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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Effects of placing a lac operator at different positions relative to a promoter for bacteriophage T7 RNA polymerase were tested. Transcription can be strongly repressed by lac repressor bound to an operator centered 15 base-pairs downstream from the RNA start, but T7 RNA polymerase initiates transcription very actively from this T7lac promoter-operator combination in the absence of repressor, or in the presence of repressor plus inducer. Sequence changes in the transcribed region were found to make transcription from some T7 promoters, including the T7lac promoter, more sensitive to inhibition by T7 lysozyme. The pET-10 and pET-11 series of plasmid vectors have been constructed to allow target genes to be placed under control of the T7lac promoter and to be expressed in BL21(DE3) or HMS174(DE3), which carry an inducible gene for T7 RNA polymerase. These vectors carry a lacI gene that provides enough lac repressor to repress both the T7lac promoter in the multicopy vectors and the chromosomal gene for T7 RNA polymerase, which is controlled by the lacUV5 promoter. Very low basal expression of target genes is achieved, but the usual high levels of expression are obtained upon induction. Addition of T7 lysozyme can reduce basal expression even further and still allow high levels of expression upon induction. Genes that are very toxic to Escherichia coli can be maintained and expressed in this system.
J Mol Biol 1991 May 05
PMID:Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. 190 22

We have studied the internalization, processing and presentation of hen egg lysozyme (HEL) attached to surface IgD (sIgD) or MHC molecules on normal murine B cells, using heterocrosslinked bispecific antibodies (HBA). Nearly all HEL attached to sIgD was internalized within one hour, with at least a portion rapidly entering a chloroquine-sensitive, acidic environment. Degradation and presentation of HEL to hybridoma T cells began several hours after internalization. Degraded HEL was found in the medium after about 6 hr incubation, but at no time were significant amounts of HEL peptides found within the cells. When HEL was attached to class I or class II MHC molecules, its rate of internalization was low. The fraction of antigen bound to MHC molecules that was inside the cell was always low, even at later stages of culture, but the internalized antigen was located in an acidic environment. Degradation and presentation of HEL internalized via MHC molecules followed internalization. No difference was observed in the processing fate of HEL attached to class I or class II MHC molecules. These results suggest that the rate limiting step in antigen processing and presentation is antigen degradation, when the antigen is bound to sIgD, and internalization when bound to MHC molecules. The slow and steady processing of bound or internalized antigen could provide a sustained presence of antigen on the B cell surface and enhance the potential for its presentation to T cells.
Mol Immunol 1991 Jul
PMID:Processing fate of protein antigen attached to IgD or MHC molecules on normal B lymphocytes using heterocrosslinked bispecific antibodies. 190 83

Packing interactions in bacteriophage T4 lysozyme were explored by determining the structural and thermodynamic effects of substitutions for Ala98 and neighboring residues. Ala98 is buried in the core of T4 lysozyme in the interface between two alpha-helices. The Ala98 to Val (A98V) replacement is a temperature-sensitive lesion that lowers the denaturation temperature of the protein by 15 degrees C (pH 3.0, delta delta G = -4.9 kcal/mol) and causes atoms within the two helices to move apart by up to 0.7 A. Additional structural shifts also occur throughout the C-terminal domain. In an attempt to compensate for the A98V replacement, substitutions were made for Val149 and Thr152, which make contact with residue 98. Site-directed mutagenesis was used to construct the multiple mutants A98V/T152S, A98V/V149C/T152S and the control mutants T152S, V149C and A98V/V149I/T152S. These proteins were crystallized, and their high-resolution X-ray crystal structures were determined. None of the second-site substitutions completely alleviates the destabilization or the structural changes caused by A98V. The changes in stability caused by the different mutations are not additive, reflecting both direct interactions between the sites and structural differences among the mutants. As an example, when Thr152 in wild-type lysozyme is replaced with serine, the protein is destabilized by 2.6 kcal/mol. Except for a small movement of Val94 toward the cavity created by removal of the methyl group, the structure of the T152S mutant is very similar to wild-type T4 lysozyme. In contrast, the same Thr152 to Ser replacement in the A98V background causes almost no change in stability. Although the structure of A98V/T152S remains similar to A98V, the combination of T152S with A98V allows relaxation of some of the strain introduced by the Ala98 to Val replacement. These studies show that removal of methyl groups by mutation can be stabilizing (Val98----Ala), neutral (Thr152----Ser in A98V) or destabilizing (Val149----Cys, Thr152----Ser). Such diverse thermodynamic effects are not accounted for by changes in buried surface area or free energies of transfer of wild-type and mutant side-chains. In general, the changes in protein stability caused by a mutation depend not only on changes in the free energy of transfer associated with the substitution, but also on the structural context within which the mutation occurs and on the ability of the surrounding structure to relax in response to the substitution.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1991 Sep 20
PMID:Structural and thermodynamic analysis of the packing of two alpha-helices in bacteriophage T4 lysozyme. 192 Apr 39

