Histidine Patch Thioredoxins

Abstract

A cluster of surface amino acid residues on Esche- richia coli thioredoxin were systematically mutated in order to provide the molecule with an ability to chelate metal ions. The combined effect of two histidine mu- tants, E30H and Q62H, gave thioredoxin the capacity to bind to nickel ions immobilized on iminodiacetic acid- and nitrilotriacetic acid-Sepharose resins. Even though these two histidines were more than 30 residues apart in thioredoxin’s primary sequence, they were found to sat- isfy the geometric constraints for metal ion coordina- tion as a result of the thioredoxin tertiary fold. A third histidine mutation, S1H, provided additional metal ion chelation affinity, but the native histidine at position 6 of thioredoxin was found not to participate in binding. All of the histidine mutants exhibited decreased thermal stability as compared with wild-type thioredoxin; how- ever, the introduction of an additional mutation, D26A, increased their melting temperatures beyond that of wild-type thioredoxin. The metal chelating abilities of these histidine mutants of thioredoxin were success- fully utilized for convenient purifications of human in- terleukin-8 and -11 expressed in E. coli as soluble thiore- doxin fusion proteins.