Sequence Of Lysozyme Research Articles

First Report of Lysozyme Amyloidosis with p.F21L/T88N Amino Acid Substitutions in a Russian Family.

Lysozyme amyloidosis is caused by an amino acid substitution in the sequence of this protein. In our study, we described a clinical case of lysozyme amyloidosis in a Russian family. In our work, we described in detail the histological changes in tissues that appeared as a result of massive deposition of amyloid aggregates that affected almost all organ systems, with the exception of the central nervous system. We determined the type of amyloidosis and mutations using mass spectrometry. Using mass spectrometry, the protein composition of tissue samples of patient 1 (autopsy material) and patient 2 (biopsy material) with histologically confirmed amyloid deposits were analyzed. Amino acid substitutions p.F21L/T88N in the lysozyme sequence were identified in both sets of samples and confirmed by sequencing of the lysozyme gene of members of this family. We have shown the inheritance of these mutations in the lysozyme gene in members of the described family. For the first time, we discovered a mutation in the first exon p.F21L of the lysozyme gene, which, together with p.T88N amino acid substitution, led to amyloidosis in members of the studied family.

Learning to Read and Write in the Language of Proteins

Learning to Read and Write in the Language of Proteins

Competitive adsorption of bovine serum albumin and lysozyme on a beta-tricalcium phosphate nanocoating

The competitive and cooperative adsorptions of proteins on the surface of implanted materials can affect the biocompatibility and osteogenesis of the material. The adsorption processes are related to the properties of the materials, as well as to the nature of the proteins. Competitive adsorption of bovine serum albumin (BSA) and lysozyme (LSZ), both sequentially, and from a mixture, was investigated on a beta-tricalcium phosphate (β-TCP) nanocoating using quartz crystal microbalance with dissipation technique. In experiments with individual proteins, the BSA adlayer showed higher adsorption quality and viscoelasticity than the LSZ on the β-TCP surface. In the sequential adsorption of LSZ + BSA, protein exchange was observed but not in the BSA + LSZ sequence, because of the larger molecular and higher surface affinity of BSA relative to LSZ. The substitution rate increased as the increasing concentration of the BSA solution. With a BSA&LSZ mixture, the total amount of protein adsorbed was greater than that for individual or sequential adsorption, owing to the formation of BSA and LSZ agglomerates. The electrostatic force and affinity were dominant factors governing the adsorption behavior between BSZ and LSZ on the β-TCP surface. This work will be beneficial to understand the protein adsorption mechanism on β-TCP surface and provide information to design better osteogenic materials.

Engineering Pichia pastoris for Efficient Production of a Novel Bifunctional Strongylocentrotus purpuratus Invertebrate-Type Lysozyme.

Lysozymes are known as ubiquitously distributed immune effectors with hydrolytic activity against peptidoglycan, the major bacterial cell wall polymer, to trigger cell lysis. In the present study, the full-length cDNA sequence of a novel sea urchin Strongylocentrotus purpuratus invertebrate-type lysozyme (sp-iLys) was synthesized according to the codon usage bias of Pichia pastoris and was cloned into a constitutive expression plasmid pPIC9K. The resulting plasmid, pPIC9K-sp-iLys, was integrated into the genome of P. pastoris strain GS115. The bioactive recombinant sp-iLys was successfully secreted into the culture broth by positive transformants. The highest lytic activity of 960U/mL of culture supernatant was reached in fed-batch fermentation. Using chitin affinity chromatography and gel-filtration chromatography, recombinant sp-iLys was produced with a yield of 94.5mg/L and purity of > 99%. Recombinant sp-iLys reached its peak lytic activity of 8560U/mg at pH 6.0 and 30°C and showed antimicrobial activities against Gram-negative bacteria (Vibrio vulnificus, Vibrio parahemolyticus, and Aeromonas hydrophila) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In addition, recombinant sp-iLys displayed isopeptidase activity which reached the peak at pH 7.5 and 37°C with the presence of 0.05M Na+. In conclusion, this report describes the heterologous expression of recombinant sp-iLys in P. pastoris on a preparative-scale, which possesses lytic activity and isopeptidase activity. This suggests that sp-iLys might play an important role in the innate immunity of S. purpuratus.

