Calorimetric studies of flavin binding protein: flavin analog binding.
The effect of flavin structure variation upon the overall binding reaction of flavin to hen egg white riboflavin binding protein (WRBP) was correlated to thermodynamic parameters obtained via titration calorimetry. This effect was measured by determining a reference binding enthalpy (delta Href) for 3-carboxymethylriboflavin and subsequently comparing delta Href to the binding enthalpies determined for all other flavin analogs in this study. The reference enthalpy change was measured in detail under a variety of temperatures (14, 25, and 38 degrees C), pH's (5.5, 6.5, 7.4, 8.5), and buffer types. The reference enthalpy for the binding of N(3)-carboxymethylriboflavin to WRBP was measured as delta H = -20.9 kcal/mol at 25.0 degrees C and pH 7.4. Evaluation of the binding thermodynamic values for the reference flavin indicates that there is no H+ flux concomitant with binding. A small negative change in heat capacity (delta Cp) occurs during the binding process suggesting a contraction of the protein. Finally, the reaction appears to be independent of pH and buffer type indicating little, if any, involvement of charged residues in the binding of flavin to apoWRBP. Thermodynamic values for the binding of an additional eight flavin analogs were then measured at pH 7.4 and 25.0 degrees C. The thermodynamic binding parameters for these analogs were evaluated by comparison to those determined for the reference flavin. The results indicate that the ribityl C2' position is a major influence in the interaction between the ribityl side chain and the protein; the C9 position of the isoalloxazine ring is sterically restricted in a manner similar to positions C7 and C8; and the positions C7 and C8 appear to be unequal with regard to enthalpy release, suggesting that the C8 position is the most restricted region in the aromatic ring. These various findings indicate the unique ability of titration calorimetry to evaluate structural variation of ligands to their corresponding binding site interactions.
D000818:Animals; D002151:Calorimetry; D002352:Carrier Proteins / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D002645:Chickens; D005260:Female; D005415:Flavins / Q000378:metabolism*; D008024:Ligands; D026901:Membrane Transport Proteins; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D005415:Flavins; D008024:Ligands; D026901:Membrane Transport Proteins; C019079:riboflavin-binding protein