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PubMed1990

Chemical modification of gamma-carboxyglutamic acid residues in prothrombin elicits a conformation similar to that of abnormal (des-gamma-carboxy)prothrombin.

Chemical modification of gamma-carboxyglutamic acid (Gla) residues in human prothrombin to gamma-methyleneglutamic acid (gamma-MGlu) residues elicited a conformation similar, if not identical, to that of des-gamma-carboxy prothrombin or PIVKA-II, i.e., prothrombin molecules induced by vitamin K antagonists or vitamin K deficiency states. The reaction seems to proceed sequentially by preferentially modifying a Gla at residue 32 that is located innermost among 10 Gla residues of human prothrombin. The initial modification resulted in nearly 50% losses of barium salt adsorption, the procoagulant activity and thrombin generation by the prothrombinase complex. The subsequent modification of two Gla residues at positions 6 and 16 gave rise to the immunoreactivity to an established monoclonal antibody that specifically recognizes the des-gamma-carboxy prothrombin. Further modification of Gla residues increased the reactivity to the antibody, indicating that the conformation recognized by the antibody was stabilized so as to more readily fit the recognition site of the antibody. The appearance of the immunoreactivity was obviously related to the modification of Gla residues in prothrombin, since all other similarly treated derivatives of prothrombin lacking the Gla-domain failed to react with the antibody. Such chemically modified prothrombins may serve as models for studying abnormal des-gamma-carboxy prothrombin produced in vitamin K deficiency states.

1-Carboxyglutamic Acid↗
PubMed1991

Chemical modification of Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase by diethyl pyrocarbonate.

Diethyl pyrocarbonate inactivates Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] by a simple bimolecular reaction. The inactivation is not reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue with a pK of 8.8. Several lines of evidence show that the inactivation is due to the modification of epsilon-amino groups of lysyl residues. Although histidyl residue is also modified, this is not directly correlated to the inactivation. No cysteinyl, tyrosyl, or tryptophyl residue or alpha-amino group is significantly modified. The modification of three lysyl residues per enzyme subunit results in the complete loss of aldolase activity toward various 4-hydroxy-2-oxo acid substrates, whereas oxaloacetate beta-decarboxylase activity associated with the enzyme is not inhibited by this modification. Statistical analysis suggests that only one of the three lysyl residues is essential for activity. l-4-Carboxy-4-hydroxy-2-oxoadipate, a physiological substrate for the enzyme, strongly protects the enzyme against inactivation. Pi as an activator of the enzyme shows no specific protection. The molecular weight of the enzyme, Km for substrate or Mg2+, and activation constant for Pi are virtually unaltered after modification. These results suggest that the modification occurs at or near the active site and that the essential lysyl residue is involved in interaction with the hydroxyl group but not with the oxal group of the substrate.

Amino Acids↗
PubMed1993

Probing the determinants of protein solubility with amino acid modification.

Chemical modification was used as a probe to study the effect of structural features of serum albumin (charge, conformation, surface hydrophobicity, etc.) on its solubility behavior in concentrated ammonium sulfate solutions. Four different acetylated derivatives of goat serum albumin namely 18% acetylated, 40% acetylated, 53% acetylated, and 93% acetylated albumins were prepared. The homogeneity of these preparations was established by gel chromatography and polyacrylamide gel electrophoresis. Hydrodynamic data on the Stokes radius of native and acetylated albumins suggested gradual change in conformation on increasing modification. Solubility experiments performed in concentrated ammonium sulfate solutions at pH 7.0 and at 30 degrees C showed a slight decrease in salting-out parameter, Ks, up to 40% modification, whereas a significant decrease was obtained at higher modification. However, the salting-out parameter, beta, decreased monotonously. Similar decrease in these parameters was also observed with different modified albumins at other pH values viz. pH 5.5, 4.5, and 3.6. From these results we conclude that the decrease in solubility of serum albumin on increasing modification was primarily due to change in conformation.

