ABSTRACT
The methods of circular dichroism, scanning microcalorimetry, electron microscopy, and proteolysis were used to study the ability of wild-type Cry3A-delta-endotoxin and three mutant toxins with cysteine substitutions in helices alpha3 and alpha4 (domain I) to form oligomeric structures in acidic alcohol solutions that reproduce the premembrane environment. At pH 2-2.2 and 20% ethanol, the mutant toxins with single substitutions E132C (alpha3) and E160C (alpha4), as well as the double mutant E132C/S160C with a cysteine bridge connecting helices alpha3 and alpha4, form short linear oligomers specific for Cry3A with a high content of the beta-structure and enhanced sensitivity to proteolysis with pepsin. The data obtained show that the formation of oligomeric structures of this type does not require any divergence of helices alpha3 and alpha4 in domain I of the Cry3A toxin. It has been demonstrated that, at higher pH values in 20% solution of ethanol, the proteins studied are in a metastable state, and their ability to form oligomeric structures depends on temperature.
Subject(s)
Bacterial Proteins/chemistry , Endotoxins/chemistry , Ethanol/chemistry , Hemolysin Proteins/chemistry , Mutation, Missense , Amino Acid Substitution , Bacillus thuringiensis Toxins , Bacterial Proteins/genetics , Calorimetry, Differential Scanning , Circular Dichroism , Endotoxins/genetics , Hemolysin Proteins/genetics , Hydrogen-Ion Concentration , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
The effect of some amino acid residues in A, B, G, and H helices on the folding nucleus and folding intermediate state formation was estimated. For four apomyoglobin mutant forms with point replacements of hydrophobic amino acid residues by Ala, the influence of the substitutions on the stability of native (N) protein and its folding intermediate state (I) was studied, as well as on the protein folding/unfolding rates. Equilibrium and kinetic studies on mutant proteins over a wide range of urea concentrations have shown that the protein native state was strongly destabilized in comparison with that of the wild type protein. At the same time, stability of the intermediate state changed insignificantly. It was shown that amino acid residues of A, G, and H helices make a small contribution to apomyoglobin folding nucleus stabilization in the rate-limiting I reversible N transition, which occurred after the intermediate state was formed. But the amino acid residue of B-helix was very important for the folding nucleus stabilization in the transition state upon the I reversible N transition.
Subject(s)
Amino Acids/chemistry , Apoproteins/chemistry , Models, Chemical , Myoglobin/chemistry , Protein Folding , Animals , Kinetics , Protein Structure, Secondary/physiology , Protein Structure, Tertiary/physiology , Sperm WhaleABSTRACT
The problems of protein aggregation and protein misfolding in the cell are connected with the appearance of many genetic diseases. Both processes can be a consequence of substitutions of certain amino acid residues in proteins. The substitutions can influence the protein stability and protein folding rates in both the intermediate and the native states. We have studied equilibrium urea unfolding of mutant forms of apomyoglobin with substitutions of conserved nonfunctional residues by Ala to estimate their influence on protein stability. These residues include Val10, Trp14, Ilel11, Leu115, Met131 and Leu135. Conformational transitions were monitored by intrinsic Trp fluorescence and by circular dichroism spectra in the far UV region. Free energy changes upon the transition from the native to intermediate state and from the intermediate to unfolded state were determined. It was shown that all substitutions used lead to an appreciable decrease of the apomyoglobin native state stability, whereas the stability of the intermediate state is affected substantially smaller.
Subject(s)
Apoproteins/chemistry , Apoproteins/genetics , Conserved Sequence , Myoglobin/chemistry , Myoglobin/genetics , Alanine/chemistry , Alanine/genetics , Amino Acid Substitution , Animals , Circular Dichroism , Fluorescence , Mutation , Protein Conformation , Protein Denaturation , Protein Folding , ThermodynamicsABSTRACT
Apomyoglobin kinetic and equilibrium unfolding and folding processes were studied at pH 6.2, 11 degrees C by stopped-flow tryptophan fluorescence. There are two distinct consecutive processes in apomyoglobin folding process, namely, the protein fast transition between the unfolded (U) and an intermediate (I) states (U <----> I) and slow transition between the intermediate and the native (N) states (I <----> N). Accumulation of the intermediate state was observed in the wide range of urea concentrations. The presence of the intermediate state was shown even beyond the middle transition on the unfolding limb. The dependence of observed folding/unfolding rates on urea concentration (chevron plot) was obtained. The shape of this dependence was compared with that of two-state proteins, folding from the U to N state.
Subject(s)
Apoproteins/chemistry , Myoglobin/chemistry , Protein Folding , Urea/chemistry , Animals , Kinetics , Protein DenaturationABSTRACT
We have analyzed the proteins whose structures were determined both by X-ray analysis (X-ray) and nuclear magnetic resonance (NMR) on condition that these structures do not differ greatly when spatially superimposed on each other (61 pairs of protein structures). Atom-atomic contacts (contact distances varied from 2 to 8 A) have been analyzed and it has been found that NMR structures (in comparison with X-ray ones) have more contacts in the range below 3.5 A and above 5.5 A. In the case of residue-residue contacts NMR structures have more contacts below 3 A and between 4.5 and 6.5 A. At all the other contact distances analyzed the X-ray structures have more contacts. The difference in the number of atom-atomic and residue-residue contacts is greater for internal residues, that are concealed from water, as compared to the surface residues. The other, not less important difference deals with the number of hydrogen bonds in the main chain: it is larger for the X-ray structures. The correlation between the hydrogen bonds identified in the structures obtained by both methods is no more than 32%. The consideration of a complete set of protein structures obtained by NMR results in the fact that the number of hydrogen bonds grows 1.2 times as compared to those obtained with the X-ray analysis, whereas the correlation increases only by 65%. We have also demonstrated that alpha-helices in the NMR structures are more distorted in comparison with the ideal alpha-helix, than alpha-helices in the X-ray structures.
Subject(s)
Models, Molecular , Proteins/chemistry , Animals , Computer Simulation , Crystallography, X-Ray , Humans , Hydrogen Bonding , Nuclear Magnetic Resonance, Biomolecular , Protein ConformationABSTRACT
It was shown that gamma-irradiation of water-ethanol and water-n-propanol solutions of DNA with doses of 10-30 Gy leads to a fall of the specific volume of the macromolecule upon the cooperative transition at a critical concentration of alcohol in solution at which the destroying of water structure by a nonelectrolyte occurs.
Subject(s)
DNA/radiation effects , Gamma Rays , Water/chemistry , 1-Propanol/chemistry , Birefringence , Circular Dichroism , DNA/chemistry , Ethanol/chemistry , Nucleic Acid Conformation , Solutions , Solvents , Urea/chemistryABSTRACT
The results of surgical treatment of inguinal hernias in 315 war veterans, including 260 the elderly and senile, have been analysed. All the patients underwent the elective operations. Four (1.2%) patients developed complications after the operation, there were no lethal outcomes. The authors came to a conclusion about the expediency to perform elective herniotomy in elderly and senile patients even in presence of concomitant diseases.
