Measuring Intracellular Secondary Structure of a Cell-Penetrating Peptide in Situ

Cell-penetrating peptides (CPPs) are short peptide sequences that can translocate across cellular plasma membranes and are thus potential delivery vectors for diagnostic and therapeutic applications. Many CPPs exhibit some sort of structural polymorphism, where the secondary structure of the peptide is altered strongly by its local environment, which is believed to facilitate membrane translocation and uptake. However, much less is known about the fate and structure of CPPs within cells largely due to measurement difficulty. Here we employ isotopic labeling combined with hyperspectral, quantitative coherent Raman microscopy to localize a model CPP-penetratin-and determine its secondary structure in different cellular compartments. Our results show that penetratin is mostly alpha-helical in the cytosol and acquires a more beta-sheet and random coil character in the nucleus. The increased helicity in the cytosol is similar to that seen in previous studies with model lipid membranes, suggesting that the peptide is associated with membranes in, e.g., endosomes (or lysosomes) in the cytosol. The ability to both localize and determine the secondary structure of a CPP within cells is critical for clarifying the mechanism of peptide-mediated translocation and delivery of cargo molecules to specific cellular destinations.