Engineering a Functional Blue-Wavelength-Shifted Rhodopsin Mutant

Biochemistry. 2001 Jun 19;40(24):7219-27. doi: 10.1021/bi002937i.

Abstract

We report an effort to engineer a functional, maximally blue-wavelength-shifted version of rhodopsin. Toward this goal, we first constructed and assayed a number of previously described mutations in the retinal binding pocket of rhodopsin, G90S, E122D, A292S, and A295S. Of these mutants, we found that only mutants E122D and A292S were like the wild type (WT). In contrast, mutant G90S exhibited a perturbed photobleaching spectrum, and mutant A295S exhibited decreased ability to activate transducin. We also identified and characterized a new blue-wavelength-shifting mutation (at site T118), a residue conserved in most opsin proteins. Interestingly, although residue T118 contacts the critically important C9-methyl group of the retinal chromophore, the T118A mutant exhibited no significant perturbation other than the blue-wavelength shift. In analyzing these mutants, we found that although several mutants exhibited different rates of retinal release, the activation energies of the retinal release were all approximately 20 kcal/mol, almost identical to the value found for WT rhodopsin. These latter results support the theory that chemical hydrolysis of the Schiff base is the rate-limiting step of the retinal release pathway. A combination of the functional blue-wavelength-shifting mutations was then used to generate a triple mutant (T118A/E122D/A292S) which exhibited a large blue-wavelength shift (absorption lambda(max) = 453 nm) while exhibiting minimal functional perturbation. Mutant T118A/E122D/A292S thus offers the possibility of a rhodopsin protein that can be worked with and studied using more ambient lighting conditions, and facilitates further study by fluorescence spectroscopy.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine / genetics
  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Animals
  • Aspartic Acid / genetics
  • COS Cells
  • Cattle
  • Glutamic Acid / genetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Photochemistry / methods
  • Retinaldehyde / metabolism
  • Rhodopsin / chemical synthesis*
  • Rhodopsin / genetics*
  • Rhodopsin / metabolism
  • Rhodopsin / physiology
  • Serine / genetics
  • Spectrometry, Fluorescence / methods
  • Spectrophotometry, Ultraviolet
  • Thermodynamics
  • Threonine / genetics
  • Transducin / metabolism

Substances

  • Threonine
  • Aspartic Acid
  • Glutamic Acid
  • Serine
  • Rhodopsin
  • Transducin
  • Alanine
  • Retinaldehyde