Improving the performance of a sol-gel-entrapped metal-binding protein by maximizing protein thermal stability before entrapment

We show that the performance of a sol-gel-entrapped protein can be improved substantially by maximizing the protein thermodynamic stability so that it can withstand the harsh conditions associated with entrapment. Two mutants of the calcium-binding protein oncomodulin were entrapped into optically clear tetraethyl orthosilicate-derived monoliths which were prepared by a two-step sol-gel processing method. The first mutant contained a single tryptophan residue at position 57 of the native CD binding loop in place of the native tryrosine (Y57W), while the second, more stable mutant contained a higher affinity engineered CD binding loop (CDOM33, with a single Trp residue at position 57). The thermodynamic stability of both proteins could be manipulated by adjusting the level of Ca2+ present during entrapment. Intrinsic tryptophan fluorescence and Tb3+ luminescence (resulting from energy transfer from the Trp residue) were monitored during binding of Tb3+ to examine terbium-binding capacity and response times for both entrapped proteins. Tryptophan fluorescence was also used to study the thermal stability of the entrapped proteins at different calcium levels. Improvements in binding ability, thermal stability, and response times were obtained when CDOM33 or Y57W was entrapped with increasing levels of Ca2+ present during entrapment.