Peptide Probe for Multiwalled Carbon Nanotubes: Electrophoretic Assessment of the Binding Interface and Evaluation of Surface Functionalization

Noncovalent interactions of peptides and proteins with carbon nanotubes play a key role in sensing, dispersion, and biocompatibility. Advances in these areas require that the forces which contribute to physical adsorption are understood in order that the carbon nanotubes present a degree of functionalization appropriate to the desired application. Affinity analyses of peptides are employed to evaluate the role of tryptophan and arginine residues in physical adsorption to carboxylated multiwalled carbon nanotubes. Peptides containing arginine and tryptophan, WR(W) n, are used with affinity capillary electrophoresis to identify factors that lead to the formation of peptide-carbon nanotube complexes. The effects of changing the amino acid composition and residue length are evaluated by measuring dissociation constants. Electrostatic interactions contribute significantly to complexation, with the strongest interaction observed using the peptide WRWWWW and carboxylated carbon nanotube. Stronger interaction is observed when the tryptophan content is successively increased as follows: WR(W)4 > WR(W)3 > WR(W)2 > WRW > WR. However, as observed with polytryptophan (W5, W4, W3, and W2), removing the arginine residue significantly reduces the interaction with carbon nanotubes. Increasing the arginine content to WRWWRW does not improve binding, whereas replacing the arginine residue in WRWWWW with lysine (WKWWWW) reveals that lysine also contributes to surface adsorption, but not as effectively as arginine. These observations are used to guide a search of the primary sequence of lysozyme to identify short regions in the peptide that contain a single cationic residue and two aromatic residues. One candidate peptide sequence (WMCLAKW) from this search is analyzed by capillary electrophoresis. The dissociation constant of carboxylated multiwalled carbon nanotubes is measured for the peptide, WMCLAKW, to demonstrate the utility of affinity capillary electrophoresis analysis.

Identification and characterization of a novel goose-type and chicken-type lysozyme genes in Chinese rare minnow (Gobiocypris rarus) with potent antimicrobial activity

Lysozymes act as important innate immunity molecule against various pathogen infections. In the present study, a new c-type and g-type lysozymes were isolated from rare minnow Gobiocypris rarus. The deduced amino acid sequence of c-type lysozyme contained 145 residues and possessed conserved catalytic residues (Glu53 and Asp72). The deduced g-type lysozyme contained 185 residues and possessed three conserved catalytic residues (Glu73, Asp86 and Asp97). qPCR analysis revealed that GrlysG and GrlysC were constitutively expressed in all examined tissues. GrlysG was most abundant in kidney and liver, while GrlysC was predominantly expressed in liver. The transcripts of GrlysG and GrlysC genes could be significantly up-regulated after Aeromonas hydrophila and poly I:C infection in the kidney and liver. The recombinant GrlysG and GrlysC proteins were successfully produced and purified. The optimal pH and temperature for rGrlysG and rGrlysC protein lytic activities was determined to be 6.5 and 32°C, respectively. From the minimal inhibitory concentration test, the rGrlysG and rGrlysC exhibited apparent antibacterial activities against both Gram-positive and Gram-negative bacteria at different concentrations. In conclusion, the present study indicated that c-type and g-type lysozymes participated the innate immune response to A. hydrophila and virus infections in fish.

Molecular characteristics of lysozyme G in Euryglossa orientalis; cDNA cloning, phylogenic analysis, physicochemical properties and tissue gene expression.

