Resonance assignments and secondary structure prediction of secretory protein Rv0603 from Mycobacterium tuberculosis H37Rv.

We report the NMR resonance assignments of N-terminal signal sequence deleted secretory protein Rv0603 (∆1-28-Rv0603) from Mycobacterium tuberculosis H37Rv. ∆1-28-Rv0603 displayed good peak yield and signal dispersion in 2D [15N-1H] HSQC spectrum, which prompted us to proceed for resonance assignments on this construct. Standard triple-resonance experiments for resonance assignments were recorded on [U-15N]-∆Rv0603 and [U-15N, 13C]-∆Rv0603 samples. We obtained 97% of backbone 1HN, 98% of 13Cα, 98% of 1Hα, 96% of 13C´, 100% of 13Cß, 100% of 1Hß and 98% of side-chain 1H chemical shifts. This protein does not show any sequence similarity to any other protein of known structure. Determination of its solution structure would facilitate understanding of its biological function.

Structural characterization of peptidyl-tRNA hydrolase from Mycobacterium smegmatis by NMR spectroscopy.

BACKGROUND: Accumulation of toxic peptidyl-tRNAs in the bacterial cytoplasm is averted by the action of peptidyl-tRNA hydrolase (Pth), which cleaves peptidyl-tRNA into free tRNA and peptide. NMR studies are needed for a protein homolog with a complete crystal structure, for comparison with the NMR structure of Mycobacterium tuberculosis Pth. METHODS: The structure and dynamics of Mycobacterium smegmatis Pth (MsPth) were characterized by NMR spectroscopy and MD simulations. The thermal stability of MsPth was characterized by DSC. RESULTS: MsPth NMR structure has a central mixed seven stranded ß-sheet that is enclosed by six α-helices. NMR relaxation and MD simulations studies show that most of the ordered regions are rigid. Of the substrate binding segments, the gate loop is rigid, the base loop displays slow motions, while the lid loop displays fast timescale motions. MsPth displays high thermal stability characterized by a melting temperature of 61.71°C. CONCLUSION: The NMR structure of MsPth shares the canonical Pth fold with the NMR structure of MtPth. The motional characteristics for the lid region, the tip of helix α3, and the gate region, as indicated by MD simulations and NMR data, are similar for MsPth and MtPth. However, MsPth has relatively less rigid base loop and more compactly packed helices α5 and α6. The packing and the dynamic differences appear to be an important contributing factor to the thermal stability of MsPth, which is significantly higher than that of MtPth. SIGNIFICANCE: MsPth structure consolidates our understanding of the structure and dynamics of bacterial Pth proteins.

Oral insulin-delivery system for diabetes mellitus.

Current insulin therapy for diabetes mellitus involves frequent dosing of subcutaneous injections, causing local discomfort, patient noncompliance, hypoglycemia and hyperinsulinemia, among others. While noninvasive therapy through oral delivery is greatly desired, there are challenges that include low bioavailability due to rapid enzymatic degradation in the stomach, inactivation and digestion by proteolytic enzymes in the intestinal lumen, poor permeability across the intestinal epithelium and poor stability. This article reviews patents that provide novel approaches for oral insulin delivery to the bloodstream through the GI tract.

Solution structure and dynamics of ADF from Toxoplasma gondii.

Toxoplasma gondii ADF (TgADF) belongs to a functional subtype characterized by strong G-actin sequestering activity and low F-actin severing activity. Among the characterized ADF/cofilin proteins, TgADF has the shortest length and is missing a C-terminal helix implicated in F-actin binding. In order to understand its characteristic properties, we have determined the solution structure of TgADF and studied its backbone dynamics from ¹5N-relaxation measurements. TgADF has conserved ADF/cofilin fold consisting of a central mixed ß-sheet comprised of six ß-strands that are partially surrounded by three α-helices and a C-terminal helical turn. The high G-actin sequestering activity of TgADF relies on highly structurally and dynamically optimized interactions between G-actin and G-actin binding surface of TgADF. The equilibrium dissociation constant for TgADF and rabbit muscle G-actin was 23.81 nM, as measured by ITC, which reflects very strong affinity of TgADF and G-actin interactions. The F-actin binding site of TgADF is partially formed, with a shortened F-loop that does not project out of the ellipsoid structure and a C-terminal helical turn in place of the C-terminal helix α4. Yet, it is more rigid than the F-actin binding site of Leishmania donovani cofilin. Experimental observations and structural features do not support the interaction of PIP2 with TgADF, and PIP2 does not affect the interaction of TgADF with G-actin. Overall, this study suggests that conformational flexibility of G-actin binding sites enhances the affinity of TgADF for G-actin, while conformational rigidity of F-actin binding sites of conventional ADF/cofilins is necessary for stable binding to F-actin.

Solution structure and dynamics of ADF/cofilin from Leishmania donovani.

Leishmania donovani ADF/cofilin (LdCof) is a novel member of ADF/cofilin family. LdCof depolymerizes, but does not co-sediment with, rabbit muscle actin filaments. Its F-actin depolymerizing activity is pH independent. Further, it possesses weak F-actin severing activity. In order to better understand its characteristic properties, we have determined the solution NMR structure of LdCof and have analyzed protein backbone dynamics from (15)N-relaxation measurements. The structure of LdCof possesses a conserved ADF/cofilin fold with a central mixed ß-sheet consisting of six ß-strands which is surrounded by five α-helices. LdCof structure has conserved G/F-actin binding site which includes the characteristic long kinked α-helix (α3). LdCof binds to rabbit muscle ADP-G-actin with 1:1 stoichiometry (K(d)∼0.2µM). The F-actin binding site is not well formed and analysis of (15)N-relaxation data shows that residues in the ß4-ß5 loop region and C-terminal are relatively flexible, which seems to be a determinant for the low F-actin severing activity of LdCof.

NMR assignment of actin depolymerizing and dynamics regulatory protein from Leishmania donovani.

Leishmania donovani cofilin displays low sequence similarity to other mammalian cofilins and also possesses characteristic activity of its own. Determination of its solution structure would facilitate understanding of the molecular mechanism of actin dynamics regulation in this disease causing pathogen.

Solution structure and dynamics of peptidyl-tRNA hydrolase from Mycobacterium tuberculosis H37Rv.

Eubacterial peptidyl-tRNA hydrolase is an essential enzyme that hydrolyzes peptidyl-tRNAs that are released into the cytoplasm because of premature termination of translation, expression of minigenes, and action of lincosamide and macrolide antibiotics. This averts the arrest of protein synthesis caused by depletion of free tRNA. Recently, we demonstrated that Mycobacterium tuberculosis peptidyl-tRNA hydrolase (MtPth) is present in the cytosol of mycobacterium and is capable of hydrolyzing peptidyl-tRNA. Here, we present the solution structure of MtPth, which is the first solution structure for this family of proteins. MtPth typically consists of seven-stranded mixed beta-sheet surrounded by six alpha-helices. The backbone dynamics for this enzyme were probed by measuring (15)N relaxation parameters and these were analyzed with model-free formalism and reduced spectral density mapping analysis. Overall, the protein molecule has tau(m) of 9.67+/-0.02 ns. The (15)N relaxation data analysis reveals that while majority of the protein backbone is rigid to motions, a short segment consisting of enzymatically critical residue H22, the loop-helix cover over the active site crevice, and the C-terminal helical hairpin exhibit motions on the milli-to microsecond timescale, all of which are linked to interaction with the substrate peptidyl-tRNA.