Acetylation↗
PubMed1980

Stopped-flow studies on the chemical modification with N-bromosuccinimide of model compounds of tryptophan residues.

The aim of this work was to study the selective modification of tryptophan residues with NBS. To accomplish this, a specific method to determine tryptophan was required initially. NBS reacts with both tryptophan and tyrosine residues, which are found in most enzyme proteins, and static spectrophotometric observation, which is usually employed to follow the progress of modification, is not selective for tryptophan. However, discrimination of tryptophan from tyrosine was achieved by the kinetic method with a stoppeed-flow apparatus. The rate of modification of tryptophan residues is 10(3) times larger than that of tyrosine, so rapid stopping of the reaction of NBS brings about the selective modification of tryptophan residues. Using fluorescence-spectrophotometric and kinetic methods, the modification with NBS of model compounds of the tryptophan residue could be simply followed as a single phase, even though the reaction is complex when followed by the static spectrophotometric method.

Bromosuccinimide↗
PubMed1981

The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. VII. A chemical modification of colonic smooth myosin B with N-ethylmaleimide.

The reactivity of the sulfhydryl groups in colonic myosin B to N-ethylmaleimide (NEM) was studied under various conditions. A higher concentration of NEM was required to alter the ATPase activity as compared with skeletal myosin B. A chemical modification of colonic myosin B at a low ionic strength brought about the marked inhibition of EDTA-ATPase and 30-40% activation of Mg2+-ATPase, but had little effect on Ca2+-ATPase. The same results were obtained with NEM modification at a high ionic strength. The presence of 10 mM MgCl2 in the reaction medium for NEM modification caused the activation of Ca2+-ATPase and remarkable inhibition of EDTA-ATPase, suggesting that SH1 is here reactive to NEM. NEM modification of colonic myosin B in the presence of ATP resulted in the inhibition of not only EDTA-ATPase but also Ca2+- and Mg2+-ATPases although the degree of inhibition was different. Addition of 2.0 M urea to the modification medium caused the simultaneous inhibition of EDTA- and Ca2+-ATPase but little change in Mg2+-ATPase. Based on these results, the presence of three specific sulfhydryl groups, SHa, SH1, and SH2, is discussed.

Adenosine Triphosphatases↗
PubMed1983

Studies on the chemical modification of tryptophan residues in thermolysin and in talopeptin (MKI) with N-bromosuccinimide.

Tryptophan residues in thermolysin (3 Trp/molecule) and in its specific inhibitor, talopeptin (1 Trp/molecule), were modified with N-bromosuccinimide (NBS). The decrease in the absorption at 280 nm and the fluorescence intensity above 310 nm (excited at 280 nm) accompanying the modification were followed by the stopped-flow method as a function of time. When the sole tryptophan residue of talopeptin was modified with NBS, its inhibitory activity against thermolysin was almost completely destroyed. For thermolysin, the decrease in molar absorptivity corresponds to the modification of one of its three tryptophan residues, and the enzyme activity does not decrease significantly with the modification (remaining activity was 96% at [NBS]/[E] = 6). The results obtained for the modification of EI complex suggested that the formation of EI complex remarkably reduces the rate constant for the modification of the tryptophan residue in talopeptin, but does not affect that of the tryptophan residue(s) in thermolysin.

Bromosuccinimide↗
PubMed2001

The chemical modification of alpha-chymotrypsin with both hydrophobic and hydrophilic compounds stabilizes the enzyme against denaturation in water-organic media.

We considered alpha-chymotrypsin (CT) in homogeneous water-organic media as a model system to examine the influence of enzyme chemical modification with hydrophilic and hydrophobic substances on its stability, activity and structure. Both types of modifying agents may lead to considerable stabilization of the enzyme in water-ethanol and water-DMF mixtures: (i) the range of organic cosolvent concentration at which enzyme activity (Vm) is at least 100% of its initial value is broadened and (ii) the range of organic cosolvent concentration at which the residual enzyme activity is observed is increased. We found that for both types of modification the stabilization effect can be correlated with the changes in protein surface hydrophobicity/hydrophilicity brought about by the modification. Circular dichroism studies indicated that the effects of these two types of modification on CT structure and its behavior in water-ethanol mixtures are different. Differential scanning calorimetry studies revealed that after modification two or three fractions or domains, differing in their stability, can be resolved. The least stable fractions (or domains) have properties similar to native CT.

