Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

We have solved the 2.0-A resolution crystal structures of four cavity-creating Ile/Leu-->Ala mutations in the hydrophobic core of barnase and compare and contrast the structural responses to mutation with those found for Leu-->Ala mutations in T4 lysozyme. First, there are rearrangements of structure of barnase that cause the cavities to collapse partly, and there is an approximately linear relationship between the changes in stability and the volume of the cavity similar to that found for the mutants of T4 lysozyme. Second, although it is currently accepted that hydrophobic cavities formed on the mutation of large hydrophobic side chains to smaller ones are not occupied by water molecules, we found a buried water molecule in the crystal structure of the barnase mutant Ile76-->Ala. A single hydrogen bond is formed between the water molecule and a polar atom, the carbonyl oxygen of Phe7, in the hydrophobic cavity that is formed on mutation. A survey of hydrophobic cavities produced by similar mutations in different proteins reveals that they all contain a proportion of polar atoms in their linings. The availability of such polar sites has implications for understanding folding pathways because a solvated core is presumed present in the transition state for folding and unfolding. Notably, the hydrogen bond between the cavity-water and the carbonyl group of Phe7 is also a marked early feature of very recent molecular dynamics simulations of barnase denaturation [Caflisch, A., & Karplus, M. (1995) J. Mol. Biol. 252, 672-708]. It is possible that cavities engineered into the hydrophobic cores of other proteins may contain water molecules, even though they cannot be detected by crystallographic analysis.
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PMID:Structural and energetic responses to cavity-creating mutations in hydrophobic cores: observation of a buried water molecule and the hydrophilic nature of such hydrophobic cavities. 860 78

Adaptive evolution of lysozyme has involved remodelling of amino acid sequences and changes in patterns of gene expression and in gene number. Following an outline of the phenomena likely to be indicative of adaptive evolution and how one can assess them, this chapter focuses on four cases in which lysozyme c has been recruited as a digestive enzyme in the stomachs of creatures needing to retrieve nutrients from microorganisms in fermented food. For each case-ruminant artiodactyls, leaf-eating monkeys, a leaf-eating bird, and fruit flies-the factors likely to be of primary importance in lysozyme's adaptation are examined. Additional examples of apparent adaptation for digestion or antimicrobial defense in animals as diverse as mice, moths, and molluscs are summarized. This chapter considers also the case of three internally clustered residues which among galliform bird lysozymes c occur either as Thr 40, Ile 55, and Ser 91 (TIS) or as Ser 40, Val 55, and Thr 91 (SVT). Reconstruction and testing of six possible intermediate proteins and development of the concept of a neutral corridor of protein traits are described.
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PMID:Adaptive evolution of lysozyme: changes in amino acid sequence, regulation of expression and gene number. 876 7

To clarify the contribution of the hydrophobic effect to the conformational stability of human lysozyme, a series of Val to Ala mutants were constructed. The thermodynamic parameters for the denaturation of these nine mutant proteins were determined using differential scanning calorimetry (DSC), and the crystal structures were solved at high resolution. The denaturation Gibbs energy (delta delta G) and enthalpy (delta delta H) values of the mutant proteins ranged from +2.2 to- 6.3 kJ/mol and from +7 to -17 kJ/mol, respectively. The structural analyses showed that the mutation site and/or the residues around it in some proteins shifted toward the created cavity, and the substitutions affected not only the mutations site but also other parts far from the site, although the structural changes were not as great. Correlation between the changes in the thermodynamic parameters and the structural features of mutant proteins was examined, including the five Ile to Val mutant human lysozymes [Takano et al. (1995) J. Mol. Biol. 254, 62-76]. There was no simple general correlation between delta delta G and the changes in hydrophobic surface area exposed upon denaturation (delta delta ASAHP). We found only a new correlation between the delta delta G and delta delta ASAHP of all of the hydrophobic residues if the effect of the secondary structure propensity was taken into account.
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PMID:Contribution of the hydrophobic effect to the stability of human lysozyme: calorimetric studies and X-ray structural analyses of the nine valine to alanine mutants. 902 Jul 66

Surface tension kinetics exhibited by the wild type and selected stability mutants of T4 lysozyme at the air-water interface were monitored with DuNouy tensiometry. Mutant lysozymes were produced by substitution of the isoleucine at position 3 with cysteine, leucine, glycine, and tryptophan. Each substitution resulted in an altered structural stability quantified by a change in the free energy of unfolding. Surface pressure kinetics were compared to the kinetic model evolving from a simple model for protein adsorption. This model allowed for parallel, irreversible adsorption into two states directly from solution, where state 2 molecules were more tightly bound to the surface and occupied greater interfacial area than state 1 molecules. Moreover, the model allowed state 2 molecules to increase spreading pressure more than state 1 molecules occupying the same interfacial area. The model indicated that less stable variants of T4 lysozyme have a greater tendency to adsorb in state 2, and state 2 molecules increase spreading pressure more than state 1 molecules occupying the same interfacial area. While agreement between the model and experimental data was very good at low concentration, these results suggest that a more comprehensive two-state model should account for the influence of surface coverage on the adsorption rate constants.
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PMID:Surface Tension Kinetics of the Wild Type and Four Synthetic Stability Mutants of T4 Phage Lysozyme at the Air-Water Interface 902 84

