Picosecond time-resolved X-ray crystallography: probing protein function in real time

A detailed mechanistic understanding of how a protein functions requires knowledge not only of its static structure, but also how its conformation evolves as it executes its function. The recent development of picosecond time-resolved X-ray crystallography has allowed us to visualize in real time and with atomic detail the conformational evolution of a protein. Here, we report the photolysis-induced structural evolution of wild-type and L29F myoglobin over times ranging from 100 ps to 3 micros. The sub-ns structural rearrangements that accompany ligand dissociation in wild-type and the mutant form differ dramatically, and lead to vastly different ligand migration dynamics. The correlated protein displacements provide a structural explanation for the kinetic differences. Our observation of functionally important protein motion on the sub-ns time scale was made possible by the 150-ps time resolution of the measurement, and demonstrates that picosecond dynamics are relevant to protein function. To visualize subtle structural changes without modeling, we developed a novel method for rendering time-resolved electron density that depicts motion as a color gradient across the atom or group of atoms that move. A sequence of these time-resolved images have been stitched together into a movie, which allows one to literally "watch" the protein as it executes its function.     

Crystallization and preliminary X-ray diffraction analysis of staphylococcal enterotoxin type C.

The Type C staphylococcal enterotoxin produced by Staphylococcus aureus strain FRI-909 has been crystallized using a combination of two precipitants, ammonium sulfate and polyethylene glycol 400, with the addition of small amounts of detergent. Two related crystal forms have been obtained, one triclinic, and one tetragonal, both with one toxin molecule per asymmetric unit. These crystals are stable for at least 75 hr in the X-ray beam and diffract to at least 2.2 and 2.6 A, respectively. The triclinic crystals have unit cell parameters a = 38.5 A, b = 43.7 A, c = 36.9 A, and interaxial angles alpha = 99.9 degrees, beta = 95.8 degrees, and gamma = 98.5 degrees. The tetragonal crystals are of space group P4(1)22 with unit cell parameters a = 43.4 A and c = 278.0 A.     

Synthesis and biological evaluation of (+)-neopeltolide analogues: Importance of the oxazole-containing side chain

We describe the synthesis and biological evaluation of (+)-neopeltolide analogues with structural modifications in the oxazole-containing side chain. Evaluation of the antiproliferative activity of newly synthesized analogues against A549 human lung adenocarcinoma cells and PANC-1 human pancreatic carcinoma cells have shown that the C19–C20 and C26–C27 double bonds within the oxazole-containing side chain and the terminal methyl carbamate group are essential for potent activity.     

Synthesis and biological evaluation of isoprenylated coumarins as potential anti-pancreatic cancer agents

A series of isoprenylated coumarins has been designed, synthesized, and evaluated against human pancreatic adenocarcinoma cell line PANC-1 under nutrient-rich and nutrient-deprived conditions. The compounds described investigate the effect of isoprenyl chain length and positioning on cell growth inhibition. The majority of these compounds displayed cytotoxicity against PANC-1 cells selectively in the absence of essential amino acids, glucose, and serum, and showed no cytotoxicity under nutrient-rich conditions. In this study, compound 6 exhibited the highest cytotoxic activity with an LC₅₀ value of 4 μM and induced apoptosis-like morphological changes in PANC-1 cells after a 24-h incubation. The evaluated structure–activity relationships show that substitution at the 6-position and the presence of a farnesyl isoprenyl tail are important structural features for enhanced preferential cytotoxicity. These findings provide important information to designing other structural analogues for potential application as novel pancreatic antitumor agents.     

