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Both epitope mapping and other in vitro selection techniques produce short polypeptides that tightly and specifically bind to any of a wide range of macromolecular targets. Here, we demonstrate a potentially general means of converting such polypeptides into optical biosensors. The sensing architecture we have developed, termed peptide beacons, is based on the observation that, whereas short peptides are almost invariably unfolded and highly dynamic, they become rigid when complexed to a macromolecular target. Using this effect to segregate a long-lived fluorophore from an electron transfer based, contact quencher (both covalently attached to the peptide), we have produced a robust optical sensor for anti-HIV antibodies. The binding-induced segregation of the fluorophore-quencher pair produces a 6-fold increase in sensor emission, thus allowing us to readily detect as low as ∼250 pM of the target …