Aggregation Behavior of pHLIP in Aqueous Solution at Low Concentrations: A Fluorescence Study.

pH (low) insertion peptide (pHLIP) is a 36-residue peptide derived from the third transmembrane helix of the membrane protein bacteriorhodopsin. The hydrophobicity of this peptide makes it prone to aggregation even at low concentrations, but this has not been studied in detail. In this work, we characterized monomeric and aggregated forms of pHLIP in aqueous solution (pH 8) at low concentrations (~μM) using fluorescence-based approaches, complemented by circular dichroism (CD) spectroscopy. We show here that monomeric and aggregated pHLIP display differential red edge excitation shift (REES) and CD spectra. These spectroscopic features allowed us to show that pHLIP aggregates even at low concentrations. A detailed knowledge of the aggregation behavior of pHLIP under these conditions will be useful for monitoring and quantifying its interaction with membranes.