Conservation of Dark Recovery Kinetic Parameters and Structural Features in the Pseudomonadaceae “Short” Light, Oxygen, Voltage (LOV) Protein Family: Implications for the Design of LOV-Based Optogenetic Tools

In bacteria and fungi, various light, oxygen, voltage (LOV) sensory systems that lack a fused effector domain but instead contain only short N- and C-terminal extensions flanking the LOV core exist. In the prokaryotic kingdom, this so-called “short” LOV protein family represents the third largest LOV photoreceptor family. This observation prompted us to study their distribution and phylogeny as well as their photochemical and structural properties in more detail. We recently described the slow and fast reverting “short” LOV proteins PpSB1-LOV and PpSB2-LOV from <i>Pseudomonas putida</i> KT2440 whose adduct state lifetimes varied by 3 orders of magnitude [Jentzsch, K., Wirtz, A., Circolone, F., Drepper, T., Losi, A., Gärtner, W., Jaeger, K. E., and Krauss, U. (2009) <i>Biochemistry 48</i>, 10321–10333]. We now present evidence of the conservation of similar fast and slow-reverting “short” LOV proteins in different <i>Pseudomonas</i> species. Truncation studies conducted with PpSB1-LOV and PpSB2-LOV suggested that the short N- and C-terminal extensions outside of the LOV core domain are essential for the structural integrity and folding of the two proteins. While circular dichroism and solution nuclear magnetic resonance experiments verify that the two short C-terminal extensions of PpSB1-LOV and PpSB2-LOV form independently folding helical structures in solution, bioinformatic analyses imply the formation of coiled coils of the respective structural elements in the context of the dimeric full-length proteins. Given their prototypic architecture, conserved in most more complex LOV photoreceptor systems, “short” LOV proteins could represent ideally suited building blocks for the design of genetically encoded photoswitches (i.e., LOV-based optogenetic tools)

Aaptamine Derivatives from the Indonesian Sponge <i>Aaptos suberitoides</i>

Four new aaptamine derivatives (<b>1</b>–<b>4</b><b></b>) along with aaptamine (<b>5</b>) and three related compounds (<b>6</b>–<b>8</b>) were isolated from the ethanol extract of the sponge <i>Aaptos suberitoides</i> collected in Indonesia. The structures of the new compounds were unambiguously determined by one- and two-dimensional NMR and by HRESIMS measurements. Compounds <b>3</b>, <b>5</b>, and <b>6</b> showed cytotoxic activity against the murine lymphoma L5178Y cell line, with IC₅₀ values ranging from 0.9 to 8.3 μM

New Cytotoxic 1,2,4-Thiadiazole Alkaloids from the Ascidian <i>Polycarpa aurata</i>

Two new alkaloids, polycarpathiamines A and B (<b>1</b> and <b>2</b>), were isolated from the ascidian <i>Polycarpa aurata</i>. Their structures were unambiguously determined by 1D, 2D NMR, and HRESIMS measurements and further confirmed by comparison with a closely related analogue, 3-dimethylamino-5-benzoyl-1,2,4-thiadiazole (<b>4</b>), that was prepared by chemical synthesis. Compounds <b>1</b> and <b>2</b> both feature an uncommon 1,2,4-thiadiazole ring whose biosynthetic origin is proposed. Compound <b>1</b> showed significant cytotoxic activity against L5178Y murine lymphoma cells (IC₅₀ 0.41 μM)

Conformational Sampling of the Intrinsically Disordered C‑Terminal Tail of DERA Is Important for Enzyme Catalysis

2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes the reversible conversion of acetaldehyde and glyceraldehyde-3-phosphate into deoxyribose-5-phosphate. DERA is used as a biocatalyst for the synthesis of drugs such as statins and is a promising pharmaceutical target due to its involvement in nucleotide catabolism. Despite previous biochemical studies suggesting the catalytic importance of the C-terminal tyrosine residue found in several bacterial DERAs, the structural and functional basis of its participation in catalysis remains elusive because the electron density for the last eight to nine residues (i.e., the C-terminal tail) is absent in all available crystal structures. Using a combination of NMR spectroscopy and molecular dynamics simulations, we conclusively show that the rarely studied C-terminal tail of E. coli DERA (<i>ec</i>DERA) is intrinsically disordered and exists in equilibrium between open and catalytically relevant closed states, where the C-terminal tyrosine (Y259) enters the active site. Nuclear Overhauser effect distance restraints, obtained due to the presence of a substantial closed state population, were used to derive the solution-state structure of the <i>ec</i>DERA closed state. Real-time NMR hydrogen/deuterium exchange experiments reveal that Y259 is required for efficiency of the proton abstraction step of the catalytic reaction. Phosphate titration experiments show that, in addition to the phosphate-binding residues located near the active site, as observed in the available crystal structures, <i>ec</i>DERA contains previously unknown auxiliary phosphate-binding residues on the C-terminal tail which could facilitate in orienting Y259 in an optimal position for catalysis. Thus, we present significant insights into the structural and mechanistic importance of the <i>ec</i>DERA C-terminal tail and illustrate the role of conformational sampling in enzyme catalysis

