FIG. 6.
BIAcore3000 sensorgram illustrating dosage-dependent binding of MV ribonucleoprotein core particles (RNP or nucleocapsid) to 250 RU of immobilized Hsp72. The RNP-Hsp72 responses were corrected for nonspecific interactions with the sensor surface by subtracting the signal recorded when RNP was passed over sensor flow channels lacking Hsp72. The corrected signal was then expressed as a response differential (Resp. Diff.) in RU. Sensorgrams for the interaction between Hsp72 and RNP N protein concentrations of 5, 10, 20, 40, and 80 nM allowed high-quality global fitting to a 1:1 Langmuir binding model, allowing the computation of KD and association and dissociation rate constants ka and kd. This binding reaction was compared to that of RNP subjected to selective proteolysis with S. aureus V8 protease. Western blot analysis of RNP and V8-digested RNP (RNP+V8) was done with an MV N protein-specific monoclonal antibody as a probe to show the conversion of the 60-kDa N protein to the 45-kDa amino-terminal three-fourths of the N protein (panel at lower right; numbers to the right of the panel are in kilodaltons). The affinity of binding between V8-digested RNP and Hsp72 was calculated as described for RNP.