The creation of novel enzymatic function is of great interest, but remains a challenge because of the large sequence space of proteins. We have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity. The Stoffel fragment (SF) of Thermus aquaticus DNA polymerase I is displayed on a filamentous phage by fusing it to a pIII coat protein, and the substrate DNA template͞primer duplexes are attached to other adjacent pIII coat proteins. Phage particles displaying SF polymerases, which are able to extend the attached oligonucleotide primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized to streptavidin-coated magnetic beads and subsequently recovered. After four rounds of screening an SF library, three SF mutants were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wildtype enzyme incorporates dNTP substrates.
Nucleic acid polymerases are the most important reagents in biotechnology. Unfortunately, their high substrate specificity severely limits their applications. Polymerases with tailored substrate repertoires would significantly expand their potential and allow enzymatic synthesis of unnatural polymers for in vivo and in vitro applications. For example, the ability to synthesize 2'-O-methyl-modified polymers would provide access to materials possessing properties that make them attractive for biotechnology and therapeutic applications, but unfortunately, no known polymerases are capable of efficiently accepting these modified substrates. To evolve such enzymes, we have developed an activity-based selection method which isolates polymerase mutants with the desired property from libraries of the enzyme displayed on phage. In this report, mutants that could efficiently synthesize an unnatural polymer from 2'-O-methyl ribonucleoside triphosphates were immobilized and isolated by means of their activity-dependent modification of a DNA oligonucleotide primer attached to the same phage particle. In each case, directed evolution resulted in relocating a critical side chain to a different position in the polypeptide, thus re-engineering the overall active site while preserving critical protein-DNA interactions. Remarkably, one evolved polymerase is shown to incorporate the modified substrates with an efficiency and fidelity equivalent to that of the wild-type enzyme with natural substrates.
Plasminogen activator inhibitor type 1 (PAI-1) is an important physiological inhibitor of the plasminogen activator system. To investigate the structure-functional aspects of this inhibitor, we have taken advantage of the lack of cysteine residues in the PAI-1 molecule and substituted Ser344 (P3) and Met347 (P1'), in the reactive center loop, with cysteines, thereby creating unique attachment sites for extrinsic fluorescent probe. Both cysteine mutants were purified and labeled with a sulfhydryl specific fluorophore, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen yl-3-propionyl)-N- (iodoacetyl)ethylenediamine (BDYIA). The labeled mutants were found to reveal biochemical characteristics very similar to those of wild type PAI-1. Time-resolved fluorescence spectroscopy was used to examine orientational freedom of BDYIA in the reactive center loop of PAI-1. The orientational freedom of the probe was found to be greater in the latent form than in the active form of PAI-1, suggesting that the reactive center has a more relaxed conformation in the latent form than in the active form. Complex formation with target proteases, tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), caused decreased orientational freedom of BDYIA in the P3 position, while the orientational freedom of BDYIA in position P1' increased to a level similar to that of BDYIA in reactive center-cleaved PAI-1. In contrast, complex formation with modified anhydro-uPA, which is unable to cleave its substrate, largely restricted the orientational freedom of BDYIA probe in the P1' position.(ABSTRACT TRUNCATED AT 250 WORDS)
A new fluorescence spectroscopic method is presented for determining intramolecular and intermolecular distances in proteins and protein complexes, respectively. The method circumvents the general problem of achieving specific labeling with two different chromophoric molecules, as needed for the conventional donor-acceptor transfer experiments. For this, mutant forms of proteins that contain one or two unique cysteine residues can be constructed for specific labeling with one or two identical fluorescent probes, so-called donors (d). Fluorescence depolarization experiments on double-labeled Cys mutant monitor both reorientational motions of the d molecules, as well as the rate of intramolecular energy migration. In this report a model that accounts for these contributions to the fluorescence anisotropy is presented and experimentally tested. Mutants of a protease inhibitor, plasminogen activator inhibitor type-1 (PAI-1), containing one or two cysteine residues, were labeled with sulfhydryl specific derivatives of 4,4-difluoro-4-borata-3a-azonia-4a-aza-s-indacence (BODIPY). From the rate of energy migration, the intramolecular distance between the d groups was calculated by using the Forster mechanism and by accounting for the influence of local anisotropic orientation of the d molecules. The calculated intramolecular distances were compared with those obtained from the crystal structure of PAI-1 in its latent form. To test the stability of parameters extracted from experiments, synthetic data were generated and reanalyzed.
The data clearly demonstrate that serpin inhibition involves reactive-centre cleavage followed by full-loop insertion whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one.
Inhibitors that belong to the serine protease inhibitor or serpin family have reactive centers that constitute a mobile loop with P1-P1' residues acting as a bait for cognate protease. Current hypotheses are conflicting as to whether the native serpin-protease complex is a tetrahedral intermediate with an intact inhibitor or an acyl-enzyme complex with a cleaved inhibitor P1-P1' peptide bond. Here we show that the P1' residue of the plasminogen activator inhibitor type 1 mutant (P1' Cys) became more accessible to radiolabeling in complex with urokinase-type plasminogen activator (uPA) compared with its complex with catalytically inactive anhydro-uPA, indicating that complex formation with cognate protease leads to a conformational change whereby the P1' residue becomes more accessible. Analysis of chemically blocked NH2 termini of serpin-protease complexes revealed that the P1-P1' peptide bonds of three different serpins are cleaved in the native complex with their cognate protease. Complex formation and reactive center cleavage were found to be rapid and coordinated events suggesting that cleavage of the reactive center loop and the subsequent loop insertion induce the conformational changes required to lock the serpin-protease complex.
In an effort to expand the genetic alphabet, a number of unnatural, predominantly hydrophobic, nucleoside analogues have been developed which pair selectively in duplex DNA and during enzymatic synthesis. Significant progress has been made toward the efficient in vitro replication of DNA containing these base pairs. However, the in vivo expansion of the genetic alphabet will require that the unnatural nucleoside triphosphates be available within the cell at sufficient concentrations for DNA replication. We report our initial efforts toward the development of an unnatural in vivo nucleoside phosphorylation pathway that is based on nucleoside salvage enzymes. The first step of this pathway is catalyzed by the D. melanogaster nucleoside kinase, which catalyzes the phosphorylation of nucleosides to the corresponding monophosphates. We demonstrate that each unnatural nucleoside is phosphorylated with a rate that should be sufficient for the in vivo replication of DNA.
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