ABSTRACT
Nicking endonucleases are a new type of enzymes. Like restriction endonucleases, they recognize short specific DNA sequence and cleave DNA at a fixed position relatively to the recognition sequence. However, unlike restriction endonucleases, nicking endonucleases cleave only one predetermined DNA strand. Until recently, nicking endonucleases were suggested to be naturally mutated restriction endonucleases which had lost their ability to dimerize and as a result the ability to cleave the second strand. We have shown that nicking endonucleases are one of the subunits of heterodimeric restriction endonucleases. Mechanisms used by various restriction endonucleases for double-stranded cleavage, designing of artificial nicking endonucleases on the basis of restriction endonucleases, and application of nicking endonucleases in molecular biology are reviewed.
Subject(s)
DNA Breaks, Single-Stranded , Deoxyribonuclease I/metabolism , Amino Acid Sequence , Base Sequence , DNA Restriction Enzymes/genetics , DNA Restriction Enzymes/metabolism , Deoxyribonuclease I/chemistry , Deoxyribonuclease I/genetics , Models, Molecular , Molecular Sequence Data , Protein Structure, TertiaryABSTRACT
The dynamics of the side groups of amino acid residues and local conformational changes in the lysozyme molecule upon dehydration and rehydration of lysozyme crystals were studied by the methods of spin label, X-ray diffraction, and molecular dynamics. The His15 residue of lysozyme from chicken egg white was modified by spin label, and spin-labeled tetragonal crystals of the protein were grown. The spatial structure of the covalently bound spin label and its immediate surroundings in the lysozyme tetragonal crystal was determined. The conformation of a fragment of the lysozyme molecule with the spin label on His15, optimized by the method of molecular dynamics, closely agreed with X-ray data. It was found by the X-ray diffraction analysis that a decrease in relative humidity to 40% is accompanied by both a decrease in the unit cell volume by 27% and a change in the diffraction field of roentgenograms from 0.23 to 0.60 HM. The dehydration of spin-labeled lysozyme crystals leads to an anomalous widening of EPR peaks without changes in their position. The dehydration in the humidity range studied has a two-stage character. The decrease in humidity to 75% is accompanied by a sharp change in the parameters measured, and on further decrease in humidity to 40% they change insignificantly. The first stage is caused by the removal of the greater part of molecules of bulk water, and the second stage is due to the removal of the remaining bulk water and possible changes in the dynamics of weakly bound water molecules and their position. The simulation of experimental EPR spectra showed that the anomalous broadening of the spectrum upon dehydration is related to an increase in the dispersion of spin label orientations induced by changes in the network of hydrogen bonds generated by water molecules in the vicinity of the spin label and a possible turn (by no more than 5 degrees) of the entire protein molecule. After rehydration, the physical state of the lysozyme crystal did not return to the starting point.
Subject(s)
Muramidase/chemistry , Spin Labels , Water , Crystallization , Electron Spin Resonance Spectroscopy , Histidine/chemistry , Models, Molecular , Protein Conformation , X-Ray DiffractionABSTRACT
The crystal structures of myoglobin in the deoxy- and carbon monoxide-ligated states at a resolution of 1.15 angstroms show that carbon monoxide binding at ambient temperatures requires concerted motions of the heme, the iron, and helices E and F for relief of steric inhibition. These steps constitute the main mechanism by which heme proteins lower the affinity of the heme group for the toxic ligand carbon monoxide.
Subject(s)
Carbon Monoxide/metabolism , Myoglobin/analogs & derivatives , Myoglobin/chemistry , Animals , Binding Sites , Carbon Monoxide/chemistry , Crystallography, X-Ray , Heme/chemistry , Heme/metabolism , Histidine/chemistry , Histidine/metabolism , Hydrogen Bonding , Iron/chemistry , Iron/metabolism , Ligands , Metmyoglobin/chemistry , Models, Molecular , Myoglobin/metabolism , Nitrogen/chemistry , Nitrogen/metabolism , Protein Conformation , Protein Structure, Secondary , Temperature , Valine/chemistry , Valine/metabolismABSTRACT
Micromethods for measurements of electric conductivity, transference numbers and concentrations of inorganic ions within immobilized protein crystals have been developed and applied to study tetragonal lysozyme crystals cross-linked with glutaraldehyde. Donnan equilibria and mobilities of ions in this crystal were calculated using the data of these methods and the data of crystal pH titration. Taken together these results characterize the lysozyme crystal as an ion exchanger whose electrical properties and ion composition differ greatly from those of the external solution. Although anions transfer most of the current in the crystals, anion mobility is considerably lower than that of cations. Mobility of all ions in the crystal is considerably lower than in solution (3.5-50 times for cations and 120-330 times for anions) and depends on steric restrictions and charges of both ions and lysozyme molecules. Similar features in behavior of crystalline and biological channels are discussed.
Subject(s)
Muramidase/chemistry , Chemical Phenomena , Chemistry, Physical , Cross-Linking Reagents/chemistry , Crystallization , Electric Conductivity , Enzyme Activation , Glutaral/chemistry , Hydrogen-Ion Concentration , Ion Channels/chemistry , Ions , Osmolar Concentration , Sodium Chloride/chemistryABSTRACT
The method of the studying of the ionic content in the protein crystals by X-ray fluorescence spectroscopy is proposed. The ionic content of the tetragonal glutaraldehyde--cross-linked lysozyme crystals in the 2- 11 pH range and upon the low and high ionic strengths is studied. The acid-base titration of the lysozyme crystals is carried out for determination of the pH-dependence of the net charge of the lysozyme molecule within protein crystal. It has been shown that ionic content in the protein crystal channels is determined mainly by the Donnan potential. The specific binding of the bromide-ions with lysozyme molecules is revealed and possible binding sites are discussed.
