To What Extent a Synthetic Strategy Can Influence the Structure, Molecular Dynamics, and Biological Properties of Cyclic Tryptophan Cage Miniproteins.

This paper presents structural and biological studies of two cyclic oligopeptides obtained as a result of macrocyclization of linear precursors containing the same amino acid composition, but arranged in reverse order. The first of them with an amino acid sequence (G1D2A3Y4A5Q6W7L8A9D10G11G12P13S14S15G16R17P18P19P20S21G22-) belongs to a group of oligopeptides known in the literature as Tryptophan Cage (Trp_C), often used as models to test and understand the mechanism of protein folding. The knowledge about second precursor (-G1S2P3P4P5R6G7S8S9P10G11G12D13A14L15W16Q17A18 Y19A20D21G22-) is not so extensive. The cyclic forms of both compounds labeled as cTrp_C and Inv-cTrp_C were studied by mass spectrometry (ESI MS and ESI MS/MS), electronic circular dichroism (ECD) and 1D and 2D NMR spectroscopy. ECD and NMR measurements were performed in a different solvents and a wide temperature range. The experimental data clearly demonstrated differences in thermal stability and conformational changes for the two samples. The nature and mechanism of these changes were investigated by means of Molecular Dynamics calculations. Theoretical studies have shown that for cTrp_C and Inv-cTrp_C the length and structure of the -helix, which is the core stabilizing the geometry of cyclic oligopeptides, are different. In the case of Inv-cTrp_C, two edge models with a indole ring of tryptophan (W16) inside the cage (Inv-cTrp_C1) and outside the cage (Inv-cTrp_C2) are postulated. Based on theoretical calculations, it is speculated that the formation of these two structures is due to the large-amplitude reorientation of the unstructured chain around the -helix.