Abstract
Trp-cage, NLYIQWLKDGGPSSGRPPPS, a synthetic 20 residue polypeptide, is obtained from the truncation and mutation of a 39 residue peptide, HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS, called exendin-4 (EX4). It has been reported that folding/unfolding of the trp-cage is not a simple two-state process. Toward clarifying this issue, we have synthesized two spin-labeled trp-cage peptides and studied the effects of the spin-label on the tryptophan fluorescence in the peptides, circular dichroism, and the electron paramagnetic resonance spectrum. The one of the spin-labels is 2,2,6,6- tetramethylpiperidine-1-oxyl-4-amino-4- carboxylic acid (TOAC) coupled to the trp-cage at the N-terminal, and the other, (1-oxyl-2,2,5,5-tetramethyl- pyrroline-3-methyl)-methanethiosulfonate (MTSSL), at the C-terminal. We probe the secondary structure of the peptides using steady-state CD spectroscopy. We find that the turn structure always exists but the helix is lost at temperature above 30℃. In the refolding of the trp-cage, the turn appears to be formed first. These measurements indicate that the spin-labels stabilize the α-helix structure. Besides, from quenching of the tryptophan fluorescence by the spin-labels, we conclude that MTSSL at C-terminal is closer to the tryptophan than TOAC. Finally, we have prepared a doubly-labeled spin-label trp-cage and have used EPR to probe the magnetic dipole-dipole interaction between the two spin-labels. The EPR signal of the diradical trp-cage is broad, interacting onsite of magnetic dipole-dipole interaction between two spin-labels, and thus the distance is within 20A.