Callyspongiolide, a Cytotoxic Macrolide from the Marine Sponge <i>Callyspongia</i> sp.

A novel macrolide, callyspongiolide, whose structure was determined by comprehensive analysis of the NMR and HRMS spectra, was isolated from the marine sponge <i>Callyspongia</i> sp. collected in Indonesia. The compound features a carbamate-substituted 14-membered macrocyclic lactone ring with a conjugated structurally unprecedented diene-ynic side chain terminating at a brominated benzene ring. Callyspongiolide showed strong cytotoxicity against human Jurkat J16 T and Ramos B lymphocytes

Size and Compositional Effects on Contrast Efficiency of Functionalized Superparamagnetic Nanoparticles at Ultralow and Ultrahigh Magnetic Fields

Magnetic resonance imaging (MRI) systems at ultralow and ultrahigh field have been developed for biomedical imaging. We synthesized and functionalized superparamagnetic (SPM) nanoparticles (NPs) and studied the spin–lattice (<i>T</i>₁) and spin–spin (<i>T</i>₂) relaxation time of protons in water surrounding these NPs at ultralow and at ultrahigh magnetic fields. In these fields, size and compositional effects of SPM NPs were observed contributing to the spin–lattice (<i>r</i>₁) and spin–spin (<i>r</i>₂) relaxivities as well as <i>r</i>₂/<i>r</i>₁ ratios. These results reveal the relationship between magnetic characteristics of SPM NPs and relaxation behavior of water proton at ultralow or ultrahigh field

Far-UV CD spectra of pEAβ(3–40) and Aβ(1–40).

<p>Peptides were dissolved in buffer (20–25% TFE in 50 mM potassium phosphate, pH 2.8). CD spectra were recorded at 20°C from 260 to 187 nm, accumulated 10 times and background corrected. (a) CD spectra of 25 μM pEAβ(3–40) in 25%, 23%, 22%, 21% and 20% TFE showed a shift from α-helical structure towards β-sheets with decreasing TFE concentrations. (b) The CD spectrum of 25 μM Aβ(1–40) indicated α-helices in 20% TFE while the spectrum of 25 μM pEAβ(3–40) in 20% TFE showed mainly β-sheet rich structures.</p

Chemical shift changes Δδ(¹H,¹⁵N) = ((10*Δδ1H^N)² + (Δδ¹⁵N)²)^1/2 between pEAβ(3–40) and Aβ(1–40) plotted as a function of the pEAβ(3–40) sequence.

<p>Pyroglutamate formation affects the N-terminal amino acids with decreasing effect towards the C-terminus and, interestingly, also the C-terminal V40.</p

Aggregation kinetics of pEAβ(3–40) and Aβ(1–40) in TFE.

<p>(a) 25 μM of monomerized pEAβ(3–40) were dissolved in buffer with various TFE contents (25%, 23%, 22%, 21% and 20% TFE in 50 mM potassium phosphate, pH 2.8) including 10 μM ThT. pEAβ(3–40) aggregated in 20% and 21% TFE but was significantly decreased in aqueous solution with higher TFE concentration. (b) 25 μM of monomerized pEAβ(3–40) and Aβ(1–40) were dissolved in buffer (20% TFE in 50 mM potassium phosphate, pH 2.8) including 10 μM ThT. An increase in ThT fluorescence was observed for pEAβ(3–40) but not for Aβ(1–40) within 72 h.</p

TEM image of pEAβ(3–40) in 50 mM potassium phosphate pH 2.8 containing 20% TFE.

<p>Monomerized pEAβ(3–40) (25 μM) was incubated for fibrillation at 20°C for five days and grids were prepared by negative staining. pEAβ(3–40) incubated in aqueous TFE solution formed large twisted fibrils up to several hundred nm in size which accumulate into large aggregates ranging from 1–5 μm in diameter.</p