Mutagenesis of L35 and Q98 on human retinol-binding protein and effects on binding with retinol

Retinol-binding protein(RBP)is a retinol-specific binding protein.To satisfy the need for cell differentiation,vision maintenance,reproduction and individual maturity,retinol is transported to target cells in a highly specific and regulated manner by combination with RBP and transthyretin.To determine amino acid residues in RBP which might be important for the binding of all trans-retinol,comparative model-building studies were performed.Based on three-dimensional X-ray crystallagraphic structure analysis,two specific amino acids were predicted to be important in retinol binding.The site-directed mutagenesis was performed to alter the L35 to G35,Q98 to R98 respectively.After expression and purification of mutant protein,flurescene binding assay was performed.L35G was found to have no apparent influence on the affinity for all trans-retinol,but the affinity of Q98R had been increased dramatically.