Subject(s)
Muramidase/chemistry , Animals , Binding Sites , Bromides/chemistry , Chick Embryo , Crystallization , Glutaral/chemistry , Hydrogen-Ion Concentration , Ions , Spectrometry, X-Ray EmissionABSTRACT
The hen egg-white lysozyme was modified by the spin label (2,2,6,6-tetramethylpiperidine-N1-oxyl-4-iodacetamide) at the single histidine residue His-15. The rotational correlation time of the molecular carrier was found to be defined by the mobility of the histidine-bearing domain and not influenced by the protein monomer shape at pH 4.7 and dimer shape at pH 7.1. The dependence of viscosity at 1 degree C on the distance between outer wide peaks in the immobilized EPR spectra enabled us to evaluate rotational correlation time of the domain. The molecular mass of the latter was close to the data obtained by X-ray analysis. The spin label was highly mobile at room temperature, as the EPR spectrum displayed the triple shape; at 1 degree C it was immobilized. The new general approach to the EPR spectra simulation was applied to all experimental EPR spectra. This approach is based on a substitution of an undefined stochastic process of the spin label reorientation relative to the lysozyme domain by the defined modelled stochastic processes: axial rotation of the nitroxide relative to the preferable axis and angular oscillations of the nitroxide relative to axes of the molecular coordinate system. Each of the modelled stochastic processes leads to a relative partial averaging of the magnetic tensor components. A set of discrete partially averaged states is introduced with the relative cluster of the spin-labelled molecules. The resulting EPR spectrum is assumed to be the sum of EPR spectra from all the clusters. A good fitting of all simulated EPR spectra is obtained.
Subject(s)
Histidine/chemistry , Muramidase/chemistry , Animals , Chickens , Cold Temperature , Electron Spin Resonance Spectroscopy , Hydrogen-Ion Concentration , Protein Conformation , Spin Labels , ViscosityABSTRACT
We have written a package called CHANNEL for determining and analyzing the channels and cavities within protein crystals. By using CHANNEL, the intermolecular space within a crystal lattice can be divided into closed cavities and channels. This package is certainly useful in determining the channel topological structure and quantitative characteristics. The package allows also the volume, and maximal and minimal areas of channels along a required direction to be calculated.
Subject(s)
Algorithms , Protein Conformation , Software , CrystallizationABSTRACT
The salt composition of the solution has been found, which does not interact with glutaraldehyde and myoglobin at an ionic strength corresponding to that of the mother liquid, i.e., 4.5 M (NH4)2SO4. Cross-linking conditions have been elaborated when crystalline myoglobin retains its native structure: myoglobin in 3 M Cs2SO4 pH 5.4 is cross-linked by diffusion in 5% glutaraldehyde vapours in the same salt solution (5 days, room temperature).
Subject(s)
Glutaral/chemistry , Myoglobin/chemistry , Animals , Cross-Linking Reagents , Crystallization , Diffusion , Osmolar Concentration , Spectrum Analysis , WhalesABSTRACT
A high resolution structure of hen egg-white lysozyme containing 36 +/- 1 mol H2O per mol of protein has been obtained using triclinic (P1) crystals cross-linked with glutaraldehyde. Analysis of dehydration-induced structural changes has revealed displacement in relative position of domains and numerous small displacements in positions of individual atoms with r.m.s. deviation of main atoms 0.60 A, and that of all atoms 0.97 A. An increase in the average packing density of atoms in dry lysozyme by 4-6% seems to be the most probable reason for the loss of its activity and mobility.
Subject(s)
Muramidase/chemistry , Animals , Chickens , Egg White , Models, Molecular , X-Ray DiffractionABSTRACT
The influence of substitution of the isotopic composition of the medium on the mechanical properties of immobilized crystals and films from bovine pancreatic ribonuclease and hen egg white lysozyme was investigated. The order of magnitude of the observed effects indicates that the contribution of the electrostatic interaction to the observed isotopic effect may be considered inessential. The absence of aggregation in the H2O and D2O medium under experimental conditions is demonstrated by the method of the low angle dispersion of X-rays. The observed effects of D2O on the mechanical behavior of crystals and films of proteins may be accounted for by the strengthening of molecular interactions in the samples.
Subject(s)
Deuterium , Proteins/analysis , Water , Animals , Cattle , Crystallization , Deuterium Oxide , Muramidase/analysis , Protein Conformation , Ribonuclease, Pancreatic/analysis , Tensile StrengthABSTRACT
The fluorescent properties of the complexes of apoMb-PPIX and dimethyl-PPIX were studied in a wide range of pH and ionic strength. The quenching of fluorescence of PPIX in the complex by I- is shown to be of dynamic type Increasing of the Stern--Volmer constant was observed in the acidic region of pH. From the sections of space-filled model of Mb the static accessibility of porphine macrocyte was calculated. It is proposed that the revealed peculiarities of the myoglobin structure allow to interprete the data on the quenching of fluorescence of PPIX by iodid ion.
