Featured Article - February 2013
Short description: Structure-guided mutagenesis of a protein-chromophore complex enables engineering of prediction-defying red shift.
Biological processes such as color vision and photosynthesis harness light energy by embedding chromophores in protein scaffolds. Protein-chromophore interactions modulate the electrostatic environment, resulting in distinct absorption wavelength maxima (λmax), even for the same chromophore bound to homologous proteins. A red shift (also termed “opsin shift”) quantifies the energy difference from a λmax of 440 nm for a model system to a theoretical maximum of 620 nm. Modulation of λmax can result from the introduction of charged or polar amino acids in the binding pocket or conformational perturbation of the planar, polyene structure of the chromophore.
Borhan, Geiger and colleagues have engineered the human cellular retinol binding protein II (hCRBPII) to investigate the biophysical basis of spectral tuning. The hCRBPII protein is an ideal model system due to its high tolerance to mutations and potential for complete encapsulation of the bound chromophore. Although retinol is the usual hCRBPII ligand, a Q108K mutant allowed formation of the characteristic protonated Schiff base (PSB) linkage to retinal. A further K40L substitution removed a positive charge inhibitory to Schiff base protonation. The resulting double mutant binds retinal with high affinity, and its crystal structure confirmed that a counteranion to the Schiff base was successfully omitted, in contrast to rhodopsins and other systems. Removal of the counterion allowed more charge delocalization throughout the chromophore, resulting in a λmax of 508 nm.
With this powerful experimental system in place, the authors sought to rationally manipulate the electrostatic environment surrounding the chromophore, focusing on amino acids close to different zones of the polyene. They found that anionic residues were not required, given that the absence of the PSB counterion resulted in a more distributed and responsive conjugated polyene system. Examination of the structures correctly predicted that the introduction of a tryptophan residue at the entrance to the chromophore binding pocket would shield the chromophore environment from the aqueous solvent, leading to a substantial red shift. The PSB charge delocalization enabled further optimization of the binding pocket by disrupting a water-mediated hydrogen-bonding network in the PSB region. A final mutant replaced an alanine with another tryptophan to completely encapsulate the binding pocket, yielding a complex with a λmax of 644 nm, the most red-shifted retinylidene PSB complex reported to date. Interestingly, this extreme red shift was achieved with no apparent conformational perturbation to the chromophore. This work will help chemical biologists to design experiments for challenging systems such as dynamic, membrane-bound channelrhodopsins, and also provides a benchmark system for theoreticians to study wavelength regulation.
W. Wang et al. Tuning the electronic absorption of protein-embedded all-trans-retinal.
Science. 338, 1340-1343 (2012). doi:10.1126/science.1226135