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Chemistry of periodate-mediated cross-linking of 3,4-dihydroxylphenylalanine-containing molecules to proteins.

ABSTRACT: Chemical cross-linking is an attractive approach to map peptide-protein and protein-protein complexes. Previously, we explored 3,4-dihydroxylphenylalanine (DOPA) as a protein cross-linking agent upon periodate oxidation (Burdine, L.; Gillette, T. G.; Lin, H.-J.; Kodadek, T. J. Am. Chem. Soc. 2004, 126, 11442-11443). We report here a study on the chemistry of DOPA-protein cross-linking. First, using a peptide nucleic acid templated system, we identified the alpha-amino, epsilon-amino of Lys, imidazole of His, and thiol of Cys as functional groups capable of attacking DOPA ortho-quinone. Second, we demonstrated that periodate-induced DOPA-protein cross-linking could be carried out efficiently at neutral pH in the presence of excess aliphatic 1,2-diols such as ethylene glycol, lactose, and adenosine triphosphate. This result indicated that DOPA-protein cross-linking and 1,2-diol oxidative cleavage proceed via different mechanisms and that carbohydrates will not interfere with this process when carried out in crude cell extracts or on intact cells.

SUBMITTER: Liu B

PROVIDER: S-EPMC2568988 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

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Chemistry of periodate-mediated cross-linking of 3,4-dihydroxylphenylalanine-containing molecules to proteins.

Liu Bo B Burdine Lyle L Kodadek Thomas T

Journal of the American Chemical Society 20061101 47

Chemical cross-linking is an attractive approach to map peptide-protein and protein-protein complexes. Previously, we explored 3,4-dihydroxylphenylalanine (DOPA) as a protein cross-linking agent upon periodate oxidation (Burdine, L.; Gillette, T. G.; Lin, H.-J.; Kodadek, T. J. Am. Chem. Soc. 2004, 126, 11442-11443). We report here a study on the chemistry of DOPA-protein cross-linking. First, using a peptide nucleic acid templated system, we identified the alpha-amino, epsilon-amino of Lys, imid ...[more]

PMID: 17117875

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Project description:Reversible covalent conjugation chemistries that allow site- and condition-specific coupling and uncoupling reactions are attractive components in nanotechnologies, bioconjugation methods, imaging, and drug delivery systems. Here, we compare three heterobifunctional cross-linkers, containing both thiol- and amine-reactive chemistries, to form pH-labile hydrazones with hydrazide derivatives of the known and often published water-soluble polymer, poly[N-(2-hydroxypropyl methacrylamide)] (pHPMA), while subsequently coupling thiol-containing molecules to the cross-linker via maleimide addition. Two novel cross-linkers were prepared from the popular heterobifunctional cross-linking agent, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), modified to contain either terminal aldehyde groups (i.e., 1-(N-3-propanal)-4-(N-maleimidomethyl) cyclohexane carboxamide, PMCA) or methylketone groups (i.e., 1-(N-3-butanone)-4-(N-maleimidomethyl) cyclohexane carboxamide, BMCA). A third cross-linking agent was the commercially available N-4-acetylphenyl maleimide (APM). PMCA and BMCA exhibited excellent reactivity toward hydrazide-derivatized pHPMA with essentially complete hydrazone conjugation to polymer reactive sites, while APM coupled only ?60% of available reactive sites on the polymer despite a 3-fold molar excess relative to polymer hydrazide groups. All polymer hydrazone conjugates bearing these bifunctional agents were then further reacted with thiol-modified tetramethylrhodamine dye, confirming cross-linker maleimide reactivity after initial hydrazone polymer conjugation. Incubation of dye-labeled polymer conjugates in phosphate buffered saline at 37 °C showed that hydrazone coupling resulting from APM exhibited the greatest difference in stability between pH 7.4 and 5.0, with hydrolysis and dye release increased at pH 5.0 over a 24 h incubation period. Polymer conjugates bearing hydrazones formed from cross-linker BMCA exhibited intermediate stability with hydrolysis much greater at pH 5.0 at early time points, but hydrolysis at pH 7.4 was significant after 5 h. Hydrazones formed with the PMCA cross-linker showed no difference in release rates at pH 7.4 and 5.0.

Project description:The incubation of lens capsules with glucose in vitro resulted in changes in the mechanical and thermal properties of type-IV collagen consistent with increased cross-linking. Differential scanning calorimetry (d.s.c.) of fresh lens capsules showed two major peaks at melting temperatures Tm 1 and Tm 2 at approx. 54 degrees C and 90 degrees C, which can be attributed to the denaturation of the triple helix and 7S domains respectively. Glycosylation of lens capsules in vitro for 24 weeks caused an increase in Tm 1 from 54 degrees C to 61 degrees C, while non-glycosylated, control incubated capsules increased to a Tm 1 of 57 degrees C. The higher temperature required to denature the type-IV collagen after incubation in vitro suggested increased intermolecular cross-linking. Glycosylated lens capsules were more brittle than fresh samples, breaking at a maximum strain of 36.8 +/- 1.8% compared with 75.6 +/- 6.3% for the fresh samples. The stress at maximum strain (or 'strength') was dramatically reduced from 12.0 to 4.7 N.mm.mg-1 after glycosylation in vitro. The increased constraints within the system leading to loss of strength and increased brittleness suggested not only the presence of more cross-links but a difference in the location of these cross-links compared with the natural lysyl-aldehyde-derived cross-links. The chemical nature of the fluorescent glucose-derived cross-link following glycosylation was determined as pentosidine, at a concentration of 1 pentosidine molecule per 600 collagen molecules after 24 weeks incubation. Pentosidine was also determined in the lens capsules obtained from uncontrolled diabetics at a level of about 1 per 100 collagen molecules. The concentration of these pentosidine cross-links is far too small to account for the observed changes in the thermal and mechanical properties following incubation in vitro, clearly indicating that another as yet undefined, but apparently more important cross-linking mechanism mediated by glucose is taking place.

Dataset Information

Chemistry of periodate-mediated cross-linking of 3,4-dihydroxylphenylalanine-containing molecules to proteins.

Publications

Chemistry of periodate-mediated cross-linking of 3,4-dihydroxylphenylalanine-containing molecules to proteins.

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