Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:[Dynamic mobility of the histidine-containing domain of spin-labeled lysozyme]. 772 55

The cDNA, coding for the first metal-binding domain (MBD1) of Menkes protein, was cloned into the T7-system based vector, pCA. The T7 lysozyme-encoding plasmid, pLysS, is shown to be crucial for expression, suggesting that the protein is toxic to the cells. Adding copper to the growth medium did not affect the plasmid stability. MBD1 is purified in two steps with a typical yield of 12 mg.L-1. Menkes protein, a P-type ATPase, contains a sequence GMXCXSC that is repeated six times, at the N-terminus. The paired cysteine residues are involved in metal binding. MBD1 has only two cysteine residues, which can exist as free thiol groups (reduced), as a disulphide bond (oxidized) or bound to a metal ion [e.g. Cu(I)-MBD1]. These three MBD1 forms have been investigated using CD. No major spectral change was seen between the different MBD1 forms, indicating that the folding is not changed upon metal binding. A copper-bound MBD1 was also studied by EPR, and the lack of an EPR signal suggests that the oxidation state of copper bound to MBD1 is Cu(I). Cu(I) binding studies were performed by equilibrium dialysis and revealed a stoichiometry of 1 : 1 and an apparent Kd = 46 microM. Oxidized MBD1, however, is not able to bind copper. Different copper complexes were investigated for their ability to reconstitute apo-MBD1. Given the same total copper concentration CuCl43- was superior to Cu(I)-thiourea (structural analogue of metallothionein) and Cu(I)-glutathione (used at fivefold higher copper concentration) although the latter two were able to partially reconstitute apo-MBD1. Cu(II) was not able to reconstitute apo-MBD1, presumably due to Cu(II)-induced oxidation of the thiol groups. Based on our results, glutathione and/or metallothionein are likely candidates for the in vivo incorporation of copper to Menkes protein.
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PMID:Expression, purification and copper-binding studies of the first metal-binding domain of Menkes protein. 1049 Nov 37

Glycation, the nonenzymatic reaction between protein amino groups and reducing sugars, induces protein damage that has been linked to several pathological conditions, especially diabetes, and general aging. Here we describe the direct identification of a protein-bound free radical formed during early glycation of histone H1 in vitro. Earlier EPR analysis of thermal browning reactions between free amino acids and reducing sugars has implicated the sugar fragmentation product glycolaldehyde in the generation of a 1,4-disubstituted pyrazinium free radical cation. In order to evaluate the potential formation of this radical in vivo, the early glycation of BSA, lysozyme, and histone H1 by several sugars (D-glucose, D-ribose, ADP-ribose, glycolaldehyde) under conditions of physiological pH and temperature was examined by EPR. The pyrazinium free radical cation was identified on histone H1 glycated by glycolaldehyde (g = 2.00539, aN = 8.01 [2N], aH = 5.26 [4H], aH = 2.72 [4H]), or ADP-ribose. Reaction of glycoaldehyde with poly-L-lysine produced an identical signal, whereas reaction with BSA or lysozyme produced only a minor unresolved singlet signal. In the absence of oxygen the signal was stable over several days. Our results raise the possibility that pyrazinium radicals may form during glycation of histone H1 in vivo.
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PMID:Formation of a protein-bound pyrazinium free radical cation during glycation of histone H1. 1102 99

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.
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PMID:[Effect of various humidity on local dynamic structure of lysozyme in a spin-labeled tetragonal crystal]. 1239 48

The method of spin labeling was used to monitor quick movements of side residues in protein monocrystals. The EPR spectra of monocrystals of spin-labeled lysozyme at different orientations of the tetrahonal crystal relative to the direction of the magnetic field were interpreted using the molecular dynamics method. A simple model was proposed, which enables one to calculate the trajectory of movements of the spin label by the molecular dynamic method over a relatively short period of time. The entire "frozen" protein molecule and a "defrozen" spin-labeled amino acid residue were considered in the framework of the model. To calculate the trajectories in vacuum, a model of spin-labeled lysozyme was constructed, and the parameters of force potentials for the atoms of the protein molecule and the spin label were specified. It follows from the calculations that the protein environment sterically hinders the range of eventual angular reorientations of the reporter NO-group of nitroxyl incorporated into the spin label, thereby affecting the shape of the EPR spectrum. However, the scatter in the positions of the reporter group in the angular space turned out to correspond to the Gauss distribution. Using the atomic coordinates of the spin label, obtained in a chosen time interval by the method of molecular dynamics, and taking into account the distribution of the states of the spin label in the ensemble of spin-labeled macromolecules in the crystal, we simulated the EPR spectra of monocrystals of spin-labeled lysozyme. The theoretical EPR spectra coincide well with the experimental.
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PMID:[Role of rapid movement of spin labels in interpreting EPR spectra for spin-labelled macromolecules]. 1451 78