Design,synthesis, and biological evaluation of 1,2,4-triazole bearing 5-substituted biphenyl-2-sulfonamide derivatives as potential antihypertensive candidates

A series of novel 1,2,4-triazole bearing 5-substituted biphenyl-2-sulfonamide derivatives were designed and synthesized to develop new angiotensin II subtype 2 (AT₂) receptor agonists as novel antihypertensive candidates. It was found that 14f (IC₅₀ = 0.4 nM) and 15e (IC₅₀ = 5.0 nM) displayed potent AT₂ receptor affinity and selectivity in binding assays. Biological evaluation in vivo suggested that 14f is obviously superior to that of reference drug losartan in RHRs, and meanwhile, 14f has no significant impact on heart rate. The interesting activities of these compounds may make them promising candidates as antihypertensive agents.     

Synthesis and biological evaluation of novel bis-aromatic amides as novel PTP1B inhibitors

A series of bis-aromatic amides was designed, synthesized, and evaluated as a new class of inhibitors with IC₅₀ values in the micromolar range against protein tyrosine phosphatase 1B (PTP1B). Among them, compound 15 displayed an IC₅₀ value of 2.34 ± 0.08 μM with 5-fold preference over TCPTP. More importantly, the treatment of CHO/HIR cells with compound 15 resulted in increased phosphorylation of insulin receptor (IR), which suggested extensive cellular activity of compound 15. These results provided novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.     

X-ray diffraction studies of lipid phase transitions in hydrated mixtures of cholesterol and diacylphosphatidylcholines and their relevance to the structure of biological membranes

Previous X-ray diffraction data on the effects of temperature on hydrated cholesterol/dimyristoylphosphatidylcholine mixtures have been confirmed and equivalent new data on cholesterol/stearolyoleoylphosphatidylcholine obtained. Molecular interpretations are discussed and related to previous studies of cholesterol/dioleoylphosphatidylcholine and of cholesterol-rich biological membranes.     

Synthesis and biological evaluation of Oblongifolin C derivatives as c-Met inhibitors

Oblongifolin C, one of the polyprenylated benzoylphloroglucinol natural products (PPAPs) isolated from the fruits of Garcinia yunnanensis Hu, was recently discovered to be a potent anti-tumor agent. A collection of 12 derivatives with modifications on the benzophenone moieties were synthesized and tested for c-Met kinase inhibition and cytotoxicity against the HepG2, Miapaca-2, HCC827, Hela, A549, AGS, and HT-29 cell lines in vitro. An oxidized derivative, 10, was found to possess strong inhibition and anti-migration properties in the HCC827 cell line and serves as a potential lead compound for the development of new anticancer drugs. In addition, structure–activity relationships (SAR) were also evaluated to provide key information for future anticancer drug development.     

Synthesis and biological evaluation of enantiomerically pure glyceric acid derivatives as LpxC inhibitors

Inhibitors of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represent a promising class of novel antibiotics, selectively combating Gram-negative bacteria. In order to elucidate the impact of the hydroxymethyl groups of diol (S,S)-4 on the inhibitory activity against LpxC, glyceric acid ethers (R)-7a, (S)-7a, (R)-7b, and (S)-7b, lacking the hydroxymethyl group in benzylic position, were synthesized. The compounds were obtained in enantiomerically pure form by a chiral pool synthesis and a lipase-catalyzed enantioselective desymmetrization, respectively. The enantiomeric hydroxamic acids (R)-7b (K_i = 230 nM) and (S)-7b (K_i = 390 nM) show promising enzyme inhibition. However, their inhibitory activities do not substantially differ from each other leading to a low eudismic ratio. Generally, the synthesized glyceric acid derivatives 7 show antibacterial activities against two Escherichia coli strains exceeding the ones of their respective regioisomes 6.     

Crystallization and preliminary X-ray diffraction studies of HutP protein: an RNA-binding protein that regulates the transcription of hut operon in Bacillus subtilis

HutP is an RNA-binding protein and regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on hut mRNA. HutP and its mutant, which has increased affinity for the regulatory sequences, were purified and crystallized by the hanging-drop vapor diffusion method. The space group was P2(1)3 with unit cell dimensions a=b=c=95.6A for HutP and a=b=c=96.8A for the mutant. Complete data sets of 3.0-A resolution for wild-type HutP and of 2.70-A resolution for the mutant HutP were collected.     

