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 Ca²⁺-binding sites (EF hands). The optimum analytical CaM-F sensitivity to Ca²⁺ occurs between ~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 ~20% EtOH and retains one water of hydration between ~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°) 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 ~20-30% EtOH but then appears to denature as the Et0H volume percent increases. The fluorescein reporter group semiangle within holo-CaM-F decreases from 43 ± 1° in neat aqueous buffer to 36 ± 1°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.