Interaction of porin from Yersinia pseudotuberculosis with lipopolysaccharides. Effect of ionic strength, pH, and divalent cations on the binding parameters.
Interaction of the pore-forming protein (porin) from Yersinia pseudotuberculosis with S- and R-forms of the endogenous lipopolysaccharide (LPS) was studied at various ionic strengths (20-600 mM NaCl), concentrations of divalent cations (5-100 mM CaCl2, MgCl2), and pH values from 3.0 to 9.0. The interaction of the R-LPS with porin has been shown in all experimental conditions to be in consensus with the model suggesting binding at independent sites of two types. S-LPS binds to interacting sites of relatively high affinity and to independent sites of low affinity at all pH values examined and at low NaCl concentration. The cooperative interaction of the S-LPS and porin is not observed at high ionic strength and in divalent cation-free medium. The number of binding sites of porin and association constants (Ka) for both LPS forms decrease significantly on increasing the solution ionic strength. The Ka values for the R- and S-LPS change oppositely on changing the pH: the Ka value for the R-LPS is maximal (Ka = 6.7 x 10(5) M-1), but that for S-LPS is minimal (Ka = 0.4 x 10(5) M(-1) at pH 5.0-5.5. The number of high-affinity and low-affinity binding sites for both LPS forms is maximal at pH 5.0-5.5. In this case, the numbers of high- and low-affinity sites for R-LPS are 3 and 10, respectively, and those for the S-LPS are 7 and 20, respectively. These data suggest an important role of electrostatic interactions on binding of LPS to porin. The contribution of conformational changes of the ligand and protein and hydrophobic interactions are discussed.
Naberezhnykh
G A
GA
Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Vladivostok, 690022, Russia.
Kim
N Y
NY
Glazunov
V P
VP
Bakholdina
S I
SI
Krasikova
I N
IN
Khomenko
V A
VA
Solov'eva
T F
TF
D001665:Binding Sites; D002122:Calcium Chloride / Q000494:pharmacology; D002413:Cations, Divalent; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes / Q000378:metabolism; D007477:Ions; D007700:Kinetics; D008070:Lipopolysaccharides / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology; D018272:Porins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012235:Rhodamines / Q000494:pharmacology; D012685:Sepharose / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D015011:Yersinia pseudotuberculosis / Q000737:chemistry*
D002413:Cations, Divalent; D005456:Fluorescent Dyes; D007457:Iodine Radioisotopes; D007477:Ions; D008070:Lipopolysaccharides; D018272:Porins; D012235:Rhodamines; D015636:Magnesium Chloride; D012965:Sodium Chloride; D012685:Sepharose; D002122:Calcium Chloride