Electron paramagnetic resonance backbone dynamics studies on spin‐labelled neuropeptide Y analogues

Abstract

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammalians. NPY acts by binding to at least five G-protein coupled receptors (GPCRs) which have been named Y1, Y2, Y4, Y5 and Y6. Three spin-labelled NPY analogues containing the nitroxide group of the amino acid TOAC (2.2.6.6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe were synthesized by solid-phase peptide synthesis. Synthetic problems owing to the sensitivity of nitroxide towards acidic and reducing conditions have been overcome by using a cleavage cocktail that contains anisole and cresol scavengers. Concerning the receptor binding preferences, the analogues [TOAC34]-pNPY and [Ala31, TOAC32]-pNPY showed a marked selectivity for the Y5 receptor, while [TOAC2]-pNPY maintained a significant binding also to the Y2 receptor subtype. The modifications of the native peptide structure caused by the introduction of TOAC were examined by circular dichroism. In order to determine the rotational correlation time of the spin probes, electron paramagnetic resonance measurements were performed in solution and in the presence of liposomes. This allowed us to evaluate the backbone dynamics of the different parts of the NPY molecule in the free and membrane bound states. The results of these studies showed that NPY Interacts with liposomes by using the C-terminal alpha-helix while the N-terminal tail retains a flexibility that is comparable to that of the peptide in solution as already shown by NMR studies on DPC micelles. Furthermore, we demonstrated that TOAC-labelllng is a valuable tool to investigate changes in the backbone conformation and dynamics. This may be of major importance for peptides and small proteins when they bind to cell membranes.

Automated solid-phase peptide synthesis to obtain therapeutic peptides

“…The paramagnetic substance TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) (Fig. 5) was manually coupled to NPY during SPPS allowing EPR (electron paramagnetic resonance) studies to investigate conformational changes during receptor binding [129]. Here, synthesis on solid support could be easily realized.…”

“…Here, synthesis on solid support could be easily realized. Nevertheless, conditions for cleavage of the peptide conjugates from the resin had to be optimized owing to the sensitivity of the nitroxide group of TOAC [129]. Koglin et al used the photo-cleavable protecting group Nvoc for selective 3 H-labeling of full length NPY.…”

Paramagnetic Tools in Protein NMR

“…b based on PRE. , the nitroxide-containing amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC, 2) [75], and the metal chelators usable as paramagnetic tags; diethylenetriaminepentaacetic acid cyclic anhydride (3) [85], 2,2 0 ,2 00 ,2 000 -((((4-amino-1,2-phenylene)bis(oxy))bis(ethane-2,1-diyl))bis(azanetriyl))tetraacetic acid (4) [82], S-mesyl-cysteine-EDTA (5) [91], CLaNP-1 (6) [95], ClaNP-3 (7) (10), CLaNP-5 (8) [92], and 4-mercaptomethyl-dipicolinic acid (9) [93]. Probes 1, 5, 6, 7 and 8 are cysteine-reactive by their one or two methanethiosulphonate leaving groups.…”

“…When defining PRE restraints derived from spin labels this has to be taken into account (see step 5 of the protocol at the end of this section) [74]. In the case of peptides it is possible to use TOAC, an amino acid with a stable radical in a rigid conformation ( Figure 6.4), which can be built into peptides using standard solid phase synthesis methodology [75]. Again, it is important to check the effect of TOAC incorporation on the structure and activity of the peptide.…”

Functional assessment of angiotensin II and bradykinin analogues containing the paramagnetic amino acid TOAC

“…From this point onwards, the literature has witnessed many types of application of this piperidine-based cyclic paramagnetic probe ranging from those that investigated in depth the characteristics of this type of C α -tetrasubstituted α-amino acid [10,11] until those related to some biological relevant peptides [12][13][14][15]. Taking into account the unique advantage of this conformationally constrained marker, i.e., of being rigidly bound to the molecule or system and therefore, of possessing greater sensitivity in monitoring dynamics of the attaching-site [16], other applications have been designed for its use, including membrane-related investigations [17][18][19], solvation process of peptide-polymers and polymers [20,21] and more recently, EPR-monitoring of an enzymatic hydrolysis of TOAC-bearing substrates [22].…”