Acetylation↗
PubMed2004

Bacterial lipid modification of proteins for novel protein engineering applications.

Functioning of proteins efficiently at the solid-liquid interface is critical to not only biological but also modern man-made systems such as ELISA, liposomes and biosensors. Anchoring hydrophilic proteins poses a major challenge in this regard. Lipid modification, N-acyl-S-diacylglyceryl-Cys, providing an N-terminal hydrophobic membrane anchor is a viable solution that bacteria have successfully evolved but remains unexploited. Based on the current understanding of this ubiquitous and unique bacterial lipid modification it is possible to use Escherichia coli, the popular recombinant protein expression host, for converting a non-lipoprotein to a lipoprotein with a hydrophobic anchor at the N-terminal end. We report two strategies applicable to non-lipoproteins (with or without signal sequences) employing minimal sequence change. Taking periplasmic Shigella apyrase as an example, its signal sequence was engineered to include a lipobox, an essential determinant for lipid modification, or its mature sequence was fused to the signal sequence of abundant outer membrane lipoprotein, Lpp. Lipid modification was proved by membrane localization, electrophoretic mobility shift and mass spectrometric analysis. Substrate specificity and specific activity measurements indicated functional integrity after modification. In conclusion, a convenient protein engineering strategy for converting non-lipoprotein to lipoprotein for commercial application has been devised and tested successfully.

Amino Acid Sequence↗
PubMed2006

Isoelectric points and post-translational modifications of connexin26 and connexin32.

The isoelectric points of the gap junction proteins connexin26 (Cx26) and connexin32 (Cx32) were determined by isoelectric focusing in free fluids. The isoelectric points were significantly more acidic than predicted from amino acid sequences and different from each other, allowing homomeric channels to be resolved separately. The isoelectric points of the homomeric channels bracketed the isoelectric points of heteromeric Cx26/Cx32 channels. For heteromeric channels, Cx26 and Cx32 were found in overlapping, pH-focused fractions, indicating quaternary structure was retained. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify post-translational modifications of Cx26 and Cx32 cytoplasmic domains, including the first reported post-translational modifications of Cx26. Suspected modifications were hydroxylation and/or phosphorylation near the amino terminus of both connexins, gamma-carboxyglutamate residues in the cytoplasmic loop of both connexins, phosphorylation in the carboxyl-terminal domain of Cx32, and palmitoylation at the carboxyl-terminus of Cx32. These modifications contribute to the measured acidic isoelectric points of Cx26 and Cx32, whereas their low molecular masses would not appreciably change connexin SDS-PAGE mobility. Most of these modifications have not previously been identified for connexins and may be instrumental in guiding and understanding novel aspects of channel trafficking and molecular mechanisms of channel regulation.

Animals↗
PubMed2006

Effects of a lifestyle modification program in HIV-infected patients with the metabolic syndrome.

