Protein design for non-aqueous solvents

Improving protein stability in unnatural and suboptimal environmentsis a promising application of protein engineering technology.Carefully designed amino acid alterations may lead to dramaticpositive effects on the stability of proteins under highly perturbingconditions, such as in non-aqueous solvents. Applications ofbiocatalysts and proteins with specific binding capabilitiesin the chemical industry have been severely limited by constraintsplaced on the solvent environment. With the advent of convenientmethods for altering the amino acid composition and even synthesizingentirely new protein molecules, it is worthwhile to considerengineering proteins for stability in non-aqueous solvents.In order to identify the features that a protein would needfor stability in organic media, we have been studying the structureand properties of the hydrophobic protein crambin. Crambin isunique in that it is soluble and stable in very high concentrationsof polar organic solvents. Crambin and its water-soluble homologsoffer a powerful demonstration of protein engineering for non-aqueoussolvents. This paper describes the structural features thatcontribute to crambin's special properties. Based on these observationsand consideration of how non-aqueous solvents affect the interactionsimportant in protein folding, a set of rules for designing non-aqueoussolvent-stable proteins is proposed.