Table 1.

Thermodynamic Parameters of PMB and LL-32 Binding to the Bacterial Outer Membrane of S. enterica R60.

Thermodynamic parameters	Polymyxin B			LL-32
	Viable bacteriaf	Viable bacteria + NaN3 0.1 %	γ-irradiated bacteria 3h	Viable bacteria	Viable bacteria + NaN3 0.1 %f	γ-irradiated bacteria 3h f
n (ƒg/bacteria)a	75 ± 6	65 ± 14	55 ± 8	n.d	172 ± 42	152 ± 12
Peptide s.a/Bacteria s.ab	3.7 ± 0.3	3.2 ± 0.7	2.7 ± 0.4	n.d	9.6 ± 2.3	8.5 ± 0.7
Peptide/LPS Ra mol ratiob	5.2 ± 0.4	4.5 ± 1.0	3.8 ± 0.6	n.d	4.2 ± 1.0	3.7 ± 0.3
KD (nM)c	397± 326	80 ± 14	84 ± 59	n.d	958 ± 458	855 ± 401
ΔH (kJ/mol)	- 59 ± 4	- 114 ± 17	- 133 ± 17	n.d	- 64 ± 4	- 81 ± 1
-TΔS (kJ/mol)d	20 ± 7	72 ± 17	91 ± 16	n.d	28 ± 5	45 ± 1
ΔG (kJ/mol)e	- 39 ± 2	- 42 ± 1	- 42 ± 2	n.d	- 36 ± 1	- 36 ± 1

The values represent the mean of three independent experiments with the s.d.

^aThe amount of bonded peptide was calculated from the inflection point of the isotherm curves. Instead of a stoichiometry value, it represents the overall peptide needed to neutralize all the constitutive negative charges on the outer leaflet of the outer membrane. The molecular weights of LPS Ra, PMB, and LL-32 are 4300, 1385.6, and 3921.7 g/mol, respectively.

^bPeptide surface area (s.a.) was calculated assuming that a single bound peptide molecule covers an area of ∼1.0×10⁻⁶ and ∼3.5×10⁻⁶ μm² for PMB and LL-32, respectively. The bacteria s.a. was estimated to be 9.5 μm², which corresponds to a cylinder of 1 μm diameter and 3.5 μm height. The Peptide/LPS Ra mol ratio was calculated assuming an LPS Ra area of 1.5×10⁻⁶ μm² (80). We assumed a bacterial surface completely covered by LPS Ra. Based on the LPS Ra dry mass yield, we calculated 52 μg of LPS Ra in 1.5×10⁹ titrated bacteria, a value that was close to the 66 μg obtained by theoretical calculation.

^cK_D represents an apparent dissociation constant due to the high chemical diversity on the bacterial membrane.

^dEntropy changes of binding were calculated with $\Delta G=\Delta H-T\Delta S$

^eFree energy changes were calculated according to $\Delta G=-RTln{K}_A$

^fEnthalpy changes were corrected for incomplete bacterial membrane binding. As a consequence, the energy released upon the addition of PMB and LL-32 only represents the total energy minus the average residual heat of the last three titration points from the isotherms. The reason for this procedure was the possibility to use the one-side model function to get the thermodynamic values. The peptide binding to the bacterial membrane was strongly exothermic for both peptides, and the maximal values were observed for viable bacteria (Fig. 5, D and E). The binding reaction was entropically unfavorable with an overall Gibbs energy change favorable for both peptides under all conditions tested. n.d., not detected.