The spin label amino acid TOAC and its uses in studies of peptides: chemical, physicochemical, spectroscopic, and conformational aspects

Abstract

We review work on the paramagnetic amino acid 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, TOAC, and its applications in studies of peptides and peptide synthesis. TOAC was the first spin label probe incorporated in peptides by means of a peptide bond. In view of the rigid character of this cyclic molecule and its attachment to the peptide backbone via a peptide bond, TOAC incorporation has been very useful to analyze backbone dynamics and peptide secondary structure. Many of these studies were performed making use of EPR spectroscopy, but other physical techniques, such as X-ray crystallography, CD, fluorescence, NMR, and FT-IR, have been employed. The use of double-labeled synthetic peptides has allowed the investigation of their secondary structure. A large number of studies have focused on the interaction of peptides, both synthetic and biologically active, with membranes. In the latter case, work has been reported on ligands and fragments of GPCR, host defense peptides, phospholamban, and β-amyloid. EPR studies of macroscopically aligned samples have provided information on the orientation of peptides in membranes. More recent studies have focused on peptide-protein and peptide-nucleic acid interactions. Moreover, TOAC has been shown to be a valuable probe for paramagnetic relaxation enhancement NMR studies of the interaction of labeled peptides with proteins. The growth of the number of TOAC-related publications suggests that this unnatural amino acid will find increasing applications in the future.

Fig. 1

Structure of TOAC and time evolution of TOAC-related publications

Fig. 2

Chemical structures of the spin-labeled amino acids TOAC, β-TOAC, and POAC and of the C^α,α-disubstituted glycines Aib and Ac6c

Fig. 3

EPR spectra (100 G scanwidth) of hexameric peptides (Boc-TOAC-Ala_n-TOAC-Ala_4-n-OtBu; n = 0–3) as a function of solvent at 298 K. Reprinted from Hanson et al. (1996a), with permission of the American Chemical Society

Fig. 4

EPR spectra of 0.1 mM TOAC¹-AII (a, c) and TOAC³-AII (b, d) in the presence of 50 mM HPS (a, b) and SDS (c, d) in 15 mM phosphate-borate-citrate buffer at pH 4.0 (top), 7.0 (middle), 10.0 (bottom). The arrows in the spectrum of TOAC³-AII in the presence of HPS at pH 10.0 indicate an immobilized component due to HPS-bound TOAC³-AII. Reprinted from Vieira et al. (2009) with permission of John Wiley & Sons

Fig. 5

X-ray diffraction structures of the two crystallographically independent molecules a and b in the asymmetric unit of the TOAC^4,8-trichogin analogue with atom numbering. The intramolecular H-bonds are indicated by dashed lines. Reprinted from Monaco et al. (1999a) with permission of John Wiley & Sons