Engineering a functional blue-wavelength-shifted rhodopsin mutant

J. M. Janz, David Farrens

Research output: Contribution to journalArticle

33 Citations (Scopus)

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 ∼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 λmax = 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.

Original languageEnglish (US)
Pages (from-to)7219-7227
Number of pages9
JournalBiochemistry
Volume40
Issue number24
StatePublished - Jun 19 2001

Fingerprint

Rhodopsin
Wavelength
Mutation
Transducin
Opsins
Photobleaching
Schiff Bases
Fluorescence Spectrometry
Lighting
Fluorescence spectroscopy
Chromophores
Proteins
Hydrolysis
Activation energy
Engineers

ASJC Scopus subject areas

  • Biochemistry

Cite this

Engineering a functional blue-wavelength-shifted rhodopsin mutant. / Janz, J. M.; Farrens, David.

In: Biochemistry, Vol. 40, No. 24, 19.06.2001, p. 7219-7227.

Research output: Contribution to journalArticle

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