Stabilization of bovine trypsin by reductive methylation

Rice, Robert H.; Means, Gary E.; Brown, W. Duane

doi:10.1016/0005-2795(77)90082-4

Cited by 116 publications

(89 citation statements)

References 27 publications

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“…More generally, protein lysine methylation has been associated with increases in protein stability. The chemical introduction of ⑀-dimethyllysine residues in bovine trypsin results in decreased autolysis (47) and in enhanced surface contacts in crystal structures (48). The thermal stability of ␤-glycosidase from the thermophilic archaeon Sulfolobus solfataricus is dependent upon the methylation of five of the 23 lysine residues (49).…”

Section: Discussionmentioning

confidence: 99%

The Ribosomal L1 Protuberance in Yeast Is Methylated on a Lysine Residue Catalyzed by a Seven-β-strand Methyltransferase

Webb¹,

Al-Hadid²,

Zurita-Lopez

et al. 2011

Journal of Biological Chemistry

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Modification of proteins of the translational apparatus is common in many organisms. In the yeast Saccharomyces cerevisiae, we provide evidence for the methylation of Rpl1ab, a well conserved protein forming the ribosomal L1 protuberance of the large subunit that functions in the release of tRNA from the exit site. We show that the intact mass of Rpl1ab is 14 Da larger than its calculated mass with the previously described loss of the initiator methionine residue and N-terminal acetylation. We determined that the increase in mass of yeast Rpl1ab is consistent with the addition of a methyl group to lysine 46 using topdown mass spectrometry. Lysine modification was confirmed by detecting 3 H-N-⑀-monomethyllysine in hydrolysates of Rpl1ab purified from yeast cells radiolabeled in vivo with S-adenosyl-L-[methyl-3 H]methionine. Mass spectrometric analysis of intact Rpl1ab purified from 37 deletion strains of known and putative yeast methyltransferases revealed that only the deletion of the YLR137W gene, encoding a seven-␤-strand methyltransferase, results in the loss of the ؉14-Da modification. We expressed the YLR137W gene as a His-tagged protein in Escherichia coli and showed that it catalyzes N-⑀-monomethyllysine formation within Rpl1ab on ribosomes from the ⌬YLR137W mutant strain lacking the methyltransferase activity but not from wild-type ribosomes. We also showed that the His-tagged protein could catalyze monomethyllysine formation on a 16-residue peptide corresponding to residues 38 -53 of Rpl1ab. We propose that the YLR137W gene be given the standard name RKM5 (ribosomal lysine (K) methyltransferase 5). Orthologs of RKM5 are found only in fungal species, suggesting a role unique to their survival.Proteins of the eukaryotic translational apparatus are often targets for post-translational covalent modifications (1). One of the major types of these reactions is the transfer of methyl groups from S-adenosylmethionine to a variety of residues including arginine (2-5), lysine (6 -11), glutamine (12), histidine (13), and N-terminal (14, 15) residues. Ribosomal proteins (1-5, 7-11, 13, 14, 16), elongation factor 1A (1, 6, 17), and translational release factors (12) are common substrates of protein methyltransferases. These modifications are known to enhance resistance to ribosome-targeting antibiotics, facilitate ribosomal protein transport into and out of the nucleus, allow efficient ribosomal assembly, and increase translational accuracy (1, 5).We have been interested in understanding the role of protein methylation in ribosomal function and assembly in Saccharomyces cerevisiae using a proteomics-guided approach. Previous efforts have identified modifications within proteins of the large ribosomal subunit. Interestingly, mass spectrometric analysis suggested that six proteins, Rpl1ab, Rpl3, Rpl12ab, Rpl23ab, Rpl42ab, and Rpl43ab, 3 were subject to methylation (18). Further analysis has localized the methylation sites on four of these proteins. Rpl3 is modified to form a 3-methylhistidine residue at position 243 (1...

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Section: Discussionmentioning

confidence: 99%

The Ribosomal L1 Protuberance in Yeast Is Methylated on a Lysine Residue Catalyzed by a Seven-β-strand Methyltransferase

Webb¹,

Al-Hadid²,

Zurita-Lopez

et al. 2011

Journal of Biological Chemistry

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“…The urea, which at 2 M does not interfere with trypsin action, was added to assist in solubilizing proteins and released peptides. To each suspension was added bovine L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Worthington), which was stabilized by reductive methylation (14) to 1% by weight. After 6 -8 h at room temperature with constant stirring, a second equal aliquot of the methylated trypsin was added, and the samples were stirred overnight at room temperature.…”