The synthesis of 4,7-disubstituted-2H-benzo[b][1,4]-oxazin-3(4H)-ones using Smiles rearrangement and their in vitro evaluation as platelet aggregation inhibitors

A series of novel benzo[b][1,4]oxazin-3(4H)-one derivatives were synthesized as platelet aggregation inhibitors for structure–activity relationships (SAR) analysis. The synthetic pattern, involved Smiles rearrangement for the preparation of benzoxazine, was proven to be more efficient than the conventional methods. Biological evaluation demonstrated that among all the synthesized compounds, compound 9u (IC₅₀ = 9.20 μM) exhibited the most potent inhibition activity compared with aspirin, the positive control (IC₅₀ = 7.07 μM). Molecular docking revealed that these set of compounds could be the GPIIb/IIIa antagonist for that they could be situated in the binding site of GPIIb/IIIa receptor quite well.     

Nucleotide binding to the G12V-mutant of Cdc42 investigated by X-ray diffraction and fluorescence spectroscopy: two different nucleotide states in one crystal

The 2.5 A crystal structure of the full length human placental isoform of the Gly12 to Val mutant Cdc42 protein (Cdc42(G12V)) bound to both GDP/Mg2+ and GDPNH2 (guanosine-5'-diphospho-beta-amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg2+, while the other has bound GDPNH2 without a Mg2+ ion. Crystallization of the protein was induced via hydrolysis of the Cdc42 x GppNHp complex by the presence of contaminating alkaline phosphatase activity in combination with the crystallization conditions. This prompted us to compare the binding characteristics of GDPNH2 vs. GDP. The amino group of GDPNH2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg2+ ion, and apparently also increases the conformational flexibility of the protein as seen in the crystal. Both the switch I and switch II regions are visible in the electron density of the GDP-bound molecule, but not in the molecule bound to GDPNH2. The C-terminus containing the CaaX-motif is partly ordered in both molecules due to an intramolecular disulfide bond formed between Cys105/Cys188 and Cys305/Cys388, respectively.     

Design,synthesis and biological evaluation of novel human monoamine oxidase B inhibitors based on a fragment in an X-ray crystal structure

Herein we report our efforts of developing reversible selective hMAO-B inhibitors based on isatin, a fragment in an X-ray crystal structure. Five different scaffolds were designed and many compounds were synthesized. Among them, compound A3 demonstrated very high potency and isoform selectivity against hMAO-B, 11 and 13 times more potent (IC₅₀?=?3?nM) and 23.64 and 6.8 times more selective than the marked drugs, selegiline and safinamide. However, the endeavors to modify the polar 3-one group of isatin, that is in a hydrophobic environment in the binding site of hMAO-B, to small nonpolar hydrophobic groups did not bring about improved hMAO-B inhibitors, which may challenge our understanding of molecular interactions and molecular recognition in biological systems.     

Cross-correlation analysis of invasive mango mealybug and its associated natural enemies in relation to meteorological factors: implications for biological control