The conformation of the milk protein alpha-lactalbumin has been studied using vibrational circular dichroism (VCD) and compared to parallel studies on lysozyme. These proteins have been shown by Acharya et al. [(1989) J. Mol. Biol. 208, 99-127] to have very similar three-dimensional crystal structures. However, their VCD spectra in D2O solution are quite different. The VCD of lysozyme in D2O more resembles that of alpha-lactalbumin in 33% propanol/D2O, under which conditions alpha-lactalbumin has conformationally transformed to a structure with increased helical fraction. These results can be seen to be consistent with UVCD and resolution-enhanced FTIR spectra of alpha-lactalbumin and lysozyme in both D2O and H2O environments. The solvent sensitivity of the alpha-lactalbumin spectra and hence of its conformation contrasted with the lack of such sensitivity for lysozyme suggest that the alpha-lactalbumin crystal structure represents a conformation different from that which is dominant in aqueous solution.
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PMID:Comparison of alpha-lactalbumin and lysozyme using vibrational circular dichroism. Evidence for a difference in crystal and solution structures. 193 71

Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above 9.0, and an excess of nine positive charges at neutral pH. It might be expected that it would be energetically costly to bring these out-of-balance charges from the extended, unfolded, form of the protein into the compact folded state. To determine the contribution of such long-range electrostatic interactions to the stability of the protein, five positively charged surface residues, Lys16, Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic acid. Eight selected double, triple and quadruple mutants were also constructed so as to sequentially reduce the out-of-balance formal charge on the molecule from +9 to +1 units. Each of the five single variant proteins was crystallized and high-resolution X-ray analysis confirmed that each mutant structure was, in general, very similar to the wild-type. In the case of R154E, however, the Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128 as the capping residue of a nearby alpha-helix. The thermal stabilities of all 13 variant proteins were found to be fairly similar, ranging from 0.5 kcal/mol more stable than wild-type to 1.7 kcal/mol less stable than wild-type. In the case of the five single charge-change variants, for which the structures were determined, the changes in stability can be rationalized in terms of changes in local interactions at the site of the replacement. There is no evidence that the reduction in the out-of-balance charge on the molecule increases the stability of the folded relative to the unfolded form, either at pH 2.8 or at pH 5.3. This indicates that long-range electrostatic interactions between the substituted amino acid residues and other charged groups on the surface of the molecule are weak or non-existent. Furthermore, the relative stabilities of the multiple charge replacement mutant proteins were found to be almost exactly equal to the sums of the relative stabilities of the constituent single mutant proteins. This also clearly indicates that the electrostatic interactions between the replaced charges are negligibly small. The activities of the charge-change mutant lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are essentially equal to that of the wild-type lysozyme, but on a lysoplate assay the mutant enzymes appear to have higher activity.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1991 Oct 05
PMID:Cumulative site-directed charge-change replacements in bacteriophage T4 lysozyme suggest that long-range electrostatic interactions contribute little to protein stability. 194 34

Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains of Salmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active. The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161).
J Mol Biol 1991 Nov 05
PMID:Systematic mutation of bacteriophage T4 lysozyme. 194 69

Genomic blotting and enzymatic amplification show that the genome of the langur monkey (like that of other primates) contains only a single gene for lysozyme c, in contrast to another group of foregut fermenters, the ruminants, which have a multigene family encoding this protein. Therefore, the langur stomach lysozyme gene has probably evolved recently (i.e., within the period of monkey evolution) from a conventional primate lysozyme. The sequences of cDNAs for the stomach lysozyme of langur and the conventional lysozymes of three other Old World monkeys were determined. Identification of the promoter for the stomach gene and comparison to the human gene, which is expressed conventionally in macrophages, show that both lysozyme genes use the same promoter. This suggests that the difference in expression patterns is due to change(s) in enhancer or silencer regulatory elements. With the cDNA sequences the hypothesis that the langur stomach lysozyme has converged in amino acid sequence upon the stomach lysozymes of ruminants is tested. Consistent with the convergence hypothesis, only those sites that specify amino acids in the mature lysozyme are shared uniquely with ruminant lysozyme genes. None of the silent sites at third positions of codons or in noncoding regions support a link between the langur and ruminants. Statistical analysis based on silent sites rules out the possibility of horizontal transfer of a stomach lysozyme gene between the langur and ruminant lineages and supports the close relationship of the langur lysozyme gene to that of other monkeys.
J Mol Evol 1991 Nov
PMID:Stomach lysozyme gene of the langur monkey: tests for convergence and positive selection. 196 Jul 39

