Binding study of riboflavin-binding protein with riboflavin and its analogues by differential scanning calorimetry.
Thermal unfolding parameters of hens' egg-white riboflavin-binding-protein (RBP) were measured by differential scanning calorimetry. Thermal denaturation scans of apoRBP and RBP complexes with riboflavin and its analogues (FMN, N10 DL-glyceryl isoalloxazine, and N10 omega-hydroxypentyl isoalloxazine) have been measured. It was found that ligand binding causes increase of RBP thermal stability, as manifested by a change of denaturation temperature from 60.8 degrees C for apoRBP to 72.8 degrees C for RBP-Rf complex. For RBP-FMN complex, the denaturation temperature of 73.0 degrees C was even higher than for the RBP-Rf complex. The other two flavin analogues showed transition temperatures in between 66.9 degrees C and 68.8 degrees C, respectively. Analysis of excess heat capacity data showed that the best fit was the sum of two independent thermal transitions. One of the transitions, which contributed approximately 70% to the total heat effect, has transition temperature in the broad range of 60.5-73.2 degrees C; the other transition temperature is in the narrower range of 65.4-71.1 degrees C. The observed transitions can be related to RBP domains.
Wasylewski
M
M
Department of Physical Biochemistry, Institute of Molecular Biology, Jagiellonian University, Kraków, Poland. mwasyle@mol.uj.edu.pl
D002152:Calorimetry, Differential Scanning; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002942:Circular Dichroism; D026901:Membrane Transport Proteins; D011485:Protein Binding; D011489:Protein Denaturation; D012256:Riboflavin / Q000031:analogs & derivatives / Q000737:chemistry*; D013816:Thermodynamics
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; C019079:riboflavin-binding protein; D012256:Riboflavin