Background and aims: The extremely high affinity of (strep)avidin towards biotin (Kd≈10-15-10-16) and stability enables its use in a variety of applications; for example, in targeted drug therapy, affinity chromatography and in a variety of analytical methods. The rapidly developing technologies require biotin binding proteins with novel characteristics that are not found among naturally occurring proteins. The main aim of the study was to characterize biophysically four novel biotin binding proteins created with DNA shuffling and phage display from avidin (AVD) and avidin related protein 2 (AVR2).

Methods:

Isothermal titration calorimetry was used to study d-biotin binding affinity. Surface plasmon resonance was used to study binding kinetics towards 2-iminobiotin, ssDNA and cysteine. Differential scanning calorimetry was used to study thermal stability of the mutants and also their ligand binding affinities. Homology models were built with MODELLER.

Results:

All the mutants studied have a lowered affinity towards d-biotin and distorted biotin binding site when compared to wtAVDs. The DNA binding abilities varied with wtAVDs bound irreversibly to DNA whereas mutant with neutral pI had no affinity towards DNA. Mutants with higher local charge around biotin binding pocket seemed to have a lower affinity towards 2-iminobiotin. Mutants having wtAVD charge distribution around biotin binding pocket were less affected. Mutant with neutral pI was not able to bind 2-iminobiotin. Thermal stability of the mutants was enhanced by the S16Y mutation. A novel activity among biotin binding proteins was found. A mutant with S16Y mutation was able to bind L- cysteine. The S16Y mutation alone was not able to induce this binding activity.

Conclusions:

A biophysical characterization of studied proteins was successful. It seems that charge distribution in the proximity of biotin binding pocket is important for 2-iminobiotin binding with pI having a smaller effect. In DNA binding, pI is a more important parameter: only biotin binding proteins having a total positive charge seem able to bind DNA. 

Asiasanat:Avidin, DNA shuffling, phage display, d-biotin, 2-iminobiotin, DNA binding proteins, avidin, avidin related protein 2 (AVR2), Biotin binding protein A