Effects of ethanol volume percent on fluorescein-labeled spinach apo- and holocalmodulin.
We report the effects of EtOH volume percent (0-70%) on spinach apo- and holocalmodulin that have been site-selectively labeled with fluorescein (F). In these experiments, calmodulin (CaM) has one F reporter group attached to Cys-26, and this site is located immediately adjacent to one of the four primary Ca(2+)-binding sites (EF hands). The optimum analytical CaM-F sensitivity to Ca2+ occurs between approximately 10 and 30% EtOH. Our results also show that added EtOH causes changes in CaM and these changes are surprisingly different for apo- and holo-CaM. Apo-CaM-F appears to lose one of its two waters of hydration at approximately 20% EtOH and retains one water of hydration between approximately 20 and 70% EtOH. In apo-CaM-F, the semiangle that describes the range over which the fluorescein reporter group can precess remains essentially constant (42 +/- 2 degrees) between 0 and 70% EtOH. This shows that the fluorescein reporter group precessional freedom in apo-CaM-F is not affected significantly by EtOH. Holo-CaM-F also appears to lose one water of hydration at approximately 20-30% EtOH but then appears to denature as the EtOH volume percent increases. The fluorescein reporter group semiangle within holo-CaM-F decreases from 43 +/- 1 degrees in neat aqueous buffer to 36 +/- 1 degrees at 70% EtOH. This shows that holo-CaM-F is less nativelike and the EF hand "closes down" about the fluorescein reporter group in holo-CaM-F as the EtOH volume percent increases.
Doody
M A
MA
Department of Chemistry, Natural Sciences Complex, University at Buffalo, State University of New York 14260-3000, USA.
Baker
G A
GA
Pandey
S
S
Bright
F V
FV
D001059:Apoproteins / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D000431:Ethanol / Q000737:chemistry*; D019793:Fluorescein / Q000737:chemistry*; D005454:Fluorescence Polarization; D010940:Plant Proteins / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D018724:Spinacia oleracea
D001059:Apoproteins; D002147:Calmodulin; D010940:Plant Proteins; D012997:Solvents; D000431:Ethanol; D019793:Fluorescein