Lysozymes are the key molecules of innate immune system against bacterial infections. In the present study, we identified the molecular characteristics, physicochemical properties, antibacterial activity, evolutionary relationship and tissue expression pattern of g-type lysozyme in Euryglossa orientalis (EuOr LysG). The full-length EuOr LysG cDNA is composed of 588 nucleotides and an open reading frame encoding a protein with 195 amino acids with more than 65% identity to g-type lysozyme of Solea senegalensis (73%) and Scophthalmus rhombus (64%). Amino acid sequence alignment showed that EuOr LysG protein possessed a conserved catalytic motif (Glu71-Asp84-Asp101) and one predicted disulfide bond between Cys32 and Cys122. Phylogenetic analysis based on the g-type lysozyme sequences indicated that E. orientalis and other fish of Pleuronectiformes were diverged together in the evolutionary history. The K m and V max values of the recombinant EuOr LysG were 0.266mg/ml of Micrococcus lysodeikticus as substrate and 667U/mg of protein, respectively. The optimum temperature and pH of recombinant EuOr LysG were 45 and 6°C, respectively. Real-time PCR analysis showed that EuOr LysG transcript was most abundant in head kidney and gill and less in muscle. We also showed that the EuOr LysG had potent lytic activity against major fish bacterial pathogens with the highest activity against Bacillus cereus and Aeromonas hydrophila. Bacterial challenge with Vibrio parahaemolyticus could upregulate LysG in immune-related tissues. Our results help to understand the molecular and physicochemical characteristics of g-type lysozyme in E. orientalis which might play an important role in host defense against the bacterial infection.

A novel minisequencing single-nucleotide polymorphism marker of the lysozyme gene detects high hatchability of Tsaiya ducks (Anas platyrhynchos)

Lysozyme, one of the major albumen antimicrobials, can break down the polysaccharide walls of a broad spectrum of bacteria. This study presents a novel lysozyme marker of high hatchability in the form of minisequencing single-nucleotide polymorphisms (SNPs). Recently, lysozyme was identified by complementary DNA microarray analysis as one of several differentially expressed genes noted to influence hatchability and recognized as a marker candidate for animal marker-assisted selection. Higher levels (P < 0.05) of lysozyme mRNA (via real-time polymerase chain reaction analysis) and protein (in Western blotting results) were found to be associated with a high-hatchability phenotype. In the preliminary sequence analysis of this study, TsLy1-1 and TsLy1-2 primer pairs, designed according to the lysozyme sequence, were used to amplify small-scale genomic DNA samples from animals in two extreme groups of hatchability. Sequence analysis of the amplified 763-bp DNA products clearly showed that AA and GG genotypes of SNP g.390A > G were from the ducks of the low- and high-hatchability groups, respectively. The SNP g.390A > G also created a new specificity protein 1 transcription factor binding site in the lysozyme gene. Primer pairs of TsLy2-1 and TsLy2-2 then probed the amplified 763-bp DNA products to produce a shorter fragment for easier minisequencing analysis to divide 114 ducks into GG, GA, and AA genotypes. The GG ducks had the highest hatchability, representing that a new lysozyme SNP marker of good hatchability performance can be used for the purpose of marker-assisted selection in Tsaiya ducks.

Molecular cloning and characterization of a lysozyme cDNA from the mole cricket Gryllotalpa orientalis (Orthoptera: Gryllotalpidae).

A full-length lysozyme cDNA from Gryllotalpa orientalis was cloned and sequenced. The deduced amino acid sequence of the lysozyme protein was 143 amino acids in length, with a calculated molecular mass of 15.84 kDa and an isoelectric point of 4.74. Sequence motifs, together with alignment and phylogenetic results, confirmed that G. orientalis lysozyme belongs to the C (chicken)-type lysozyme family of proteins. The protein sequence of lysozyme from G. orientalis showed high identity to that of Drosophila melanogaster (51.7 %); however, in contrast to D. melanogaster lysozyme, G. orientalis lysozyme was immune inducible and expressed in a wide range of tissues. Expression of G. orientalis lysozyme mRNA was highest at 8 h post-infection and subsequently decreased with time after bacterial infection. We also expressed G. orientalis lysozyme protein in vitro using the pET expression system. Compared with the negative control, over-expressed G. orientalis lysozyme showed antimicrobial activity against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Bacillus subtilis by radial diffusion assay, with minimal inhibitory concentration values of 30.3 and 7.55 µM, respectively. These results indicate that G. orientalis lysozyme may have stronger antimicrobial activity than other lysozymes against a broad range of microorganisms.

