Detection of threonine structural changes upon formation of the M-intermediate of bacteriorhodopsin: evidence for assignment to Thr-89.
The behavior of threonine residues in the bacteriorhodopsin (bR) photocycle has been investigated by Fourier transform infrared difference spectroscopy. L-Threonine labeled at the hydroxyl group with 18O (L-[3-(18)O]threonine) was incorporated into bR and the bR-->M FTIR difference spectra measured. Bands are assigned to threonine vibrational modes on the basis of 18O induced isotope frequency shifts and normal mode calculations. In the 3500 cm-1 region, a negative band is assigned to the OH stretch of threonine. In the 1125 cm-1 region, a negative band is assigned to a mixed CH3 rock/CO stretch mode. The frequency of both these bands indicates the presence of at least one hydrogen bonded threonine hydroxyl group in light adapted bR which undergoes a change in structure by formation of the M intermediate. Spectral changes induced by the substitution Thr-89-->Asn but not Thr-46-->Asn or Asp-96-->Asn are consistent with the assignment of these bands to Thr-89. These results along with another related study on the mutant Thr-89-->Asn indicate that the active site of bR includes Thr-89 and that its interaction with the retinylidene Schiff base and Asp-85 may play an important role in regulating the color of bacteriorhodopsin and the transfer of a proton to the Schiff base.
Liu
X
X
Physics Department, Boston University, MA 02215, USA.
Lee
M J
MJ
Coleman
M
M
Rath
P
P
Nilsson
A
A
Fischer
W B
WB
Bizounok
M
M
Herzfeld
J
J
Karstens
W F
WF
Raap
J
J
Lugtenburg
J
J
Rothschild
K J
KJ
D001436:Bacteriorhodopsins / Q000737:chemistry*; D008027:Light; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013912:Threonine / Q000737:chemistry*
D013912:Threonine; D001436:Bacteriorhodopsins