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Biophys J, December 1999, p. 3277-3286, Vol. 77, No. 6
*Max-Planck-Institut für Strahlenchemie, D-45413 Mülheim an der Ruhr, and #Max-Planck-Institut für Molekulare Physiologie, D-44139 Dortmund, Germany
Purified wild-type sensory rhodopsin II from
Natronobacterium pharaonis (pSRII-WT) and its
histidine-tagged analog (pSRII-His) were studied by laser-induced
optoacoustic spectroscopy (LIOAS) and flash photolysis with optical
detection. The samples were either dissolved in detergent or
reconstituted into polar lipids from purple membrane (PML). The quantum
yield for the formation of the long-lived state M400 was
determined as 
M = 0.5 ± 0.06 for both
proteins. The structural volume change accompanying the production of
K510 as determined with LIOAS was
VR,1 
10 ml for both proteins,
assuming K 
M, indicating that the
His tag does not influence this early step of the photocycle. The
medium has no influence on VR,1, which is
the largest so far measured for a retinal protein in this time range
(<10 ns). This confirms the occurrence of conformational movements in
pSRII for this step, as previously suggested by Fourier transform
infrared spectroscopy. On the contrary, the decay of K510
is an expansion in the detergent-dissolved sample and a contraction in
PML. Assuming an efficiency of 1.0, VR,2 = 
3 ml/mol for pSRII-WT and
4.6 ml/mol for pSRII-His were calculated in PML, indicative of a
small structural difference between the two proteins. The energy
content of K510 is also affected by the tag. It is
EK = (88 ± 13) for pSRII-WT and
(134 ± 11) kJ/mol for pSRII-His. A slight difference in the
activation parameters for K510 decay confirms an influence
of the C-terminal His on this step. At variance with
VR,1, the opposite sign of
VR,2 in detergent and PML suggests the
occurrence of solvation effects on the decay of K510, which
are probably due to a different interaction of the active site with the
two dissolving media.
Biophys J, December 1999, p. 3277-3286, Vol. 77, No. 6
© 1999 by the Biophysical Society 0006-3495/99/12/3277/10 $2.00
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