OBJECTIVES: A large percentage of HIV-infected patients receiving HAART develop the metabolic syndrome. In this study, we sought to determine whether lifestyle modification improves metabolic syndrome criteria, including waist circumference, blood pressure, fasting blood sugar, triglycerides, and HDL-cholesterol among HIV-infected patients with the metabolic syndrome. DESIGN: We conducted a randomized, 6-month study in HIV-infected patients with metabolic syndrome as defined by the National Cholesterol Education Program. Subjects were randomly assigned to an intensive lifestyle modification program, which included weekly one-on-one counseling sessions with a registered dietician, or observation (control group). METHODS: Metabolic syndrome criteria and cardiovascular parameters, including blood pressure, body composition, submaximal stress testing, lipids and other biochemical parameters were determined. RESULTS: Thirty-four patients were randomly assigned and 28 subjects completed the study. Compared with the control group, subjects randomly assigned to the lifestyle modification program demonstrated significant decreases in waist circumference (-2.6 +/- 1.1 versus 1.2 +/- 1.0 cm, P = 0.022), systolic blood pressure (-13 +/- 4 versus 4 +/- 4 mmHg, P = 0.008), hemoglobin A1C (-0.1 +/- 0.1 versus 0.2 +/- 0.1%, P = 0.017), lipodystrophy score (-1.2 +/- 0.3 versus 0.9 +/- 0.6, P = 0.006) and increased activity (17.7 +/- 14.3 versus -33.1 +/- 12.7 metabolic equivalents, P = 0.014) as measured by the Modifiable Activity Questionnaire, but lipid levels did not improve. CONCLUSION: These data demonstrate that intensive lifestyle modification significantly improved important cardiovascular risk indices in HIV-infected patients with the metabolic syndrome. Lifestyle modification may be a useful strategy to decrease cardiovascular risk in this population.

Adolescent↗
PubMed2025

A survey of dietary effects on tRNA abundance and modifications.

Transfer RNAs (tRNAs) play a central role in protein translation and are increasingly recognized as dynamic regulators of gene expression. Both physiological and environmental signals can modulate tRNA abundance and chemical modifications, yet the impact of dietary cues on the tRNA landscape remains poorly understood. Here, we investigated the effects of two distinct dietary interventions-low-protein and high-fat diets-on tRNA abundance and modification profiles across multiple mouse tissues. We conducted a comprehensive analysis of tRNA abundance and modification changes in response to these nutritional challenges using RNA mass spectrometry and Ordered Two-Template Relay sequencing (OTTR-seq), a modified-base-sensitive tRNA sequencing method. Our results reveal both shared and tissue-specific alterations in abundance and modifications of specific nuclear and mitochondrial genome-encoded tRNAs in response to dietary conditions at isotype, isoacceptor, and isodecoder levels. As many of the tissue-specific or diet-responsive tRNA modifications have been previously reported to affect decoding efficiency or translational fidelity, these results have implications for understanding translational adaptation in response to dietary conditions.

Journal Article↗
PubMed2004

The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote.

The covalent modification of nucleosomal histones has emerged as a major determinant of chromatin structure and gene activity. To understand the interplay between various histone modifications, including acetylation and methylation, we performed a genome-wide chromatin structure analysis in a higher eukaryote. We found a binary pattern of histone modifications among euchromatic genes, with active genes being hyperacetylated for H3 and H4 and hypermethylated at Lys 4 and Lys 79 of H3, and inactive genes being hypomethylated and deacetylated at the same residues. Furthermore, the degree of modification correlates with the level of transcription, and modifications are largely restricted to transcribed regions, suggesting that their regulation is tightly linked to polymerase activity.

Acetylation↗
PubMed2006

Histone sumoylation is a negative regulator in Saccharomyces cerevisiae and shows dynamic interplay with positive-acting histone modifications.

Covalent histone post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitylation play pivotal roles in regulating many cellular processes, including transcription, response to DNA damage, and epigenetic control. Although positive-acting post-translational modifications have been studied in Saccharomyces cerevisiae, histone modifications that are associated with transcriptional repression have not been shown to occur in this yeast. Here, we provide evidence that histone sumoylation negatively regulates transcription in S. cerevisiae. We show that all four core histones are sumoylated and identify specific sites of sumoylation in histones H2A, H2B, and H4. We demonstrate that histone sumoylation sites are involved directly in transcriptional repression. Further, while histone sumoylation occurs at all loci tested throughout the genome, slightly higher levels occur proximal to telomeres. We observe a dynamic interplay between histone sumoylation and either acetylation or ubiquitylation, where sumoylation serves as a potential block to these activating modifications. These results indicate that sumoylation is the first negative histone modification to be identified in S. cerevisiae and further suggest that sumoylation may serve as a general dynamic mark to oppose transcription.

