The phototransformation of phytochrome probed by 360 MHz proton NMR spectra

The 360 MHz proton NMR spectra of the Pr and Pfr forms of phytochrome have been recorded to probe the nature of the phytochrome phototransformation (Pr → Pfr). The NMR spectra of aliphatic protons in Pr and Pfr proteins are similar, suggesting that the conformation of both proteins are not drastically different. However, the NMR spectrum of aromatic and the -NH- proton resonance region of Pfr differs significantly from that of Pr, including the absence of a resonance at 6.15 ppm in the former. Differences in the NMR spectra of small and large mol wt phytochromes have also been noted. © 1982 Academic Press, Inc

Characterization of Site-Directed Mutants in the lac Permease of Escherichia coli. 1. Replacement of Histidine Residues

Wild-type lac permease from Escherichia coli and two site-directed mutant permeases containing Arg in place of His35 and His39 or His322 were purified and reconstituted into proteoliposomes. H35-39R permease is indistinguishable from wild type with regard to all modes of translocation. In contrast, purified, reconstituted permease with Arg in place of His322 is defective in active transport, efflux, equilibrium exchange, and counterflow but catalyzes downhill influx of lactose without concomitant H+ translocation. Although permease with Arg in place of His205 was thought to be devoid of activity, sequencing of lac Y in pH205R reveals the presence of two additional mutations in the 5' end of the gene, and replacement of this portion of lac Y with a restriction fragment from the wild-type gene yields permease with normal activity. Permeases with Asn, Gln, or Lys in place of His322, like H322R permease, catalyze downhill influx of lactose without H+ translocation but are unable to catalyze active transport, equilibrium exchange, or counterflow. Unlike H322R permease, however, the latter mutants catalyze efflux at rates comparable to that of wild-type permease, although the reaction does not occur in symport with H+. Finally, as evidenced by flow dialysis and photoaffinity labeling experiments, replacement of His322 appears to cause a marked decrease in the affinity of the permease for substrate. The results confirm and extend the contention that His322 is the only His residue in the permease involved in lactose/H+ symport and that an imidazole moiety at position 322 is obligatory. In addition, the observations are consistent with the idea that His322 functions as a component of a catalytic triad that is important for lactose/H+ symport. Next, the role of Glu325 is examined in detail, and in the near future, evidence is presented supporting the hypothesis that His322 and Glu325 are ion-paired.

A novel spirooxindole derivative inhibits the growth of Leishmania donovani parasite both in vitro and in vivo by targeting type IB topoisomerase

Visceral Leishmaniasis is a fatal parasitic disease and there is an emergent need for development of effective drugs against this neglected tropical disease. We report here development of a novel spirooxindole derivative N-benzyl 2, 2’ α 3, 3’, 5’, 6’, 7’, 7α,α'-octahydro-2methoxycarbonyl-spiro [indole-3, 3’ -pyrrolizidine]-2 one (Compound 4c) which inhibits Leishmania donovani topoisomerase IB (LdTopIB) and kills the wild type as well as drug-resistant parasite strains. This compound inhibits catalytic activity of LdTopIB in competitive manner. Unlike Camptothecin, the compound does not stabilize the DNA-topoisomerase IB cleavage complex; rather, they hinder drug-DNA-enzyme covalent complex formation. Fluorescence studies show stoichiometry of this compound binding to LdTopIB is 2:1 (mole/mole) with a dissociation constant of 6.65 μM. Molecular docking with LdTopIB using the stereoisomers of Compound 4c produced two probable hits for binding site: one in small subunit and the other in the hinge region of the large subunit of LdTopIB. This spirooxindole is highly cytotoxic to promastogotes of L. donovani and also induces apoptosis-like cell death in parasite. Treatment with compound 4c causes depolarization of mitochondrial membrane potential, formation of reactive oxygen species inside parasites and ultimately fragmentation of nuclear DNA. Compound 4c also effectively clears amastigote forms of wild type and drug-resistant parasites from infected mouse peritoneal macrophages but has less effect on host macrophages. Moreover compound 4c showed strong antileishmanial efficacies in BALB/c mice model of leishmaniasis. Potentially this compound can be used as a lead for developing excellent anileishmanial agent against emerging drug resistant strains of the parasite