The pH-selective insertion and folding of a membrane peptide, pHLIP [pH (low) insertion peptide], can be used to target acidic tissue in vivo, including acidic foci in tumors, kidneys, and inflammatory sites. In a mouse breast adenocarcinoma model, fluorescently labeled pHLIP finds solid acidic tumors with high accuracy and accumulates in them even at a very early stage of tumor development. The fluorescence signal is stable for >4 days and is approximately five times higher in tumors than in healthy counterpart tissue. In a rat antigen-induced arthritis model, pHLIP preferentially accumulates in inflammatory foci. pHLIP also maps the renal cortical interstitium; however, kidney accumulation can be reduced significantly by providing mice with bicarbonatecontaining drinking water. The peptide has three states: soluble in water, bound to the surface of a membrane, and inserted across the membrane as an ␣-helix. At physiological pH, the equilibrium is toward water, which explains its low affinity for cells in healthy tissue; at acidic pH, titration of Asp residues shifts the equilibrium toward membrane insertion and tissue accumulation. The replacement of two key Asp residues located in the transmembrane part of pHLIP by Lys or Asn led to the loss of pH-sensitive insertion into membranes of liposomes, red blood cells, and cancer cells in vivo, as well as to the loss of specific accumulation in tumors. pHLIP nanotechnology introduces a new method of detecting, targeting, and possibly treating acidic diseased tissue by using the selective insertion and folding of membrane peptides.cancer targeting ͉ imaging ͉ peptide insertion M any pathological conditions such as cancer, ischemic stroke, inflammation, atherosclerotic plaques, and others are associated with increased metabolic activity and hypoxia resulting in an elevated extracellular acidity (1-6). Hypoxia and acidity have emerged as important factors in tumor biology and responses to cancer treatment. Imaging of hypoxic and acidic regions could provide new information about disease localization and progression and might enhance diagnosis and therapy. Here we describe the use of a peptide, pHLIP [pH (low) insertion peptide], to selectively accumulate in and label acidic tissues. We previously reported that the pHLIP bionanosyringe, a 36-aa peptide derived from the bacteriorhodopsin C helix, has three states: soluble in water, bound to the surface of a membrane, and inserted across the membrane as an ␣-helix. At physiological pH the water-soluble form is favored, whereas at acidic pH the transmembrane ␣-helix predominates (Fig. 1a) (7). We show that at low pH, pHLIP can translocate cell-impermeable cargo molecules that are disulfide-linked to the C terminus across a cell membrane, where they are released in the cytoplasm by reduction of the disulfide bond (8, 9). Among the successfully translocated molecules are organic dyes, phalloidin (a polar, cyclic peptide), and a 12-mer peptide nucleic acid.

The folding of K K-helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable K K-helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization. Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process. Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure. ß

In the Fluid Mosaic Model for biological membrane structure, proposed by Singer and Nicolson in 1972, the lipid bilayer is represented as a neutral two-dimensional solvent in which the proteins of the membrane are dispersed and distributed randomly. The model portrays the membrane as dominated by a membrane lipid bilayer, directly exposed to the aqueous environment, and only occasionally interrupted by transmembrane proteins. This view is reproduced in virtually every textbook in biochemistry and cell biology, yet some critical features have yet to be closely examined, including the key parameter of the relative occupancy of protein and lipid at the center of a natural membrane. Here we show that the area occupied by protein and lipid at the center of the human red blood cell (RBC) plasma membrane is at least Ϸ23% protein and less than Ϸ77% lipid. This measurement is in close agreement with previous estimates for the RBC plasma membrane and the recently published measurements for the synaptic vesicle. Given that transmembrane proteins are surrounded by phospholipids that are perturbed by their presence, the occupancy by protein of more than Ϸ20% of the RBC plasma membrane and the synaptic vesicle plasma membrane implies that natural membrane bilayers may be more rigid and less fluid than has been thought for the past several decades, and that studies of pure lipid bilayers do not fully reveal the properties of lipids in membranes. Thus, it appears to be the case that membranes may be more mosaic than fluid, with little unperturbed phospholipid bilayer.fluid mosaic model ͉ membrane lipid bilayer T he protein-to-lipid ratio has often been measured, but such measures do not address the question we raise here, because the proteins usually extend well beyond the bilayer and often occupy surface area-the finding from the recent modeling of the synaptic vesicle is that there is little lipid exposed, being covered by extrabilayer protein domains (1). There is great variability in the measured protein-to-lipid ratios, from myelin, with Ϸ20% protein, to the inner mitochondrial membrane at 76% protein; the plasma membranes of most animal cells are Ϸ50% protein. Nonetheless, with the exception of the purple membrane from Halobacterium salinarium, there is little accurate information concerning the area occupancy of protein and lipid at the center of natural membranes (2, 3).