Detection of Intercatechol Cross-Links in Insect Cuticle by Solid-State Carbon-13 and Nitrogen-15 NMR

Abstract

Solid-state NMR has been used to detect an intercatechol covalent bond in sclerotized cuticle of the tobacco hornworm (Manduca sexta). Rotational-echo, double-resonance (REDOR) 13C and 15N NMR, in combination with 1H−13C dipolar modulation and 15N−15N dipolar restoration at the magic angle, were used to examine hornworm pupal exuviae labeled with β-[15N]alanine and either [β-13C]dopamine or [α-13C,15N]dopamine. The REDOR spectra showed the incorporation of N-β-alanyldopamine into insect cuticle by the formation of a variety of covalent bonds. One of these bonds links the terminal nitrogen of one N-β-alanyldopamine molecule to the β carbon of another to form an intercatechol covalent bond. This result is interpreted in terms of a novel structure for stabilized cuticle that postulates protein−catechol−catechol−protein cross-links. REDOR spectra also showed oxygen substitution at both α and β carbons of N-β-alanyldopamine, consistent with the possibility of intercatechol oxygen bridges in dimeric or oligomeric forms of catecholamines in the cuticle.

Insect cuticular sclerotization: A review

“…Solid-state NMR measurements showed that isotopically labeled dopamine and b-alanine are incorporated into pupal cuticle of M. sexta by formation of covalent linkages between the imidazole ring of histidine residues and the aromatic ring or the sidechain b-position in dopamine; occurrence of linkages to chitin and to the b-alanyl residue of NBAD was also indicated (Schaefer et al, 1987;Kramer et al, 1989Kramer et al, , 1995Christensen et al, 1991;Merrit et al, 1996).…”

Coupling Amplification in 2D MAS NMR and Its Application to Torsion Angle Determination in Peptides

“…The resulting spectrum has sidebands spaced at the actual spinning speed but an intensity envelope identical to that taken at a quarter of the spinning speed. The extendability of this pulse sequence distinguishes it from methods that enhance the sidebands by dipolar decoupling for a fraction of the rotor cycle (14,22).…”

Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

“…REDOR has found additional application in detection of formation of chemical bonds [7,8] and in resonance assignment of small peptides [9]. The wide use of REDOR is primarily due to the relative simplicity and robustness of its pulse sequence and data analysis.…”