Molecular characterization, gene structure and antibacterial activity of a g-type lysozyme from the European sea bass (Dicentrarchus labrax L.)

In fish, the first line of defense is represented by the innate immune system and the lysozyme is one of the molecules involved in this mechanism of protection. Three types of lysozymes have been identified in metazoan, the c-type (chicken or conventional), the g-type (goose-type) and the i-type (invertebrate type). They are all involved in the hydrolysation of the bacterial cell wall. Our work has been focused on the molecular characterization, expression analysis by real-time PCR, both at basal condition and after in vivo challenges, and 3D structural studies on the g-type lysozyme from sea bass (Dicentrarchus labrax L.). Moreover, a recombinant sea bass lysozyme has been produced in Escherichia coli and used to investigate the activity of the enzyme at different pH and temperatures and to perform antibacterial assays against typical fish pathogens. The cloned sea bass cDNA for g-type lysozyme (accession number FN667957) consists of 742bp and translates for a putative protein of 188 amino acids. The molecular weight is 20.251, 41Da with a theoretical pI of 8.53, two cysteine residues along the sequence and no putative signal peptide. These features of the enzyme are in agreement with the expected characteristics of a proper g-type lysozyme, except for the cysteine residues that in fish are quite variable in number. An alignment between known g-type lysozyme sequences evidences that the amino acid residues thought to be involved in the enzyme catalysis (Glu71, Asp84 and Asp95 in sea bass) are quite well conserved between mammalian, avian and fish sequences. The sea bass g-type lysozyme gene is composed of four exons and three introns and this gene structure is more compact compared to other known fish lysozyme homologues. Modeling of 3D structure has been performed on the template structure of g-type lysozyme from Atlantic cod. The catalytic site appears well conserved when compared with known structures of fish g-type lysozymes (cod and salmon). The basal expression of lysozyme transcripts is highest in gills, followed by head kidney and peripheral blood leukocytes. The lysozyme expression is up regulated in head kidney leukocytes both after challenge with the fish bacterial pathogen Photobacterium damselae subsp. piscicida. The lytic activity, determined using as substrate Micrococcus lysodeikticus, was optimal at pH 5.5 and at a temperature of 30°C. In conclusion, these results suggest that the identified g-type lysozyme should be involved in the innate immune responses of sea bass.

Cysteine inhibits amyloid fibrillation of lysozyme and directs the formation of small worm‐like aggregates through non‐covalent interactions

In this article, we discuss the effects of amino acids on amyloid aggregation of lysozyme. l-cysteine (Cys) dramatically inhibited fibrillation of lysozyme, whereas other amino acids (including l-arginine) did not. In the presence of Cys, the aggregation pathway of lysozyme shifted from fibrillation to the formation of the small worm-like aggregates with unfolding. The interaction between Cys and lysozyme was observed to be non-covalent, suggesting that the thiophilic interaction between the thiol group on the side chain of Cys and the core sequence of lysozyme significantly contributes to the inhibition of amyloid aggregation. These findings provide a new basis for the design of a biocompatible additive to prevent amyloid fibrillation.

