Single-molecule studies of HIV-1 protease catalysis enabled by chemical protein synthesis

Single-molecule fluorescence studies of the proteolytic activity of the enzyme HIV-1 protease were performed using FRET-pair dye labeled peptide substrates and substrate-derived inhibitors prepared by solid phase peptide synthesis. Chemical protein synthesis was used to prepare homodimeric HIV-1 protease in soluble form and to prepare a covalent dimer 203 amino acid residue HIV-1 protease containing a biotin tether at the mid-point of the synthetic protein molecule. The biotin-tagged HIV-1 protease was immobilized on a neutravidin-coated glass slide. Total internal reflection excitation multiwavelength fluorescence spectroscopy was used to monitor substrate binding and cleavage by the synthetic enzyme molecules. Single-molecule traces for the dye-labeled peptide substrate showed distinct binding and cleavage events; the corresponding dye-labeled peptide inhibitor showed only the binding event. These results constitute strong proof-of-principle for the utility of chemical peptide and protein synthesis for single-molecule studies of enzyme catalysis.