We summarize in this communication the data supporting the two functions of ribosome recycling factor (RRF, originally called ribosome releasing factor). The first described role involves the disassembly of the termination complex which consists of mRNA, tRNA and the ribosome bound to the mRNA at the termination codon. This process is catalyzed by two factors, elongation factor G (EF-G) and RRF. RRF stimulated protein synthesis as much as eight-fold in the in vitro lysozyme synthesis system, when ribosomes were limiting. In the absence of RRF, ribosomes remain mRNA-bound at the termination codon and translate downstream codons. In the in vitro system, the site of reinitiation is the triplet codon 3' to the termination codon. RRF is an essential protein for bacterial life. Temperature sensitive (ts) RRF mutants were isolated and in vivo translational reinitiation due to inactivation of ts RRF was demonstrated using the beta-galactosidase reporter gene placed downstream from the termination codon. A second function of RRF involves preventing errors in translation. In polyphenylalanine synthesis programmed by polyuridylic acid, misincorporation of isoleucine, leucine or a mixture of amino acids was stimulated upto 17-fold when RRF was omitted from the in vitro system. RRF did not influence the large error (10-fold increase) induced by streptomycin. This means that RRF participates not only in the disassembly of the termination complex but also in peptide elongation. Extending this concept and its conventional role for releasing ribosomes from mRNA, involvement of RRF in the reinitiation in the 3A' system (a construct using S aureus protein A, a collaborative work with Dr Isaksson), in programmed frame shifting, in trans-translation with 10Sa RNA (collaborative work with Dr Muto), and in the reinitiation downstream from the ORF A of the IS 3 (insertion sequence of a transposon, collaborative work with Dr Sekine) are discussed on the basis of preliminary data to be published elsewhere. Finally, we review the known RRF sequences from various organisms including eukaryotes and discuss the possible mechanism for disassembly of the eukaryotic termination complex.
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PMID:Dual functions of ribosome recycling factor in protein biosynthesis: disassembling the termination complex and preventing translational errors. 915 Aug 73

The paper is investigating the mechanism of stabilization of proteins by polyols at the molecular level. It is addressing the interactions of sorbitol, a polyol commonly used as a protein stabilizing agent, with hen egg white lysozyme, a well studied protein. Differential scanning calorimetry shows an increase in denaturation temperature of lysozyme upon addition of sorbitol at a concentration of 250 mM and above. Increasing sorbitol concentration also caused an increase in signal intensity of the CD spectrum of lysozyme in the wavelength region of 280-300 nm. Two-dimensional nuclear magnetic resonance spectroscopy was used to examine interactions between lysozyme and sorbitol. Most significant changes are manifest in the anomalous relaxation properties of Ala and Thr methyl groups indicating modifications of local motions and possibly compression of the entire structure. This is further corroborated by new intra-protein nuclear Overhauser effects in the presence of sorbitol. There is also evidence that water is displaced from the enzyme surface close to Ile-88 upon addition of sorbitol. In combination these results reveal a complex interplay of different interactions. Comparison to NMR-spectra of lysozyme with a bound inhibitor (tri-N-acetyl-glucosamine) shows that the interaction with sorbitol affects spatially disparate regions of the protein.
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PMID:Towards a molecular level understanding of protein stabilization: the interaction between lysozyme and sorbitol. 923 31

The genetic modification of lysozyme was attempted to improve the bactericidal activity against Gram-negative bacteria E. coli. The different lengths of hydrophobic peptides were attached to the C-terminus of the hen egg white lysozyme to investigate the most effective length of the hydrophobic peptides for killing bacteria. The oligonucleotides encoding Phe-Val-Pro (H3), Phe-Phe-Val-Ala-Pro (H5) and Phe-Phe-Val-Ala-Ile-Ile-Pro (H7) were fused to the C-terminus Leu 129 of lysozyme cDNA. The reconstructed cDNAs were inserted into the yeast expression vector. The hydrophobic peptide-fused lysozymes were secreted in the yeast carrying the reconstructed cDNA. Although the hydrophobic peptide-fused lysozymes retained 75 80% lytic activity of the wild-type protein, the bactericidal action to E. coli was greatly increased with the length of hydrophobic peptides. These results suggest that the hydrophobic peptides play an important role in killing Gram-negative bacteria. To elucidate the role of catalytic domain in bactericidal action of the hydrophobic fusion lysozyme (H5-Lz), the mutant hydrophobic lysozyme (H5/E35A-Lz) whose glutamic acid was substituted with alanine at the position 35 was constructed to diminish the catalytic activity. The mutant hydrophobic lysozyme (H5/E35A-Lz) was greatly lost the bactericidal action to E. coli, suggesting that not only the length of hydrophobic peptide fused to C-terminus but also the catalytic domain is important for the bactericidal action of the hydrophobic peptide-fused lysozyme.
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PMID:Bactericidal action of lysozymes attached with various sizes of hydrophobic peptides to the C-terminal using genetic modification. 932 80

