Proteases play key roles in the regulation of normal cellular function, and thus, their deregulation leads to many disease states. Molecular beacons are promising protease-imaging probes for the detection and characterization of disease as well as for the evaluation of treatment. Inspired by this, we examined the efficiency of zipper molecular beacons (ZMBs) as imaging probes. First, we showed experimentally that the symmetrical ZMB (zip5e5r), bearing 5-arginine and 5-glutamate arms, is as efficient as the asymmetrical zip5e8r in enhancing cell uptake but without the dark toxicity exhibited by the asymmetric zipper. Also, zip5e5r was shown to dissociate more efficiently at pHs greater than 5. Using a simple two-state binding model, we attributed this to a larger number of charge-pair conformations for zip5e8r. We then measured the ability of soluble matrix metalloproteinases (MMPs) to cleave zip5e5r, and compared their cleavage efficiency with the original photodynamic molecular beacon (PMB). Finally, as a first step toward understanding our observations quantitatively, we simulated the native structures of the peptides GPLGLARK and EGPLGLARRK with charged termini NH3+ and COO- that approximate the PMB and ZMB (with one pair of arginine/glutamate electrostatic zipper), respectively. We concluded that inclusion of the zipper changes the native structure of the MBs, altering the cleavage efficiency of different MMPs.