, viscometry, gel electrophoresis, and hemolytic activity assays. Between pH 7 and 2, EqTxII does not exhibit any significant structural changes. Below pH 2, EqTxII undergoes a native-to-partially unfolded transition with a concomitant loss of its rigid tertiary structure and the formation of a non-native secondary structure containing additional α-helix. The acid-induced denatured state of EqTxII exhibits a higher intrinsic viscosity and a lower adiabatic compressibility than the native state. Above 50°C, the acid-induced denatured state of EqTxII reversibly denatures to a more unfolded state as judged by the far UV CD spectrum of the protein. At alkaline pH, EqTxII undergoes two base-induced conformational transitions. The first transition occurs between pH 7 and 10 and results in a partial disruption of tertiary structure, while the secondary structure remains largely preserved. The second transition occurs between pH 11 and 13 and results in the complete loss of tertiary structure and the formation of a non-native, more α-helical secondary structure. The acid- and base-induced partially unfolded states of EqTxII form water-soluble oligomers at low salt, while at high salt (>350 mM NaCl), the acid-induced denatured state precipitates. The hemolytic activity assay shows that the acid- and base-induced denatured states of EqTxII exhibit significantly reduced activity compared to the native state.

The unusual dodecameric ferritin from Listeria innocua dissociates below pH 2.0.

The stability of the dodecameric Listeria innocua ferritin at low pH values has been investigated by spectroscopic methods and size-exclusion chromatography. The dodecamer is extremely stable in comparison to the classic ferritin tetracosamer and preserves its quaternary assembly at pH 2.0, despite an altered tertiary structure. Below pH 2.0, dissociation into dimers occurs and is paralleled by the complete loss of tertiary structure and a significant decrease in secondary structure elements. Dissociation of dimers into monomers occurs only at pH 1.0. Addition of NaCl to the protein at pH 2.0 induces structural changes similar to those observed upon increasing the proton concentration, although dissociation proceeds only to the dimer stage. Addition of sulfate at pH values >/= 1.5 prevents the dissociation of the dodecamer. The role played by hydrophilic and hydrophobic interactions in determining the resistance to dissociation of L. innocua ferritin at low pH is discussed in the light of its three-dimensional structure.

Ultrasonic examination of the hemoglobin dissociation process in aqueous solutions of guanidine hydrochloride.

The ultrasonic absorption was determined, in aqueous solutions of hemoglobin undergoing dissociation as a function of the strong electrolyte guanidine hydrochloride (GuHCl), up to 5.4M (molar), over the frequency range 1–50 MHz. With increasing GuHCl concentration to 0.7M, the Hb molecule dissociates into half-molecules without loss of tertiary structure, and at GuHCl concentrations greater than 2.5M, the Hb molecule dissociates further into its four individual polypeptide chains with complete loss of tertiary structure. The ultrasonic absorption coefficient appears to be insensitive to the dissociation process, though a small contribution appears at the lower frequencies, owing to the expansion of the polypeptide molecule. A maximum in the absorption occurs near 2M GuHCl concentration, which does not correspond to dissociation or expansion of the Hb molecule, and it is thought to be associated with the proton transfer reaction.

Ultrasonic Examination of the Hemoglobin Dissociation Process

The ultrasonic absorption and velocity were determined in aqueous solutions of hemoglobin (Hb) undergoing dissociation. The dissociation of Hb in aqueous solutions of the strong electrolyte guanidine hydrochloride (GuHCl) has been studied extensively [K. Kawahara, A. G. Kirschner, and C. Tanford, Biochemistry 4, 1203 (1965)]. With increasing concentration of GuHCl to 0.7M (molar), the Hb molecule dissociates into half-molecules without loss of tertiary structure. At GuHCl concentrations greater than 2.5M, the Hb molecule dissociates further into its four individual polypeptide chains with complete loss of tertiary structure. The acoustical absorption coefficient within the frequency range 1–50 MHz appears to be insensitive to these conformational changes. Within the neighborhood of 2-M GuHCl concentration, a maximum in the excess ultrasonic absorption coefficient occurs which does not correspond to dissociation or expansion of the Hb molecule, and it is speculated that this maximum can be attributed to the proton transfer reaction.

USE OF OXIDIZED PROTEINS IN THE EXAMINATION OF IMMUNOCHEMICAL SPECIFICITY IN DELAYED HYPERSENSITIVITY TO HAPTEN-PROTEIN CONJUGATES.

RECENT investigations of delayed hypersensitivity to hapten–protein conjugates have established clearly the importance of the carrier protein as one of the determinants of specificity1–3. Efforts to define further such determinants are hampered to some extent by the heterogeneity of the response to immunization with a hapten–protein conjugate in which many possible antigenic determinants may play a part. In previous work4, it was shown that oxidation or reduction and alkylation of all disulphide bonds in several proteins (resulting in complete loss of tertiary structure with retention of primary structure) produced the most drastic reduction in the ability of these proteins to precipitate rabbit antibody and to sensitize guinea pigs to delayed hypersensitivity to the native proteins. It was thoughtthat the use of such open chains to serve as hapten carrier might provide valuable information about the relative contributions of folded structure and amino-acid sequence to the specificity involved in delayed hypersensitivity.