Spectral Tuning in Retinal Proteins Color Vision

hν hν

N N H H H H N all-trans N 11-cis 11-cis all-trans

Visual Receptors Light Spectral tuning in color visual receptors Color is sensed by red, green and blue rhodopsin visual receptors.

Rod Cone

G-protein signaling pathway

400nm 500nm 600nm absorption spectrum

Their chromophores

H are exactly the same! 11-cis N

How does the protein tune its Rhodopsin absorption spectrum?

Spectral Tuning in bacteriorhodpsin’s How can we change the maximal photocycle absorption of retinal chromophore?

Me Me Me Me

N

H Me

Me Me Me

N

H Me Me Me Me Me

Me Me H N

1 Excitation energy determines the Electronic Absorption

maximal absorption Me Me Me Me π π* N - H S1 Me Absorption of light in the UV-VIS region of the S0 spectrum is due to Response excitation of electrons to higher energy levels.

Me Me Me 13 7911 15 N

H Me Me

π-π* excitation in polyenes π-π* excitation in polyenes π∗ π∗ π∗ π∗ ∆E π∗ E photon E π π π π π

Ground state (S0) Excited state (S1)

∆E (excitation energy, band gap) = hν = hc/λ blue-shift red-shift

π-π* excitation in polyenes Tuning the length of the conjugated backbone

β-carotene

O O

Vitamin A2 (retinal II) Vitamin A1 (retinal I)

Longer wavelength Short wavelength

2 Retinal I Retinal II OPSIN SHIFT: how protein tunes the absorption maximum of its chromophore.

Maximal absorption of protonated retinal Schiff base in:

Water/methanol solution: 440 nm

bR: 568 nm

rod Rh: 500 nm red receptor: 560 nm green receptor: 530 nm blue receptor: 426 nm

Salmon: different retinals in different stages of life

Electrostatics and opsin shift Electrostatics and opsin shift

S S positive charge 2 positive charge 2 S2 S2 Me Me Me Me Me Me S1 S1 S1 S1 + + + + N N

H H Me O S0 Me O S0 Me O Me O C C Asp (Glu) Asp (Glu) S0 S0 no protein in protein no protein in protein counterion counterion

• The counterion stabilizes the positive charge of the chromophore. Maximal absopriton of protonated retinal Schiff base can be changed by D85N (Purple to blue shift) •The position of the counterion determines how Red shift from 568 to 605 nm at pH = 3 and how much the band gap energy changes.

Dinosaurs had red-shifted visual receptors! Microbial rhodopsins in Halobacteria

Bacteriorhodopsin (bR): purple membrane proton pump Halorhodopsin (hR): chloride pump Sensory rhodopsin I (sRI): attractant (repellent) to orange (near UV) light sRII hR Sensory rhodopsin II (sRII): bR sRI Howard Hughes Medical Institute repellent to blue-green light

at The Rockefeller University and phototaxis Yale University (color vision of halobacteria) 500nm 600nm

3 Spectral Tuning in Bacterial Structures of bR and sRII Rhodopsins

Sensory Rhodopsin II Bacteriorhodopsin X-ray crystallography shows (sRII) (bR) that structures are very similar. Phototaxis Proton pump Both include protonated all- trans retinal Schiff base

• Large blue shift of sRII bR absorption maximum in sRII (70 nm)

orange: sRII 500nm 600nm purple: bR

Binding Sites of bR and sRII QM/MM Calculation of spectral sRII shift in bR and sR-II Similar structure QM/MM helix G • Aromatic residues. • Refinement of X-ray structures by HF (retinal, 2Asp, 3H O) retinal-K205/216 • Hydrogen-bond network. 2 • Excitation energy calculations (counter-ion asparatates, for retinal D201/212 internal water molecules) bR: purple, sRII: orange Calculated spectra ∆Ε( S1-S0) bR Mutagenic substitutions Spectral shift sRII ∆Ε( T204A/V108M/G130S of bR S1-S0) : 6.1 (exp. 7.2) kcal/mol sRII produces only 20 nm ∆Ε( S2-S0 ): 1.7 (exp. 4.0) kcal/mol (30%) spectral shift. A sub-band in sRII is due to the What is the main determinant(s) of second excited state (S2). spectral tuning? 500nm 600nm

Deprotonation of the Schiff base p atomic orbitals

Me Me Me Me

N

H UV vision Me birds, honeybee

Me Me Me Me

N Planarity is essential for maximal overlap of p orbitals Me in a double bond (π molecular Strong blue shift orbital)

4 Steric interactions and Summary of Mechanisms of Spectral Tuning spectral shift? • Using a different chromophore with a longer or a shorter conjugated chain • Modifying the amino acid composition of the binding pocket (electrostatics) • Manipulating the distance and/or conformation of charged/polar groups in the vicinity of retinal • Steric interaction with the chromophore so that some of the double bonds go out of plane (a similar effect to using a shorter chromophore) • Protonation state of retinal Schiff base (Strong blue shift upon deprotonation)

Me Me Me Me

A highly twisted structure can decrease the overlap of N p orbitals and effectively decrease the length of the H conjugation, i.e., blue shift. Me

Me Me Me Me

N

H Me The chromophore retinal adopts different colors in different environments. Doesn’t it remind you of something? cameleon

5