Determination of Penetratin Secondary Structure in Live Cells with Raman Microscopy

摘要

Cell penetrating peptides (CPPs) have attracted recent interest as drug delivery tools, although the mechanisms by which CPPs are internalized by cells are not well defined. Here we report a new experimental approach for the detection and secondary structure determination of CPPs in live cells using Raman microscopy with heavy isotope labeling of the peptide. As a first demonstration of principle Penetratin, a sixteen-residue CPP derived from the Antennapedia homeodomain protein of Drosophila, was measured in single, living melanoma cells. Carbon-13 labeling of the Phe residue of penetratin was used to shift the intense aromatic ring-breathing vibrational mode from 1003 cm⁻¹ to 967 cm⁻¹, thereby enabling the peptide to be traced in cells. Difference spectroscopy and principal components analysis (PCA) were used independently to resolve the Raman spectrum of the peptide from the background cellular Raman signals. Based on the position of the amide I vibrational band in the Raman spectra, the secondary structure of the peptide was found to be mainly random coil and β-strand in the cytoplasm, and possibly assembling as β-sheets in the nucleus. The rapid entry and almost uniform cellular distribution of the peptide, as well as the lack of correlation between peptide and lipid Raman signatures, indicated that the mechanism of internalization under the conditions of study was probably non-endocytotic. This experimental approach can be used to study a wide variety of CPPs as well as other classes of peptides in living cells.