NMR structure and dynamics of Q4D059, a kinetoplastid-specific and conserved protein from Trypanosoma cruzi.

Q4D059 (UniProt accession number), is an 86-residue protein from Trypanosoma cruzi, conserved in the related kinetoplastid parasites Trypanosoma brucei and Leishmania major. These pathogens are the causal agents of the neglected diseases: Chagas, sleeping sickness and leishmaniases respectively and had recently their genomes sequenced. Q4D059 shows low sequence similarity with mammal proteins and because of its essentiality demonstrated in T. brucei, it is a potential target for anti-parasitic drugs. The 11 hypothetical proteins homologous to Q4D059 are all uncharacterized proteins of unknown function. Here, the solution structure of Q4D059 was solved by NMR and its backbone dynamics was characterized by (15)N relaxation parameters. The structure is composed by a parallel/anti-parallel three-stranded ß-sheet packed against four helical regions. The structure is well defined by ca. 9 NOEs per residue and a backbone rmsd of 0.50±0.05 Å for the representative ensemble of 20 lowest-energy structures. The structure is overall rigid except for N-terminal residues A(9) to D(11) at the beginning of ß1, K(38), V(39) at the end of helix H3 with rapid motion in the ps-ns timescale and G(25) (helix H2), I(68) (ß2) and V(78) (loop 3) undergoing internal motion in the µs-ms timescale. Limited structural similarities were found in protein structures deposited in the PDB, therefore functional inferences based on protein structure information are not clear. Q4D059 adopts a α/ß fold that is slightly similar to the ATPase sub-domain IIB of the heat-shock protein 70 (HSP70) and to the N-terminal domain of the ribosomal protein L11.

(1)H, (15)N and (13)C resonance assignments and secondary structure prediction of Q4D059, a conserved and kinetoplastid-specific hypothetical protein from Trypanosoma cruzi.

Trypanosoma cruzi is a human parasite that causes Chagas disease, an illness affecting millions of people and without an efficient treatment available. Sequencing the pathogen genome has revealed that near half of protein-coding genes correspond to hypothetical proteins of unknown function, increasing the possibilities for novel target discovery. Q4D059 is a putative essential hypothetical protein from T. cruzi and it is specific and conserved among the trypanosomatid genomes. Here, we report the sequential backbone and side chain resonance assignments and secondary structure analysis of Q4D059, as first step for protein structure determination, function elucidation and drug screening.

¹H, ¹5N and ¹³C assignments of a putative peptidyl prolyl cis-trans isomerase FKBP12 from Trypanosoma brucei.

TbFKBP12 is a putative peptidyl prolyl cis-trans isomerase from Trypanosoma brucei, causative agent of the African trypanosomiasis or sleeping sickness. It interacts with the immunosuppressive drug rapamycin inhibiting the formation of TORC2 complex leading to parasite death by inhibiting cell proliferation through cytokinesis blockade. Moreover, RNAi silencing of TbFKBP12 revealed essential function in both procyclic and bloodstream forms. Both facts make TbFKBP12 an attractive target for ligand development and thus structural data is desirable. In this work we report the NMR resonance assignments for (1)H, (15)N and (13)C nuclei in the backbone and side chains of the TbFKBP12 as basis for further studies of structure, backbone dynamics, interaction mapping and drug screening.

Purification and characterization of Hb 98-114: a novel hemoglobin-derived antimicrobial peptide from the midgut of Rhipicephalus (Boophilus) microplus.

The antimicrobial activity of hemoglobin fragments (hemocidins) has been reported in a variety of models. The cattle tick Rhipicephalus (Boophilus) microplus is a blood sucking arthropod from where the first in vivo-generated hemocidin was characterized (Hb 33-61). In the present work we identified a novel antimicrobial peptide from the midgut of fully engorged R. (B.) microplus females, which comprises the amino acids 98-114 of the alpha subunit of bovine hemoglobin, and was designated Hb 98-114. This peptide was active against several yeast and filamentous fungi, although no activity was detected against bacteria up to 50µM of the synthetic peptide. Hb 98-114 was capable of permeabilizing Candida albicans cell membrane and had a fungicidal effect against this yeast. Circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments showed that Hb 98-114 has a random conformation in aqueous solution but switches to an alpha-helical conformation in the presence of sodium dodecyl sulfate (SDS). This alpha helix adopts an amphipathic structure which may be the mechanism of cell membrane permeabilization. Importantly, Hb 98-114 may play an important role in defending the tick midgut against fungal pathogens and is the first hemocidin with specific antifungal activity to be characterized.

Spectroscopic characterization of oligoaniline microspheres obtained by an aniline-persulfate approach.

This paper investigates the structure of the products obtained from the polymerization of aniline with ammonium persulfate in a citrate/phosphate buffer solution at pH 3 by resonance Raman, NMR, FTIR, and UV-vis-NIR spectroscopies. All the spectroscopic data showed that the major product presented segments that were formed by a 1,4-Michael reaction between aniline and p-benzoquinone monoimine, ruling out the formation of polyazane structure that has been recently proposed. The characterization of samples obtained at different stages of the reaction indicated that, as the reaction progressed, phenazine units were formed and 1,4-Michael-type adducts were hydrolyzed/oxidized to yield benzoquinone. Raman mapping data suggested that phenazine-like segments could be related to the formation of the microspheres morphology.

(1)H, (15)N and (13)C assignments of the Rhipicephalus (Boophilus) microplus anti-microbial peptide microplusin.

Microplusin, a Rhipicephalus (Boophilus) microplus anti-microbial peptide (AMP) is the first member of a new family of cysteine-rich AMPs with histidine-rich regions at the N- and C-termini, which is being fully characterized by biophysical and biochemical methods. Here we report the NMR resonance assignments for (1)H, (15)N, and (13)C nuclei in the backbone and side chains of the microplusin as basis for further studies of structure, backbone dynamics and interactions mapping.

Structure and mode of action of microplusin, a copper II-chelating antimicrobial peptide from the cattle tick Rhipicephalus (Boophilus) microplus.

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha1 (residues Gly-9 to Arg-21), alpha2 (residues Glu-27 to Asn-40), alpha3 (residues Arg-44 to Thr-54), alpha4 (residues Leu-57 to Tyr-64), and alpha5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Solution structure and backbone dynamics of the Trypanosoma cruzi cysteine protease inhibitor chagasin.

A Trypanosoma cruzi cysteine protease inhibitor, termed chagasin, is the first characterized member of a new family of tight-binding cysteine protease inhibitors identified in several lower eukaryotes and prokaryotes but not present in mammals. In the protozoan parasite T.cruzi, chagasin plays a role in parasite differentiation and in mammalian host cell invasion, due to its ability to modulate the endogenous activity of cruzipain, a lysosomal-like cysteine protease. In the present work, we determined the solution structure of chagasin and studied its backbone dynamics by NMR techniques. Structured as a single immunoglobulin-like domain in solution, chagasin exerts its inhibitory activity on cruzipain through conserved residues placed in three loops in the same side of the structure. One of these three loops, L4, predicted to be of variable length among chagasin homologues, is flexible in solution as determined by measurements of (15)N relaxation. The biological implications of structural homology between chagasin and other members of the immunoglobulin super-family are discussed.