The structure and thermodynamics of inclusion complexes of seven different gamma-cyclodextrins (gamma CDs) and three biologically relevant bile salts (BS) were investigated in the present study. Natural gamma CD and six modified gamma CDs [two methyl-gamma CDs, one sulfobutyl ether-gamma CD (SBE gamma CD), and three 2-hydroxypropyl-gamma CDs (HP gamma CD)] and their complexes with BS were investigated by isothermal titration calorimetry, NMR, and molecular dynamics simulations. With the exception of the fully methylated gamma CD, which did not bind the BSs investigated, all of the gamma CDs formed 1:1 complexes with the BS, and the structures were similar to those with natural gamma CD; i.e., the modifications of the gamma CD had limited structural impact on the formation of complexes. Isothermal titration calorimetry was carried out over in the temperature interval 5-55 degrees C to enable the calculation of the stability constant (K) and the thermodynamic parameters enthalpy (Delta H degrees), entropy (Delta S degrees), and heat capacity (Delta C-p degrees). The stability constants decreased with an increased degree of substitution (DS), with methyl substituents having a lower effect on the stability constant than the sulfobutyl ether and hydroxypropyl substituents on the stability constants. Enthalpy-entropy compensation was observed, since both enthalpy and entropy increased with the degree of substitution, which may reflect dehydration of the hydrophobic surface on both CD and BS. Calculations based on Delta C-p degrees data suggested that each of the substituents dehydrated 10-20 (hydroxypropyl), 22-33 (sulfobutyl ether), and 10-15 angstrom(2) (methyl) of the BS surface area, in reasonable agreement with estimates from the molecular dynamics simulations. Combined with earlier investigations on modified beta CDs, these results indicate general trends of the substituents on the thermodynamics of complex formation.