Identification of Glucose-Derived Cross-Linking Sites in Ribonuclease A (original) (raw)
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Modification of Proteins In Vitro by Physiological Levels of Glucose
Journal of Biological Chemistry, 2003
Hyperglycemic conditions of diabetes accelerate protein modifications by glucose leading to the accumulation of advanced glycation end-products (AGEs). We have investigated the conversion of protein-Amadori intermediate to protein-AGE and the mechanism of its inhibition by pyridoxamine (PM), a potent AGE inhibitor that has been shown to prevent diabetic complications in animal models. During incubation of proteins with physiological diabetic concentrations of glucose, PM prevented the degradation of the protein glycation intermediate identified as fructosyllysine (Amadori) by 13 C NMR using [2-13 C]-enriched glucose. Subsequent removal of glucose and PM led to conversion of protein-Amadori to AGE N ⑀-carboxymethyllysine (CML). We utilized this inhibition of post-Amadori reactions by PM to isolate protein-Amadori intermediate and to study the inhibitory effect of PM on its degradation to protein-CML. We first tested the hypothesis that PM blocks Amadori-to-CML conversion by interfering with the catalytic role of redox metal ions that are required for this glycoxidative reaction. Support for this hypothesis was obtained by examining structural analogs of PM in which its known bidentate metal ion binding sites were modified and by determining the effect of endogenous metal ions on PM inhibition. We also tested the alternative hypothesis that the inhibitory mechanism involves formation of covalent adducts between PM and protein-Amadori. However, our 13 C NMR studies demonstrated that PM does not react with the Amadori. Because the mechanism of interference with redox metal catalysis is operative under the conditions closely mimicking the diabetic state, it may contribute significantly to PM efficacy in preventing diabetic complications in vivo. Inhibition of protein-Amadori degradation by PM also provides a simple procedure for the isolation of protein-Amadori intermediate, prepared at physiological levels of glucose for relevancy, to study both the biological effects and the chemistry of post-Amadori pathways of AGE formation.
Chemically crosslinked protein dimers: Stability and denaturation effects
Protein Science, 1995
Nine single substitution cysteine mutants of staphylococcal nuclease (nuclease) were preferentially crosslinked at the introduced cysteine residues using three different bifunctional crosslinking reagents; I ,6-bismaleimidohexane (BMH), 1,3-dibromo-2-propanol (DBP), and the chemical warfare agent, mustard gas (bis(2-chloroethy1)sulfide; mustard). BMH and mustard gas are highly specific reagents for cysteine residues, whereas DBP is not as specific. Guanidine hydrochloride (GuHCI) denaturations of the resulting dimeric proteins exhibited biphasic unfolding behavior that did not fit the two-state model of unfolding. The monofunctional reagent, e-maleimidocaproic acid (MCA), was used as a control for the effects of alkylation. Proteins modified with MCA unfolded normally, showing that this unusual unfolding behavior is due to crosslinking. The data obtained from these crosslinked dimers was fitted to a three-state thermodynamic model of two successive transitions in which the individual subunits cooperatively unfold. These two unfolding transitions were very different from the unfolding of the monomeric protein. These differences in unfolding behavior can be attributed in large part to changes in the denatured state. In addition to GuHCl titrations, the crosslinked dimers were also thermally unfolded. In contrast to the GuHCl denaturations, analysis of this data fit a two-state model well, but with greatly elevated van't Hoff enthalpies in many cases. However, clear correlations between the thermal and GuHCl denaturations exist, and the differences in thermal unfolding can be rationalized by postulating interactions of the denatured crosslinked proteins.
Covalent cross-linking of proteins without chemical reagents
Protein Science, 2002
A facile method for the formation of zero-length covalent cross-links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross-linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85°C for 24 h. Under these conditions, approximately one-third of the total protein present becomes cross-linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero-length cross-links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross-linked dimer has only one amide cross-link and retains the enzymatic activity of the monomer. The in vacuo cross-linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross-linking, and co-lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross-linked dimer.
Impact of Chemical Cross-Linking on Protein Structure and Function
Analytical chemistry, 2018
Chemical cross-linking coupled with mass spectrometry is a popular technique for deriving structural information on proteins and protein complexes. Also, cross-linking has become a powerful tool for stabilizing macromolecular complexes for single-particle cryo-electron microscopy. However, an effect of cross-linking on protein structure and function should not be forgotten, and surprisingly, it has not been investigated in detail so far. Here, we used kinetic studies, mass spectrometry, and NMR spectroscopy to systematically investigate an impact of cross-linking on structure and function of human carbonic anhydrase and alcohol dehydrogenase 1 from Saccharomyces cerevisiae. We found that cross-linking induces rather local structural disturbances and the overall fold is preserved even at a higher cross-linker concentration. The results establish general experimental conditions for chemical cross-linking with minimal effect on protein structure and function.
Proceedings of the National Academy of Sciences, 1984
Proteins exposed to glucose over long periods are known to undergo physicochemical changes including crosslinking and formation of brown fluorescent pigments of poorly characterized structure. Acid hydrolysis of both browned poly(L-lysine) and browned bovine serum albumin is found to release a major fluorescent chromophore, which after alkalinization is extractable into organic solvents and which can be purified by. silica gel chromatography. The fluorescence properties of this compound very closely resemble those of the bulk browned polypeptides. By NMR, mass spectroscopy, and chemical derivatization, this compound is assigned the structure 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI). Confirmation was obtained by independent chemical synthesis from furylglyoxal and ammonia. The incorporation of two peptide-derived amine nitrogens and two glucose residues in FFI strongly suggests that peptide-bound FFI precursors are implicated in the crosslinking of proteins by glucose in vivo. This reaction has potential implications in the understanding of glucose-mediated protein modifications and their role in the complications of diabetes and aging.