Damage caused by invasive downey snow line mealybug, Rastrococcus iceryoides Green (Hemiptera: Pseudococcidae) has been reported to vary between 30% to complete crop loss where no control measure is applied. The current studies seek to determine factors influencing R. iceryoides population outbreaks, parasitoid – host and predator–prey relationships as well as predict optimal management strategies through weather modelling over a period of 28 months from 2008 to 2010 in Tanzania. The highest incidence of R. iceryoides was recorded during the dry season coinciding with the major mango fruiting season. The relationship between R. iceryoides and the parasitoid was positive but not significant, which implies the influence on outbreaks was negligible probably due to low percent parasitism (<12%). However, the predator abundance was directly and significantly related to that of R. iceryoides. Average temperature, average relative humidity, rainfall, and R. iceryoides abundance were autocorrelated to each other. Cross-correlation coef?cients vary significantly from ?0.286 to 0.589 for the pair-variable between R. iceryoides, temperature, relative humidity, rainfall, parasitism and predators. Our findings showed that temperature was the key climatic variable that significantly influenced R. iceryoides outbreaks while rainfall was significantly negatively associated with the pest. Time series analyses show R. iceryoides population increased 4 months after an increase in average temperature in all the sites, 11 months after rainfall and 11 months after relative humidity in Kibaha and Dar es Salaam, respectively. Our findings revealed that R. iceryoides is an excellent target for classical biological control. Thus, the importation of promising co-evolved parasitoid specific to R. iceryoides from the aboriginal home is crucial in formulating an efficient and sustainable management approaches against the invasive mealybug pest in mango agro-ecosystems.     

Alterations of pigment composition and their interactions in response to different light conditions in the diatom Chaetoceros gracilis probed by time-resolved fluorescence spectroscopy

Maintenance of energy balance under changeable light conditions is an essential function of photosynthetic organisms to achieve efficient photochemical reactions. Among the photosynthetic organisms, diatoms possess light-harvesting fucoxanthin chlorophyll (Chl) a/c-binding protein (FCP) as peripheral antennas. However, how diatoms regulate excitation-energy distribution between FCP and the two photosystem cores during light adaptation is poorly understood. In this study, we examined spectroscopic properties of a marine diatom Chaetoceros gracilis adapted in the dark and at photosynthetic photon flux density at 30 and 300?μmol?photons?m^?2?s^?1. Absorption spectra at 77?K showed significant changes in the Soret region, and 77-K steady-state fluorescence spectra showed significant differences in the spectral shape and relative fluorescence intensity originating from both PSII and PSI, among the cells grown under different light conditions. These results suggest alterations of pigment composition and their interactions under the different light conditions. These alterations affected the excitation-energy dynamics monitored by picosecond time-resolved fluorescence analyses at 77?K significantly. The contributions of Chls having lower energy levels than the reaction center Chls in the two photosystems to the energy dynamics were clearly identified in the three cells but with presumably different roles. These findings provide insights into the regulatory mechanism of excitation-energy balance in diatoms under various light conditions.     

Performance of a time-resolved IR facility for assessment of protonation states and polarity changes in carboxyl groups in a large membrane protein,mammalian cytochrome c oxidase,under turnover conditions in a sub-millisecond time resolution

Time-resolved IR analyses for the protonation and polarity changes of carboxyl groups involved in proton pump enzymes under turnover conditions are indispensable for elucidation of their proton-pump mechanisms. We have developed a new time-resolved infrared facility by introducing a flow system for transferring highly concentrated and thus viscous protein solution to a thin (50?μm) flow cell equipped in a highly sensitive IR spectrometer constructed with the femtosecond mid-IR pulse laser with spectral width of 350?cm^?1 as an IR white light source equipped with multi-channel MCT detector. This facility equipped with O₂ supply system enables the sub-millisecond time scale infrared measurements of the O₂ reduction coupled with proton pumping by bovine cytochrome c oxidase (CcO) initiated by CO-flash photolysis in the COOH (1725–1770?cm^?1) region with the accuracy of about 10?μO.D. under the background O.D. of 1. The facility identifies a band intensity change at ~1744?cm^?1 assignable to protonation of a carboxyl group coupled with a single electron transfer to the O₂ reduction center within 1?ms after initiation of the reaction. The results suggest that the facility detects protonation of a single carboxyl group included in large proteins like as CcO (210?kDa). The present facility sensitively identifies also polarity changes in COOH group by detecting shifts of the bands near 1750?cm^?1 and 1760?cm^?1, without significant intensity changes. These findings show the performance of this facility sufficiently high for providing crucial information for understanding the proton transferring mechanisms of protein carboxyl groups.