Jane M. Kuo

A COMBINATORIAL LIBRARY FOR THE BINUCLEAR METAL CENTER OF BACTERIAL PHOSPHOTRIESTERASE

Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of an extensive array of organophosphate pesticides and mammalian acetylcholinesterase nerve agents. Although the three‐dimensional crystal structure of PTE has been solved (M. M. Benning et al., Biochemistry 34:7973–7978, 1995), the precise functions of the individual amino acid residues that interact directly with the substrate at the active site are largely unknown. To construct mutants of PTE with altered specificities for particular target substrates, a simple methodology for generating a library of mutants at specific sites was developed. In this investigation, four of the six protein ligands to the binuclear metal site (His‐55, His‐57, His‐201, and His‐230) were targeted for further characterization and investigation. Using the polymerase chain reaction (PCR) protocols, a library of modified PTE genes was generated by simultaneously creating random combinations of histidine and cysteine codons at these four positions. The 16 possible DNA sequences were isolated and confirmed by dideoxy‐DNA sequencing. The 16 mutant proteins were expressed in Escherichia coli and grown with the presence or absence of 1 mM CoCl2, ZnSO4, or CdSO4in the growth medium. When grown in the presence of CoCl2, the H57C protein cell lysate showed greater activity for the hydrolysis of paraoxon than the wild type PTE cell lysate. H201C and H230C exhibited up to 15% of the wild‐type activity, while H55C, a green protein, was inactive under all assay conditions. All other mutants had <10−5 of wild‐type activity. None of the purified mutants that exhibited catalytic activity had a significantly altered Km for paraoxon. Proteins 29:553–561, 1997.

Circular permutation of RNase T1 through PCR based site-directed mutagenesis

Publisher Summary Current methods for creating circularly permuted variants through alterations in the genetic sequence of a protein are laborious and involve a large number of recombinant DNA manipulations. This chapter discusses the utility of a general PCR approach for the creation of circularly permuted proteins through the initial characterization of cp35SL, and discusses the construction of a circularly permuted variant of ribonuclease Tl (RNase Tl). The native RNase Tl, (C2A, ClOA), and cp35Sl proteins are characterized by specific activity, polyacrylamide electrophoresis (SDS and native), thermodynamic stability, matrix-assisted laser desorption ionization (MALDI) mass spectrometry, and amino-terminal sequencing. The specific activity and thermodynamic stability of the (C2A, ClOA) mutant confirm that the Cys-2 to Cys-10 disulfide bond imparts thermodynamic stability but has little effect on catalytic activity. Hence this mutant was selected as the starting point for constructing a circularly permuted form of RNase-Tl, such that as short a linker as possible could be used to bridge the original N- and C-termini. The activity and stability of the circularly permuted variant indicate that, it adopts an overall tertiary fold very similar to that of the native protein. However, the real effect, however, may be more evident in the kinetics of the specific folding pathway.