Acetylation↗
PubMed2026

Dietary effects on cytosolic and mitochondrial tRNA abundance and modification patterns across mouse tissues.

Transfer RNAs (tRNAs) are central to protein synthesis and are increasingly recognized as dynamic regulators of gene expression whose abundance and chemical modifications are subject to precise biological control. Here, we systematically investigate how two distinct dietary interventions, low-protein and high-fat diets, reshape the tRNA landscape across multiple mouse tissues, using RNA mass spectrometry and ordered two-template relay sequencing (OTTR-seq) to comprehensively profile cytosolic and mitochondrial tRNAs at single-nucleotide resolution. We reveal pronounced tissue-specific biases in tRNA isodecoder expression, including the unexpected presence of full-length cytosolic tRNAs in mature sperm with a distinct isotype composition. In somatic tissues such as liver and heart, dietary conditions alter both tRNA abundance and key modifications known to regulate decoding efficiency, whereas in reproductive tissues diet primarily affects the abundance of select tRNAs with comparatively limited changes in modification profiles. We further demonstrate that mitochondrial tRNAs are subject to diet-responsive changes in both abundance and modification status and that even subtle differences in dietary fat composition are sufficient to alter tRNA modification signatures. Together, these findings establish the tRNA epitranscriptome as a sensitive and tissue-specific sensor of nutritional state and provide a resource for understanding how dietary cues interface with translational regulation in somatic and reproductive tissues.

Male↗
PubMed1981

Characterization of a molecular modification of phytochrome that is associated with its conversion to the far-red-absorbing form.

Phytochrome that has been photoinduced to pellet by irradiation of intact oat (cv. Garry) shoots and recovered from a pellet obtained by centrifugation of crude extracts exhibits modified behavior when compared to soluble phytochrome isolated from shoots that had never been irradiated. This modified behavior includes retarded mobility during sodium dodecyl sulfate polyacrylamide gel electrophoresis (Boeshore ML, LH Pratt 1980 Plant Physiol 66: 500-504). The electrophoretic mobility of several different kinds of phytochrome preparations were examined to study how this modification might arise.Phytochrome that was extracted in the pelletable condition from red-, far-red-irradiated tissue, but without added divalent cation so that it did not pellet, did not exhibit an altered electrophoretic mobility. Hence, this modification of phytochrome is not required for the expression in vitro of pelletability induced in vivo. Phytochrome that was extracted in the pelletable condition and in the far-red-absorbing form, but without added divalent cation so that it did not pellet, and phytochrome in the far-red-absorbing form that remained in the supernatant after collection of pellets containing pelleted phytochrome both electrophoresed with reduced mobility. Thus, this modification does not arise as a consequence of phytochrome having been pelleted. Differential sensitivity of phytochrome to different handling conditions also is not the cause of this modification since the far-red-absorbing form of phytochrome, which was extracted in the pelletable condition but by the same protocol used to extract soluble phytochrome, also exhibited reduced mobility. Furthermore, the reduced electrophoretic rate is not due to a simple differential lability of the far-red-absorbing form of phytochrome to extraction conditions, since partially purified soluble phytochrome that was exposed in the far-red-absorbing form to the isolation and extraction conditions used for preparation of soluble phytochrome did not exhibit the alteration.The data are instead consistent with the more complex interpretation that phytochrome is modified in vitro if two conditions are met: (a) that phytochrome is extracted in the far-red-absorbing form or is converted to the far-red-absorbing form in the crude extract soon after extraction and (b), that phytochrome remains in the far-red-absorbing form in the crude extract for at least a brief period.The possibility that the phytochrome modification studied here might have arisen because of a change in carbohydrate content was tested by periodic acid Schiff staining of sodium dodecyl sulfate polyacrylamide gels. No carbohydrate was detected in any of the phytochrome preparations that were examined. This inability to detect carbohydrate is in direct contrast to the report of Roux et al. (1975 Physiol Plant 35: 85-90).