Label-free bifunctional electrochemiluminescence aptasensor for detection of adenosine and lysozyme

A label-free bifunctional electrochemiluminescence (ECL) aptasensor for parallel detection of small molecule (adenosine) and protein (lysozyme) was developed based on switching structures of aptamers from DNA/DNA duplex to DNA/target complex. Thiolated single-stranded DNA (DNA1) containing adenosine aptamer and sequence complementary to DNA2 was first immobilized on the Au electrode via sulfur–gold affinity, DNA2 containing lysozyme aptamer and sequence complememtary to DNA3 was bound to the electrode via forming DNA/DNA duplex. Gold nanoparticles functionalized with DNA3 (DNA3-GNPs) were used as signal amplifier to enhance the sensitivity of the aptasensor. With Ru(phen)32+ as the signal transducer, the aptasensor exhibited high sensitivity and specificity. Under optimal conditions, a linear range from 0.5 to 7nM for adenosine (R=0.999) and from 0.5 to 9ng/mL for lysozyme (R=0.999) was obtained, respectively. The proposed method may provide a promising platform for ECL parallel detection of small molecules and protein.

Effect of polymorphism in egg white lysozyme on muramidase and antibacterial activities as well as hatchability in the Japanese quail (Coturnix japonica)

Lysozyme is one of the best characterized antimicrobial proteins in egg white. Three phenotypes of egg white lysozyme in Japanese quail, Coturnix japonica, (namely fast; slow; and the combination, FS) were observed by acid polyacrylamide gel electrophoresis. The fast phenotype showed faster mobility on Acid-PAGE than the slow phenotype. Comparison of the coding sequences for lysozyme derived from the slow and fast phenotypes revealed a nonsynonymous SNP at nucleotide position 115 from the translation initiation site, which alters AA sequence of lysozyme. This nonsynonymous SNP converted glutamine (Q) in the slow phenotype to lysine (K) in the fast phenotype at AA residue 21 of mature lysozyme (Q21K). Here, we investigated the effect of these phenotypes on muramidase activity, antibacterial activity, and hatchability. Muramidase activity toward isolated cell walls of Micrococcus lysodeikticus was in the order: fast allozyme > slow allozyme > chicken (Gallus gallus), but no significant difference was found among the 3 (P > 0.05). Antibacterial activity against live Staphylococcus aureus cells was significantly greater for the fast allozyme than the slow allozyme from 20 h after incubation (P < 0.05). For the antibacterial effects against live Escherichia coli cells, the activity of fast was significantly higher than that of slow at 16 h after incubation (P < 0.05). Hatchability was estimated for reciprocal crosses of Japanese quail with the FF (fast) and SS (slow) genotypes. Hatchability was 92.5% in FF male × SS female crosses and 87.2% in SS male × FF female crosses. A Cochran-Mantel-Haenszel test revealed a significant difference between the crosses (P < 0.05) and indicated that the female-derived slow phenotype led to improved rates of hatching. Our results suggest that the nonsynonymous SNP in Japanese quail lysozyme influences the electrophoretic migration, muramidase activity, and antibacterial activity of the protein, in addition to the hatchability of the eggs. These results demonstrate, for the first time, a significant difference in antibacterial activity and hatchability between 2 lysozyme phenotypes in Japanese quail.

Stacking of antimicrobial genes in potato transgenic plants confers increased resistance to bacterial and fungal pathogens

Solanum tuberosum plants were transformed with three genetic constructions expressing the Nicotiana tabacum AP24 osmotine, Phyllomedusa sauvagii dermaseptin and Gallus gallus lysozyme, and with a double-transgene construction expressing the AP24 and lysozyme sequences. Re-transformation of dermaseptin-transformed plants with the AP24/lysozyme construction allowed selection of plants simultaneously expressing the three transgenes. Potato lines expressing individual transgenes or double- and triple-transgene combinations were assayed for resistance to Erwinia carotovora using whole-plant and tuber infection assays. Resistance levels for both infection tests compared consistently for most potato lines and allowed selection of highly resistant phenotypes. Higher resistance levels were found in lines carrying the dermaseptin and lysozyme sequences, indicating that theses proteins are the major contributors to antibacterial activity. Similar results were obtained in tuber infection tests conducted with Streptomyces scabies. Plant lines showing the higher resistance to bacterial infections were challenged with Phytophthora infestans, Rhizoctonia solani and Fusarium solani. Considerable levels of resistance to each of these pathogens were evidenced employing semi-quantitative tests based in detached-leaf inoculation, fungal growth inhibition and in vitro plant inoculation. On the basis of these results, we propose that stacking of these transgenes is a promising approach to achieve resistance to both bacterial and fungal pathogens.

Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs

BackgroundG protein coupled receptors (GPCRs) represent the largest family of membrane proteins in the human genome and the richest source of targets for the pharmaceutical industry. A major limitation to characterizing GPCRs has been the difficulty in developing high-level heterologous expression systems that are cost effective. Reasons for these difficulties include inefficient transport and insertion in the plasma membrane and cytotoxicity. Additionally, GPCR purification requires detergents, which have a negative effect on receptor yields and stability.ResultsHere we report a detergent-free cell-free protein expression-based method to obtain pharmacologically active GPCRs in about 2 hours. Our strategy relies on the co-translational insertion of modified GPCRs into nanometer-sized planar membranes. As a model we employed an engineered β2-adrenergic receptor in which the third intracellular loop has been replaced with T4 lysozyme (β2AR -T4L). We demonstrated that nanolipoprotein particles (NLPs) are necessary for expression of active β2AR -T4L in cell-free systems. The binding specificity of the NLP- β2AR-T4L complex has been determined by competitive assays. Our results demonstrate that β2AR-T4L synthesized in vitro depends on similar oxidative conditions as those required by an in vivo-expressed receptor.ConclusionsAlthough the activation of β2AR-T4L requires the insertion of the T4 lysozyme sequence and the yield of that active protein limited, our results conceptually prove that cell-free protein expression could be used as a fast approach to express these valuable and notoriously difficult-to-express proteins.

Population of nonnative states of lysozyme variants drives amyloid fibril formation.

The propensity of protein molecules to self-assemble into highly ordered, fibrillar aggregates lies at the heart of understanding many disorders ranging from Alzheimer's disease to systemic lysozyme amyloidosis. In this paper we use highly accurate kinetic measurements of amyloid fibril growth in combination with spectroscopic tools to quantify the effect of modifications in solution conditions and in the amino acid sequence of human lysozyme on its propensity to form amyloid fibrils under acidic conditions. We elucidate and quantify the correlation between the rate of amyloid growth and the population of nonnative states, and we show that changes in amyloidogenicity are almost entirely due to alterations in the stability of the native state, while other regions of the global free-energy surface remain largely unmodified. These results provide insight into the complex dynamics of a macromolecule on a multidimensional energy landscape and point the way for a better understanding of amyloid diseases.

Co-adsorption of electrolyte and protein to Ag colloid observed by surface-enhanced Raman scattering

Protein detection using surface-enhanced Raman scattering (SERS) usually requires electrolytes to yield an enhanced SERS signal. However, the adsorption mechanism of electrolyte and protein to Ag colloid is not yet clearly understood. In this work, we have investigated co-adsorption of NO(3)(-) and lysozyme to Ag colloid using SERS. Three experimental factors including concentration of lysozyme (10(-5) and 10(-6) M), concentration of NO(3)(-) (0, 1, 2, 3 and 5 mM) and drying temperature (25 and 100 degrees C) have been studied. The results have shown that the co-adsorption of the adsorbates (lysozyme and NO(3)(-)) on a SERS substrate and the non-absorption of NO(3)(-) on the substrate can be controlled by using different experimental conditions. The co-adsorption manner of lysozyme and NO(3)(-) is consistent with the mechanism of double-layer adsorbates when a protein adsorbs on a solid/liquid interface. The variation in protein conformation, especially the main-chain conformation, seems to affect the adsorption manner of the adsorbates. It has been found that the final adsorption result is not affected by the addition sequence of lysozyme and NO(3)(-) during the sample preparation.

