Microfluidic rotating-target device capable of three-degrees-of-freedom motion for efficient in situ serial synchrotron crystallography.

There is an increasing demand for simple and efficient sample delivery technology to match the rapid development of serial crystallography and its wide application in analyzing the structural dynamics of biological macromolecules. Here, a microfluidic rotating-target device is presented, capable of three-degrees-of-freedom motion, including two rotational degrees of freedom and one translational degree of freedom, for sample delivery. Lysozyme crystals were used as a test model with this device to collect serial synchrotron crystallography data and the device was found to be convenient and useful. This device enables in situ diffraction from crystals in a microfluidic channel without the need for crystal harvesting. The circular motion ensures that the delivery speed can be adjusted over a wide range, showing its good compatibility with different light sources. Moreover, the three-degrees-of-freedom motion guarantees the full utilization of crystals. Hence, sample consumption is greatly reduced, and only 0.1 mg of protein is consumed in collecting a complete dataset.

A novel sample delivery system based on circular motion for in situ serial synchrotron crystallography.

A sample delivery system is one of the key parts of serial crystallography. It is the main limiting factor affecting the application of serial crystallography. At present, although a variety of useful sample delivery systems have been developed for serial crystallography, it still remains the focus of the field to further improve the performance and efficiency of sample delivery. In existing sample delivery technologies, samples are usually delivered in linear motion. Here we show that the samples can also be delivered using circular motion, which is a novel motion mode never tested before. In this paper, we report a microfluidic rotating-target sample delivery device, which is characterized by the circular motion of the samples, and verify the performance of the device at a synchrotron radiation facility. The microfluidic rotating-target sample delivery device consists of two parts: a microfluidic sample plate and a motion control system. Sample delivery is realized by rotating the microfluidic sample plate containing in situ grown crystals. This device offers significant advantages, including a very wide adjustable range of delivery speed, low background noise, and low sample consumption. Using the microfluidic rotating-target device, we carried out in situ serial crystallography experiments with lysozyme and proteinase K as model samples at the Shanghai Synchrotron Radiation Facility, and performed structural determination based on the serial crystallographic data. The results showed that the designed device is fully compatible with the synchrotron radiation facility, and the structure determination of proteins is successful using the serial crystallographic data obtained with the device.

Structural characterization of a Δ3, Δ2-enoyl-CoA isomerase from Pseudomonas aeruginosa: implications for its involvement in unsaturated fatty acid metabolism.

Gene PA4980 from Pseudomonas aeruginosa encodes a putative enoyl-coenzyme A hydratase/isomerase that is associated with the function of the biofilm dispersion-inducing signal molecule cis-2-decenoic acid. To elucidate the role of PA4980 in cis-2-decenoic acid biosynthesis, we reported the crystal structure of its protein product at 2.39 Å. The structural analysis and substrate binding prediction suggest that it acts as a monofunctional enoyl-coenzyme A isomerase, implicating an alternative pathway of the cis-2-decenoic acid synthesis.

Author Correction: Structural and functional studies on Pseudomonas aeruginosa DspI: implications for its role in DSF biosynthesis.

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Structural and functional studies on Pseudomonas aeruginosa DspI: implications for its role in DSF biosynthesis.

DspI, a putative enoyl-coenzyme A (CoA) hydratase/isomerase, was proposed to be involved in the synthesis of cis-2-decenoic acid (CDA), a quorum sensing (QS) signal molecule in the pathogen Pseudomonas aeruginosa (P. aeruginosa). The present study provided a structural basis for the dehydration reaction mechanism of DspI during CDA synthesis. Structural analysis reveals that Glu126, Glu146, Cys127, Cys131 and Cys154 are important for its enzymatic function. Moreover, we show that the deletion of dspI results in a remarkable decreased in the pyoverdine production, flagella-dependent swarming motility, and biofilm dispersion as well as attenuated virulence in P. aeruginosa PA14. This study thus unravels the mechanism of DspI in diffusible signal factor (DSF) CDA biosynthesis, providing vital information for developing inhibitors that interfere with DSF associated pathogenicity in P. aeruginosa.

Heat-induced unfolding of apo-CP43 studied by fluorescence spectroscopy and CD spectroscopy.

CP43 is a chlorophyll-binding protein, which acts as a conduit for the excitation energy transfer. The thermal stability of apo-CP43 was studied by intrinsic fluorescence, exogenous ANS fluorescence, and circular dichroism spectroscopy. Under heat treatment, the structure of apo-CP43 changed and existed transition state occurred between 56 and 62 °C by the intrinsic, exogenous ANS fluorescence and the analysis of hydrophobicity. Besides, the isosbestic point of the sigmoidal curve was 58.10 ± 1.02 °C by calculating α-helix transition and the Tm was 56.45 ± 0.52 and 55.59 ± 0.68 °C by calculating the unfolded fraction of tryptophan and tyrosine fluorescence, respectively. During the process of unfolding, the hydrophobic structure of C-terminal segment firstly started to expose at 40 °C, and then the hydrophobic cluster adjacent to the N-terminal segment also gradually exposed to hydrophilic environment with increasing temperature. Our results indicated that heat treatment, especially above 40 °C, has an important impact on the structural stability of apo-CP43.