Journal Article↗
PubMed2003

Two modifications of a KH2PO4.HF adduct.

The structures of two modifications, (I) and (II), of potassium dihydrogenphosphate-hydrofluoric acid (1/1), KH(2)PO(4).HF, were determined at 250 and 150 K, and at 292 and 150 K, respectively. Modifications (I) and (II) crystallize from stoichiometric aqueous solutions at 295 (1) and 308 (3) K, respectively. The H atoms were located clearly from the difference Fourier maps in each modification. The two modifications differ mainly in the arrangement of the dihydrogenphosphate anions, i.e. (I) contains looped dimeric and tetrameric units of the dihydrogenphosphate ions, whereas (II) contains two types of looped tetrameric unit. In addition, both structures contain a very short F-H.O hydrogen bond (2.38-2.40 A). The K(+) ions are coordinated by O and F atoms, with similar K.O and K.F distances in both modifications.

Journal Article↗
PubMed2004

Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms.

Covalent modification is an important strategy for introducing new functions into proteins. As engineered proteins become more sophisticated, it is often desirable to introduce multiple, modifications involving several different functionalities in a site-specific manner. Such orthogonal labeling schemes require independent labeling of differentially reactive nucleophilic amino acid side chains. We have developed two protein-mediated protection schemes that permit independent labeling of multiple thiols. These schemes exploit metal coordination or disulfide bond formation to reversibly protect cysteines in a Cys(2)His(2) zinc finger domain. We constructed a variety of N- and C-terminal fusions of these domains with maltose-binding protein, which were labeled with two or three different fluorophores. Multiple modifications were made by reacting an unprotected cysteine in MBP first, deprotecting the zinc finger, and then reacting the zinc finger cysteines. The fusion proteins were orthogonally labeled with two different fluorophores, which exhibited intramolecular fluorescene resonance energy transfer (FRET). These conjugates showed up to a threefold ratiometric change in emission intensities in response to maltose binding. We also demonstrated that the metal- and redox-mediated protection methods can be combined to produce triple independent modifications, and prepared a protein labeled with three different fluorophores that exhibited a FRET relay. Finally, labeled glucose-binding protein was covalently patterned on glass slides using thiol-mediated immobilization chemistries. Together, these experiments demonstrated that reversible thiol protection schemes provide a rapid, straightforward method for producing multiple, site-specific modifications.

Binding Sites↗
PubMed2006

Adaptational modification and ligand occupancy have opposite effects on positioning of the transmembrane signalling helix of a chemoreceptor.

Sensory systems adapt to persistent stimulation. In the transmembrane receptors of bacterial chemotaxis, adaptation is mediated by methylation at specific glutamyl residues in the cytoplasmic domain. Methylation counteracts effects of ligand binding on functional activities of that domain. Both ligand binding and adaptational modification are thought to act through conformational changes. As characterized for Escherichia coli chemoreceptors, a mechanistically crucial feature of the ligand-induced conformational change is piston sliding towards the cytoplasm of a signalling helix in the periplasmic/transmembrane domain. Adaptational modification could counteract this signalling movement by blocking its influence on the cytoplasmic domain or by reversing it. To investigate, we characterized effects of adaptational modification on the position of the signalling helix in chemoreceptor Trg using rates of disulphide formation between introduced cysteines. We utilized an intact cell procedure in which receptors were in their native, functional state. In vivo rates of disulphide formation between diagnostic cysteine pairs spanning a signalling helix interface changed as a function of adaptational modification. Strikingly, those changes were opposite those caused by ligand occupancy for each diagnostic pair tested. This suggests that adaptational modification resets the receptor complex to its null state by reversal of the conformational change generated by ligand binding.

Cysteine↗