Conformation and EPR characterization of rigid, 3₁₀‐helical peptides with TOAC spin labels: Models for short distances

Abstract

For 3D-structure determination in biophysical systems EPR is rapidly gaining ground. Proteins labeled specifically with two nitroxide spin labels can be prepared, and several EPR methods are available for distance determination, which makes it possible to determine distance constraints. However, such methods require frozen solutions, potentially causing non-physiological states of the sample. Here, we target spin- spin interaction in liquid solution at room temperature using rigid model compounds. A series of 310 -helical peptides, based on α-aminoisobutyric acid (Aib), is synthesized with pairs of spin labels separated by three, four, and five amino acids. To avoid flexibility, the noncoded nitroxyl-containing α-amino acid TOAC that is rigidly connected with the peptide backbone, is used. The EPR spectra of the peptides show a decreasing amount of coupling between the two spin labels within this series. We suggest through-bond interaction as the dominating mechanism for exchange interaction (J) and find a stronger J-coupling than in the corresponding Ala-based TOAC-peptides investigated previously (Hanson, et al., J Am Chem Soc 1996, 118, 7618-7625). We speculate that stronger coupling in Aib- vs Ala- peptides is due to intrinsically stronger through-bond interaction in the Aib-based peptides.

Integrated Computational Approach to the Electron Paramagnetic Resonance Characterization of Rigid 310-Helical Peptides with TOAC Nitroxide Spin Labels

“…The DFT geometry optimization procedure has led to a 3 10 -helical structure for all of the peptides studied in this work, consistent with previous theoretical and experimental studies. 3 − 5 For the systems OCTA 2,7 and NONA 2,8 we have found that the principal values of the hyperfine tensors ( A 1 and A 2 ) are equal.…”

“…The high fraction of monoradical in HEXA 1,5 seems too high given the chemical purity of the sample and may indicate a dynamic process, as discussed before. 5 …”

“…The high fraction of monoradical in HEXA 1,5 seems too high given the chemical purity of the sample and may indicate a dynamic process, as discussed before. 5 On the other hand, the experimental spectra of OCTA 2,7 and NONA 2,8 show only three peaks, suggesting a weak exchange interaction between the two unpaired electrons. Also, due to the large distance of the unpaired electrons (15 and 13 Å, respectively; see Table 3), the dipolar interaction is not able to contribute to the broadening of the peaks.…”

“…28 The hybrid counterpart PBE0 of the conventional functional PBE with the standard 6-31G(d) basis set has been employed. On the basis of previous studies on TOAC-labeled, Aib-rich peptides, [3][4][5]14 the backbone of the peptides is fixed in the 3 10 -helix conformation, and a twist geometry for the piperidine rings is assumed.…”

Tracking amyloid oligomerization with monomer resolution using a 13-amino acid peptide with a backbone-fixed spin label

“…The principal values of the 14 N hyperfine coupling tensor were A xx = A yy = 13 MHz and A zz = 110 MHz. 35 The spectra were simulated with a superposition of three components: a fast fraction using the ''Garlic'' function, a medium and a slow fraction using the ''Chili'' function. For the 9 GHz spectra, a Gaussian component with a linewidth of 0.12 mT was used for the fast component, and 1 mT for the medium and slow components.…”