The susceptibility of Spy's lysine and arginine residues to digestion by trypsin was measured in the presence or absence of Im7 L53A I54A. Protein samples were incubated with trypsin at a constant 100:1 mass ratio (protein: protease). At various times, aliquots were withdrawn, quenched, and analyzed by mass spectrometry. The residue numbers of trypsin-cleavable sites are shown along with their location in Spy's secondary structure (indicated by the cartoon). The bars indicate the time at which cleavage at that residue took place. A missing bar indicates that cleavage was not seen within the 8-min time frame. The unstructured termini are more accessible to trypsin in general, and many sites show apparent increased digestion in the presence of client compared to Spy alone. The interpretation of increased cleavage of these sites is not straightforward. The increased digestion might reflect some real structural rearrangement in Spy upon client binding so that these residues are more exposed to trypsin. Alternatively, they may not be involved in substrate binding and their increased digestion might be an artifact due to a higher total mass of trypsin, which was used to accommodate the addition of client. We were not able to distinguish these two possibilities. On the other hand, the sites showed decreased cleavage despite an increased amount of trypsin are more easily interpreted. Decreased cleavage suggests that they are protected, either by the substrate protein, or by other regions in Spy, which further implies a conformational change upon substrate binding. Cleavage at residues 61, 84, 102, 113, and 122 (circled) is significantly delayed when the client Im7 L53A I54A is present. We conclude that R61, K84, K102, K113, and R122 are sites but not necessarily the only sites affected by the binding events.