Antibody E225 reacts with a private idiotope of the anti-lysozyme antibody D1.3. A complex between the Fab fragments from these BALB/c monoclonal antibodies has been crystallized and the determination of the three-dimensional structure of this idiotope-anti-idiotope complex is under way. The nucleotide VH and VL sequences of E225 presented here have been determined to provide the amino acid sequence information necessary for the interpretation of the high resolution electron density maps of the complex, obtained by X-ray crystallography. The cDNAs synthesized from the Vkappa and VH mRNAs were cloned in E. coli. Both cDNA strands were sequenced by the dideoxy termination method. The translated amino acid sequence shows that Vkappa, VH correspond to groups five (V) and II(b) of mouse immunoglobulin light and heavy chains, respectively. Sequence alignments between the complementarity determining regions of E225 and the antigenic determinant of lysozyme recognized by D1.3 do not indicate whether or not the anti-idiotopic antibody structurally mimics the external antigen.
Mol Immunol 1990 May
PMID:Nucleotide sequence of the VH, VL regions of an anti-idiotopic antibody reacting with a private idiotope of the anti-lysozyme D1.3 antibody. 197 59

Amide hydrogen/deuterium exchange behaviour has been studied for all of the peptide amides of hen lysozyme by means of two-dimensional n.m.r. spectroscopy. The amides have been grouped into four categories on the basis of their rates of exchange in solution at pH 4.2 and 7.5. The distribution of the amides into the different categories has been examined in the light of the crystallographic structural information, considering the type of secondary structure, the nature of hydrogen bonding and the distance from the protein surface. None of these features was found to determine uniquely the pattern of hydrogen exchange rates within the protein. The exchange behaviour of the individual amides could, however, in general be rationalized by a combination of these features. Hydrogen exchange was also monitored in both tetragonal and triclinic crystals of lysozyme, by allowing exchange to take place in the crystals prior to dissolution and recording of n.m.r. spectra under conditions where further exchange was minimized. This enabled direct comparison to be made of the exchange behaviour in the crystals and solution. A reduction in exchange rate was observed in the crystalline state relative to solution for a substantial number of amides and distinct differences between exchange in the different crystals could be observed. These differences between the solution and the different crystal states do not, however, correlate in a simple manner with proximity to intermolecular contacts in the crystals. However, the existence of these contacts, which are on the surface of the protein molecule, have a profound effect on the exchange of amides in the interior of the protein. The results indicate that the spectrum of fluctuations giving rise to hydrogen exchange may be significantly altered by the intermolecular interactions present within the crystalline state.
J Mol Biol 1991 Mar 20
PMID:A nuclear magnetic resonance study of the hydrogen-exchange behaviour of lysozyme in crystals and solution. 201 Sep 18

Bacteriophage T7 lysozyme, a natural inhibitor of T7 RNA polymerase, can reduce basal activity from an inducible gene for T7 RNA polymerase and allow relatively toxic genes to be established in the same cell under control of a T7 promoter. Low levels of T7 lysozyme supplied by plasmids pLysS or pLysL, which are compatible with the pET vectors for expressing genes from a T7 promoter, are sufficient to stabilize many target plasmids and yet allow high levels of target protein to be produced upon induction of T7 RNA polymerase. Higher levels of lysozyme supplied by plasmids pLysE or pLysH reduce the fully induced activity of T7 RNA polymerase such that induced cells can continue to grow and produce innocuous target proteins indefinitely. Different configurations of the expression system can maintain several different steady-state levels of target gene expression. The presence of T7 lysozyme has the further advantage of facilitating the lysis of cells in preparing extracts for purification of target gene products.
J Mol Biol 1991 May 05
PMID:Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. 202 59


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