Cancer is a complex disease with a range of genetic and biochemical markers within and among tumors, but a general tumor characteristic is extracellular acidity, which is associated with tumor growth and development. Acidosis could be a universal marker for cancer imaging and the delivery of therapeutic molecules, but its promise as a cancer biomarker has not been fully realized in the clinic. We have discovered a unique approach for the targeting of acidic tissue using the pH-sensitive folding and transmembrane insertion of pH (low) insertion peptide (pHLIP). The essence of the molecular mechanism has been elucidated, but the principles of design need to be understood for optimal clinical applications. Here, we report on a library of 16 rationally designed pHLIP variants. We show how the tuning of the biophysical properties of peptide-lipid bilayer interactions alters tumor targeting, distribution in organs, and blood clearance. Lead compounds for PET/single photon emission computed tomography and fluorescence imaging/MRI were identified, and targeting specificity was shown by use of noninserting variants. Finally, we present our current understanding of the main principles of pHLIP design.membrane-associated folding | tumor acidity | thermodynamics | kinetics | diagnostics

The membrane-associated folding/unfolding of pH (low) insertion peptide (pHLIP) provides an opportunity to study how sequence variations influence the kinetics and pathway of peptide insertion into bilayers. Here, we present the results of steady-state and kinetics investigations of several pHLIP variants with different numbers of charged residues, with attached polar cargoes at the peptide's membrane-inserting end, and with three single-Trp variants placed at the beginning, middle, and end of the transmembrane helix. Each pHLIP variant exhibits a pH-dependent interaction with a lipid bilayer. Although the number of protonatable residues at the inserting end does not affect the ultimate formation of helical structure across a membrane, it correlates with the time for peptide insertion, the number of intermediate states on the folding pathway, and the rates of unfolding and exit. The presence of polar cargoes at the peptide's inserting end leads to the appearance of intermediate states on the insertion pathway. Cargo polarity correlates with a decrease of the insertion rate. We conclude that the existence of intermediate states on the folding and unfolding pathways is not mandatory and, in the simple case of a polypeptide with a noncharged and nonpolar inserting end, the folding and unfolding appears as an all-or-none transition. We propose a model for membrane-associated insertion/folding and exit/unfolding and discuss the importance of these observations for the design of new delivery agents for direct translocation of polar therapeutic and diagnostic cargo molecules across cellular membranes.

Antimicrobial peptides are naturally occurring short amphipathic proteins, innate to the immune system and shown to induce selective lytic activity towards microbial pathogens. Protegrin-1 is an 18-residue, cationic, b-sheet antimicrobial peptide stabilized by two disulfide bonds. Concentration-dependent structural transformations of supported lipid bilayer patches as a result of peptide-membrane interactions have been visualized through the use of atomic force microscopy. A three-stage concentration-dependent transformation has been characterized, which begins with edge instability, followed by pore formation and worm-like micelle formation. This suggests that protegrin-1 acts to lower the line-energy at the edge of the bilayer. Membrane and lipid characteristics, including fluidity, charge and acyl chain length, can alter the activity of antimicrobial peptides. To identify the importance of both acyl-chain length and fluidity on the activity of protegrin-1, these two variables were decoupled. When the bilayers are examined at the same relative fluidity levels, they demonstrate the three-stage transformation observed on a fluid control bilayer, in contrast to the structural transformations that were observed in the gel phase bilayers. This suggests that fluidity exhibits a large influence on the transformations that occur as a result of protegrin-1. To examine the importance of acyl-chain length, the activity of antimicrobial peptides was studied using unsaturated bilayers. Our results indicate that the longer chain bilayers are less susceptible to disruption. This could be due to the hydrophobic mismatch between protegrin-1 and the thicker hydrophobic portion of longer chain lipid bilayers . These results highlight the importance of subtle membrane characteristics in the activity of antimicrobial peptides towards bacterial cells. Lipid bilayers with cholesterol are more accurate eukaryotic cell mimics and will allow examination of the selective preference of antimicrobial peptide activity.

placed in different locations throughout the protein, including the very Cterminus. Addition of a charged, cleavable signal peptide to the N-terminus of the protein indicates that topology does not change after insertion into the membrane. These observation point to an unanticipated plasticity in pre-insertion membrane protein topology.

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