Section: Preparation Of Hair Samplesmentioning

confidence: 99%

Proteome Analysis of Human Hair Shaft

Lee

Rice

Lee

2006

Molecular & Cellular Proteomics

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111

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The human hair proteome was investigated using twodimensional LC-MS/MS. Among the 343 identified proteins, 70 were detected in high relative abundance, including keratin intermediate filament proteins, largely extractable with denaturants. Over 300 proteins were found to constitute the insoluble complex formed by transglutaminase cross-linking. The intracellular distribution of identified proteins is wide from cytoplasm to nucleus, mitochondria, ribosome, and plasma membrane. These results help rationalize ultrastructural features visible in the mature hair. Keratins and several substrates for transglutaminase were found to be posttranslationally modified by methylation and dimethylation. Evidence for ubiquitination of hair proteins was also obtained. Molecular & Cellular Proteomics 5:789 -800, 2006.Hair is synthesized in mammals by specialized follicles in the skin. In the follicles, cells undergo an intricate maturation program resulting in a hair shaft of terminally differentiated cells. In humans, a protective layer of cuticle cells surrounds the cortex, which in turn surrounds an intermittent column of medulla cells in the center of the shaft. In each of the three layers, the cells have distinctive shapes and contents (1, 2). The keratins and keratin-associated proteins (KAPs), 1 major constituents of the cortex, have been studied for many years after extraction with denaturants under reducing conditions. However, the contents of the medulla and cuticle cells have been much harder to examine because they present great difficulties in solubilization. A major advance in understanding hair structure occurred with the finding of transglutaminase activity in hair follicles (3, 4). The resulting extensive ⑀-(␥-glutamyl)lysine cross-linking accounted for the insolubility in the medulla of proteins, notably the major transglutaminase substrate, trichohyalin. A high content of isopeptide bonding has also been reported in the cuticle cells (5), rationalizing the low degree of protein extraction apparent ultramicroscopically even under harsh denaturing and reducing conditions (6).In recent years, identification of proteins participating in cross-linked structures in epidermis and appendages has benefited from the approach of peptide generation with enzymatic fragmentation followed by separation and amino acid sequencing of the peptides. Applied to the corneocyte-crosslinked envelope, a number of constituent proteins have been identified, including some where the isolated peptides were cross-linked, evidence of their participation in isopeptide bonds (7). Advances in current proteomic technology prompt application of this approach to the hair shaft where much of the protein resists extraction, and hence few data are available on the participants. It is evident, however, that much of the resistance is transglutaminase-dependent because hair from individuals afflicted by TGM1-negative lamellar ichthyosis is subject to considerable extraction (8). Present efforts using multidimensional protein identification technology (M...

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“…dues). [31][32] The autolytic degradation of trypsin was confirmed by gel chromatography in a recent report. 23 Prior to enzymatic degradation, all samples of treated and untreated purified type I collagen were lyophilized and accurately weighed (duplicate measurements).…”

Section: Enzyme Degradation Studiesmentioning

confidence: 55%

The effect of chemical modification of amino acid side‐chains on collagen degradation by enzymes

Gratzer¹,

Santerre²,

Lee³

2006

J Biomed Mater Res

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In this study, the effects of specific chemical modifications of amino acid side-chains on the in vitro enzyme degradation of type I collagen was studied. Two monofunctional epoxides of different size and chemistry were used to modify lysine and methylglyoxal was used to modify arginine. Lysine residues were modified using glycidol, a small hydrophilic reagent or n-butylglycidylether, a larger hydrophobic reagent. Amino acid analysis, swelling measurements, in vitro enzyme degradation analyses (using either collagenase, trypsin, acetyltrypsin, or cathepsin B), and gel chromatography were used to determine the effects of each chemical modification on purified type I collagen. Collagen solubilization by enzymes depended upon the size and chemistry of epoxides used to modify lysine residues. Modification of lysine residues by glycidol and arginine modification by methylglyoxal together significantly reduced collagen solubilization by acetyltrypsin and collagenase, whereas increased collagen solubilization was observed for all enzymes after lysine modification with n-butylglycidylether combined with arginine modification by methylglyoxal. Gel chromatographic analyses of collagen fragments solubilized by acetyltrypsin from type I collagen revealed that both the extent of solubilization and sites of cleavage were altered after lysine and arginine modification. In contrast, lysine and arginine modification only altered the amount of collagen solubilized by collagenase and had no effect on the amount collagen solubilized by cathepsin B. The ability to modulate the enzyme degradation of collagen-based materials as demonstrated in this study may facilitate the design of novel scaffolds for tissue regeneration or collagen-based drug/protein/gene delivery systems.

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Stabilization of bovine trypsin by reductive methylation

Cited by 116 publications

References 27 publications

The Ribosomal L1 Protuberance in Yeast Is Methylated on a Lysine Residue Catalyzed by a Seven-β-strand Methyltransferase

The Ribosomal L1 Protuberance in Yeast Is Methylated on a Lysine Residue Catalyzed by a Seven-β-strand Methyltransferase

Proteome Analysis of Human Hair Shaft

The effect of chemical modification of amino acid side‐chains on collagen degradation by enzymes

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