New variant of quail egg white lysozyme identified by peptide mapping

Two lysozymes were purified from quail egg white by cation exchange column chromatography and analyzed for amino acid sequence. The enzymes showed the same pH optimum profile for lytic activity with broad pH optima (pH 5.0–8.0) but had difference in mobility on native-PAGE. The native-PAGE immunoblot showed one or two lysozymes present in individual egg whites. The established amino acid sequence of quail egg white lysozyme A (QEWL A) was the same as quail lysozyme reported by Kaneda et al. [Kaneda, M., Kato, I., Tominaga, N., Titani, K., Narita, K., 1969. The amino acid sequence of quail lysozyme. J. Biochem. (Tokyo). 66, 747–749] and had six amino acid substitutions at position 3 (Phe to Tyr), 19 (Asn to Lys), 21 (Arg to Gln), 102 (Gly to Val) 103 (Asn to His) and 121 (Gln to Asn) compared to hen egg white lysozyme. QEWL A and QEWL B showed one substitution, at the position 21, Gln replaced by Lys, plus an insertion of Leu between position 20 and 21, being the first report that QEWL B had 130 amino acids. The amino acid differences between two lysozymes did not seem to affect antigenic determinants detected by polyclonal anti-hen egg white lysozyme, but caused them to separate well from each other by ion exchange chromatography.

Photocleavage of Lysozyme by Cobalt(III) Complexes

Photochemical reagents that cleave proteins at specific sites (photoproteases) are useful for studying protein structure and protein-ligand interactions. PolyammineCo(III) complexes are tested here as photochemical probes to cleave proteins. Irradiation of a mixture of lysozyme, a model protein, and polyammineCo(III) complexes resulted in the facile cleavage of the peptide backbone. Photocleavage yielded two fragments of molecular weights 10.6 and 3.7 kDa, and these masses sum to the molecular mass of lysozyme (14.3 kDa). No cleavage was detected in the absence of the metal complex, in the dark, or upon irradiation at wavelengths of >420 nm. The photocleavage yield increased with irradiation time and with the concentrations of the metal complex and the protein. N-terminal sequencing of the 10.6 kDa fragment indicated residues that are identical to the N-terminus of lysozyme, and sequencing of the 3.7 kDa fragment indicated Val-Ala-Trp-Arg, an internal sequence of lysozyme. From the known primary sequence of lysozyme and the sequencing data, the cleavage site was assigned to Trp108-Val109. Molecular modeling indicates that the observed cleavage site is within few angstroms from the proposed metal binding site at Glu35-Asp52. This is the first report of the successful photocleavage of proteins, with high selectivity, by transition metal complexes. This novel observation can facilitate the rational design of transition metal complexes for the photochemical footprinting of metal binding sites on proteins.

A Genetic Algorithm Approach to Detecting Lineage-Specific Variation in Selection Pressure

The ratio of nonsynonymous (dN) to synonymous (dS) substitution rates, omega, provides a measure of selection at the protein level. Models have been developed that allow omega to vary among lineages. However, these models require the lineages in which differential selection has acted to be specified a priori. We propose a genetic algorithm approach to assign lineages in a phylogeny to a fixed number of different classes of omega, thus allowing variable selection pressure without a priori specification of particular lineages. This approach can identify models with a better fit than a single-ratio model, and with fits that are better than (in an information theoretic sense) a fully local model, in which all lineages are assumed to evolve under different values of omega, but with far fewer parameters. By averaging over models which explain the data reasonably well, we can assess the robustness of our conclusions to uncertainty in model estimation. Our approach can also be used to compare results from models in which branch classes are specified a priori with a wide range of credible models. We illustrate our methods on primate lysozyme sequences and compare them with previous methods applied to the same data sets.