Figure 2.

NMR spectra of U–¹³C,¹⁵N-CF in vesicle arrays with CheA and CheW reveal a mobile fraction in the kinase-on state (CF4Q, blue) and kinase-off state (CF4Q.A411V, red). ¹³C spectra of unfrozen arrays (15 °C) were collected using (A) refocused INEPT to observe the flexible segments and (B) CP to observe the rigid segments of the receptor in functional complexes. Integrated intensities of the aliphatic region (10–80 ppm) indicate that, at this temperature, the kinase-on receptor is approximately 20% flexible and 80% rigid, while the kinase-off receptor is approximately 30% flexible and 70% rigid. (C) ¹³C spectra of frozen samples at −25 °C show the expected loss of flexibility: INEPT spectra have zero intensity (not shown) and CP frozen spectra have intensity comparable to the sum of unfrozen INEPT + CP (see Table 2). Each spectrum was recorded with 2400 scans and a recycle delay of 1 s, at 11.11 kHz MAS.