Protein design: reengineering cellular retinoic acid binding protein II into a rhodopsin protein mimic.
Vasileiou, C., Vaezeslami, S., Crist, R.M., Rabago-Smith, M., Geiger, J.H., Borhan, B.(2007) J Am Chem Soc 129: 6140-6148
- PubMed: 17447762 
- DOI: https://doi.org/10.1021/ja067546r
- Primary Citation of Related Structures:  
2G78, 2G79, 2G7B - PubMed Abstract: 
Rational redesign of the binding pocket of Cellular Retinoic Acid Binding Protein II (CRABPII) has provided a mutant that can bind retinal as a protonated Schiff base, mimicking the binding observed in rhodopsin. The reengineering was accomplished through a series of choreographed manipulations to ultimately orient the reactive species (the epsilon-amino group of Lys132 and the carbonyl of retinal) in the proper geometry for imine formation. The guiding principle was to achieve the appropriate Bürgi-Dunitz trajectory for the reaction to ensue. Through crystallographic analysis of protein mutants incapable of forming the requisite Schiff base, a highly ordered water molecule was identified as a key culprit in orienting retinal in a nonconstructive manner. Removal of the ordered water, along with placing reinforcing mutations to favor the desired orientation of retinal, led to a triple mutant CRABPII protein capable of nanomolar binding of retinal as a protonated Schiff base. The high-resolution crystal structure of all-trans-retinal bound to the CRABPII triple mutant (1.2 A resolution) unequivocally illustrates the imine formed between retinal and the protein.
Organizational Affiliation: 
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.