The spin label method was used to observe the nature of the fast motions of side chains in protein monocrystals. The EPR spectra of spin-labeled lysozyme monocrystals (with different orientations of the tetragonal protein crystal in relation to the direction of the magnetic field) were interpreted using the method of molecular dynamics (MD). Within the proposed simple model, MD calculations of the spin label motion trajectories are performed in a reasonable real time. The model regards the protein molecule as frozen as a whole and the spin-labeled amino acid residue as unfrozen. To calculate the trajectories in vacuum, a model of spin-labeled lysozyme was assembled, and the parameters of the force fields were specified for atoms of the protein molecule, including the spin label. The calculations show that the protein environment sterically limits the area of the possible angular reorientations for the NO reporter group of the nitroxide (within the spin label), and this, in turn, affects the shape of the EPR spectrum. However, it turned out that the spread in the positions of the reporter group in the angle space strictly adheres to the Gaussian distribution. Using the coordinates of the spin label atoms obtained by the MD method within a selected time range and considering the distribution of the spin label states over the ensemble of spin-labeled macromolecules in a crystal, the EPR spectra of spin-labeled lysozyme monocrystals were simulated. The resultant theoretical EPR spectra appeared to be similar to experimental ones.
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PMID:The role of the fast motion of the spin label in the interpretation of EPR spectra for spin-labeled macromolecules. 1461 32

We have identified sequence and structural determinants of oligomer size, symmetry, and polydispersity in the small heat shock protein super family. Using an insertion mutagenesis strategy that mimics evolutionary sequence divergence, we induced the ordered oligomer of Methanococcus jannaschii Hsp16.5 to transition to either expanded symmetric or polydisperse assemblies. A hybrid approach combining spin labeling EPR and cryoelectron microscopy imaging at 10A resolution reveals that the underlying plasticity is mediated by a packing interface with minimal contacts and a flexible C-terminal tether between dimers. Twenty-four dimeric building blocks related by octahedral symmetry assemble into the expanded symmetric oligomer. In contrast, the polydisperse variant has an ordered dimeric building block that heterogeneously packs to yield oligomers of various sizes. Increased exposure of the N-terminal region in the Hsp16.5 variants correlates with enhanced binding to destabilized mutants of T4 lysozyme, whereas deletion of this region reduces binding. Transition to larger intermediates with enhanced substrate binding capacity has been observed in other small heat shock proteins including lens alpha-crystallin mutants linked to congenital cataract. Together, these results provide a mechanistic perspective on substrate recognition and binding by the small heat shock protein superfamily.
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PMID:Cryoelectron microscopy and EPR analysis of engineered symmetric and polydisperse Hsp16.5 assemblies reveals determinants of polydispersity and substrate binding. 1707 34

A nitroxide side chain (R1) has been substituted at single sites along a helix-turn-helix motif in T4 lysozyme (residues 114-135). Together with previously published data, the new sites reported complete a continuous scan through the motif. Mutants with R1 at sites 115 and 118 were selected for crystallographic analysis to identify the structural origins of the corresponding two-component EPR spectra. At 115, R1 is shown to occupy two rotamers in the room temperature crystal structure, one of which has not been previously reported. The two components in the EPR spectrum apparently arise from differential interactions of the two rotamers with the surrounding structure, the most important of which is a hydrophobic interaction of the nitroxide ring. Interestingly, the crystal structure at 100 K reveals a single rotamer, emphasizing the possibility of rotamer selection in low-temperature crystal structures. Residue 118 is at a solvent-inaccessible site in the protein core, and the structure of 118R1, the first reported for the R1 side chain at a buried site, reveals how the side chain is accommodated in an overpacked core.
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PMID:Structural determinants of nitroxide motion in spin-labeled proteins: tertiary contact and solvent-inaccessible sites in helix G of T4 lysozyme. 1747 14

In order to efficiently simulate spin label behavior when attached to the protein backbone we developed a novel approach that enhances local conformational sampling. The simulated scaling (SS) approach (Li, H., et al. J. Chem. Phys. 2007, 126, 24106) couples the random walk of a potential scaling parameter and molecular dynamics in the framework of hybrid Monte Carlo. This approach allows efficient barrier crossings between conformations. The method retains the thermodynamic detailed balance allowing for determination of relative free energies between various conformations. The accuracy of our method was validated by comparison with the recently resolved X-ray crystal structure of a spin labeled T4 lysozyme in which the spin label was in the interior of the protein. Consistent potentials of mean force (PMF) are obtained for the spin label torsion angles to illustrate their behavior in various protein environments: surface, semiburied, and buried. These PMFs reflect the experimentally observed trends and provide the rationale for the spin label dynamics. We have used this method to compare an implicit and explicit solvent model in spin label modeling. The implicit model, which is computationally faster, was found to be in excellent agreement with the explicit solvent treatment. Based on this collection of results, we believe that the presented approach has great potential in the general strategy of describing the behavior of the spin label using molecular modeling and using this information in the interpretation of EPR measurements in terms of protein conformation and dynamics.
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PMID:Mapping electron paramagnetic resonance spin label conformations by the simulated scaling method. 1794 93

Site-directed spin labeling provides a means for exploring structure and dynamics in proteins. To interpret the complex EPR spectra that often arise, it is necessary to characterize the rotamers of the spin-labeled side chain and the interactions they make with the local environment in proteins of known structure. For this purpose, crystal structures have been determined for T4 lysozyme bearing a nitroxide side chain (R1) at the solvent-exposed helical sites 41 and 44 in the B helix. These sites are of particular interest in that the corresponding EPR spectra reveal two dynamic states of R1, one of which is relatively immobilized suggesting interactions of the nitroxide with the environment. The crystal structures together with the effect of mutagenesis of nearest neighbors on the motion of R1 suggest intrahelical interactions of 41R1 with the i + 4 residue and of 44R1 with the i + 1 residue. Such interactions appear to be specific to particular rotamers of the R1 side chain.
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PMID:Structural determinants of nitroxide motion in spin-labeled proteins: solvent-exposed sites in helix B of T4 lysozyme. 1809 42


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