To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non-polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the "large-to-small" substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts < or = 0.3 A); in other cases mainchain atoms moved up to 2 A. In the case of Ile 29 --> Ala the structure collapsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 --> Ala (V149A) and Met 6 --> Ala (M6A), however, are exceptions and have, respectively, one and two well-ordered water molecules within the cavity. The Val 149 --> Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild-type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 --> Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., -2.1 kcal/mol for all Leu --> Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol(-1) A(-3). Physically, this term is due to the loss of van der Waal's interactions between the bulky sidechain that is removed and the atoms that form the wall of the cavity. The overall results are consistent with the prior rationalization of Leu --> Ala mutants in T4 lysozyme by Eriksson et al. (Eriksson et al., 1992, Science 255:178-183).
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PMID:The response of T4 lysozyme to large-to-small substitutions within the core and its relation to the hydrophobic effect. 951 71

To get a general rule for the relationship between hydrophobic effect and conformational stability, five Ile to Val and nine Val to Ala mutants (3SS mutants) from 3SS (C77A/C95A) human lysozyme were constructed. As known from previous studies, the 3SS protein lacking a disulfide bond between Cys77 and Cys95 is destabilized by enthalpic factors, as revealed by a decrease of about 20 kJ/mol in the denaturation Gibbs energy change (DeltaG) value, as compared to the wild-type protein, which has four disulfide bonds. In this study, the stabilities and structures of the 3SS mutants were determined by differential scanning calorimetry and X-ray crystal analysis, respectively, and compared with those of the mutants (4SS mutants) from the wild-type (4SS) protein published previously. The stabilities of all the 3SS mutants, except for V110A-3SS were decreased as compared with that of the 3SS protein, coinciding with the results for the 4SS mutants. The change in the denaturation Gibbs energy change (DeltaDeltaG) values of the 3SS mutants relative to the 3SS protein at the denaturation temperature (49.2 degreesC) of the 3SS protein at pH 2.7 were similar to those of the equivalent 4SS mutants relative to the wild-type at 64.9 degreesC. The Delta DeltaG values of the 3SS mutants correlated with the changes in hydrophobic surface area exposed upon denaturation (Delta DeltaASAHP) for all of the hydrophobic residues when the effects of the secondary structure propensity were considered. This correlation is identical with that previously found for the 4SS mutants. The linear relation between Delta DeltaG and Delta DeltaASAHP for all of the hydrophobic residues with the same slope was found also for the mutants of T4 lysozyme already reported, indicating that this is a general relationship between changes in conformational stability and changes in ASA values of hydrophobic residues due to mutations.
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PMID:A general rule for the relationship between hydrophobic effect and conformational stability of a protein: stability and structure of a series of hydrophobic mutants of human lysozyme. 967 1

In an attempt to identify a systematic relation between the structure of a protein and its folding kinetics, the rate of folding was determined for 20 mutants of T4 lysozyme in which a bulky, buried, nonpolar wild-type residue (Leu, Ile, Phe, Val, or Met) was substituted with alanine. Methionine, which approximated the size of the original side chain but which is of different shape and flexibility, was also substituted at most of the same sites. Mutations that substantially destabilize the protein and are located in the carboxy-terminal domain generally slow the rate of folding. Destabilizing mutations in the amino-terminal domain, however, have little effect on the rate of folding. Mutations that have little effect on stability tend to have little effect on the rate, no matter where they are located. These results suggest that, at the rate-limiting step, elements of structure in the C-terminal domain are formed and have a structure similar to that of the fully folded protein. Consistent with this, two variants that somewhat increase the rate of folding (Phe104 --> Met and Val149 --> Met) are located within the carboxy-terminal domain and maintain or improve packing with very little perturbation of the wild-type structure.
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PMID:Methionine and alanine substitutions show that the formation of wild-type-like structure in the carboxy-terminal domain of T4 lysozyme is a rate-limiting step in folding. 1054 67


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