Biochemistry, 1996
Nonenzymatic glycation (Maillard reaction) of long-lived proteins is a major contributor to the pathology of diabetes and possibly aging and Alzheimer's disease. We report here kinetic studies of the glycation of the model protein ribonuclease A by glucose and ribose leading to the formation of antigenic advanced glycation end products ("AGEs"), detectable by AGE-specific polyclonal antibodies, and pentosidine, an acid-stable fluorescent AGE. As anticipated, the kinetics of glycation by ribose were considerably faster than by glucose, and the rate of AGE formation initially increased with increasing sugar concentrations. However, ribose above 0.15 M appeared to paradoxically slow the kinetics of AGE formation, suggesting ribose inhibits the conversion of "early" Amadori rearrangement products to "late" AGEs and thus favors the accumulation of reactive Amadori intermediates. The facile isolation of such protein intermediates was achieved by an "interrupted glycation" protocol in which free and reversibly bound (Schiff base) ribose was removed following a short (24 h) initial incubation with 0.5 M ribose at 37°C. The kinetics of buildup of the Amadori intermediates and the kinetics of their post-Amadori conversion to antigenic AGEs were then independently studied. A rapid and reversible inhibition of the post-Amadori kinetics by free ribose was verified by direct re-addition of ribose to the isolated, sugarfree intermediate. The pH dependence of the kinetics of antigenic AGE formation from such intermediates was measured and exhibited an unusual bell-shaped profile over the pH range of 5.0-9.5 with a maximum near pH 8.0. Aminoguanidine, a pharmacological AGE inhibitor, was found to moderately or weakly inhibit antigenic AGE formation in such post-Amadori steps. The isolation of the glycated ribonuclease intermediate thus simplifies kinetic and mechanistic studies of AGE formation, permits AGE studies in the absence of complications arising from free or Schiff base bound sugar, and provides a novel methodology for evaluating the mechanism and efficacy of therapeutic agents that may inhibit AGE formation.
Compensatory secondary structure alterations in protein glycation
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2007
Glycation, a local covalent interaction, leads to alterations in secondary and tertiary structures of hemoglobin, the changes produced by fructose being more pronounced than those caused by glucose. The Stokes diameter of hemoglobin increases upon glycation from 7 to 14 nm and a concurrent inter-chain cross-linking and heme loss are also observed, particularly in the later stage of glycation. An initial increase of tryptophan (trp) fluorescence was observed in both glucation and fructation. In case of frucation however there was a decrease in tryptophan fluorescence that was accompanied by an increase in fluorescence of the advanced glycosylation end products (AGEs). This fluorescence behavior is indicative of energy transfer between tryptophan and the AGEs formed during the late stage of glycation. Emergence of an isosbestic point in the fluorescence spectra (taken at different time intervals) implies existence of two distinct glycation stages. The late glycation stage is also marked by an increase of beta structure and random coil at the expense of alpha helix. It is further observed that this compensatory loss of alpha helix (reported for the first time) and increase in beta sheet and random coil elements depend on the number of solvent-accessible glycation sites (rather than total number of such sites) and the subunit assembly of the protein.
Clinical Chemistry and Laboratory Medicine, 1981
A specific and sensitive method for quantification of the fructose-lysine linkages present in nonenzymatically glycosylated albumin and other proteins is described. Protein is hydrolyzed for 18 h in 6 mol/1 HC1 at 95 °C to yield furosine (e-N-(2-furoylmethyl)-I-lysine) known as a specific degradation product of fructose-lysine. Furosine is then separated on HPLC and quantified by its UV-absorbance against a prepared fructose-lysine standard. The method has been successfully used for the determination of glycosyl-albumin in diabetic patients starting from 100 serum or less, as well as for various other proteins. Unlike the usually employed thiobarbituric acid assay the present procedure is truly specific for the detection of ketoamine linkages of glycosylated proteins. Spezifische Bestimmung von Protein (Lysin)-gebundener Glucose in Serumalbumin des Menschen und anderen glycosylierten Proteinen durch Hochdruckflüssigchromatographie Zusammenfassung: Es wird eine spezifische und empfindliche Methode zur Bestimmung der Fructose-Lysinbindungen von nichtenzymatisch glycosyliertem Albumin und anderen Proteinen beschrieben. Sie beruht auf der Hydrolyse des Proteins für 18 Stunden in 6 mol/1 HC1 bei 95 °C, wobei als spezifisches Abbauprodukt des Fructose-Lysins Furosin (e-N-(2-Furoylmethyl)-L-lysin) entsteht. Furosin wird dann durch HPLC abgetrennt und durch UV-Absorption gegen einen selbstsynthetisierten Fructose-Lysinstandard gemessen. Die Methode eignet sich fur die Bestimmung von glycosyliertem Albumin bei Diabetikern, ausgehend von 100 Serum und weniger, sowie auch allgemein für das Studium glycosylierter Proteine. Sie ist spezifischer als der häufig gebrauchte Thiobarbitursäure-Test, indem sie ausschließlich die Ketoaminbindungen glycosylierter Proteine erfaßt.