Table 1 Thermodynamics and kinetics of ligand binding to BtuFfluo at pH 7.5 and 23 °C.

From: Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD

BtuFfluo ligand

K d 1) or K i (M)

k off (s−1)

k on (M−1s−1)

Cbl

(8.1 ± 0.3) × 10−9  1)

(7.1 ± 0.4) × 10−1  2)

(8.8 ± 0.2) × 107  3)

Nb7

(2.4 ± 0.3) × 10−8

(1.9 ± 0.01) × 10−3

(7.9 ± 0.9) × 104

Nb9

(9.4 ± 1.5) × 10−10

(4.2 ± 0.00) × 10−3

(4.5 ± 0.7) × 106

Nb10

(6.0 ± 0.3) × 10−9

(7.1 ± 0.04) × 10−3

(1.2 ± 0.1) × 106

Nb14

(7.7 ± 0.4) × 10−7

n.d.

n.d.

Nb15

(7.9 ± 0.6) × 10−8

n.d.

n.d.

Nb17

(5.4 ± 0.4) × 10−8

(2.5 ± 0.02) × 10−3

(4.6 ± 0.3) × 104

  1. To determine the dissociation constants (K i values) of the BtuF-nanobody complexes, the competitive binding equilibria from Fig. 2B were fitted numerically with Dynafit 43,44, using the dissociation constant of the BtuF-Cbl complex (8.1 nM) as input and the K i value of the respective nanbody as open parameter. The dissociation rates (k off ) of the BtuF-nanobody complexes were derived from the data in Fig. 3, and the respective association rate constants (k on ) were calculated from the equation k on  = k off /K i . The indicated errors correspond to the standard errors derived from the fits. n.d.: not determined.
  2. 1)Dissociation constant of the BtuFfluo-Cbl complex obtained from equilibrium data (Fig. 2B).
  3. 2)Calculated from the equation k off  = K d  × k on .
  4. 3)Recorded with stopped-flow fluorescence kinetics (Supplementary Figure 2).
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