Guidelines for de novo phasing using multiple small-wedge data collection.

Intense micro-focus X-ray beamlines available at synchrotron facilities have achieved high-quality data collection even from the microcrystals of membrane proteins. The automatic data collection system developed at SPring-8, named ZOO, has contributed to many structure determinations of membrane proteins using small-wedge synchrotron crystallography (SWSX) datasets. The `small-wedge' (5-20°) datasets are collected from multiple crystals and then merged to obtain the final structure factors. To our knowledge, no systematic investigation on the dose dependence of data accuracy has so far been reported for SWSX, which is between `serial crystallography' and `rotation crystallography'. Thus, herein, we investigated the optimal dose conditions for experimental phasing with SWSX. Phase determination using anomalous scattering signals was found to be more difficult at higher doses. Furthermore, merging more homogeneous datasets grouped by hierarchical clustering with controlled doses mildly reduced the negative factors in data collection, such as `lack of signal' and `radiation damage'. In turn, as more datasets were merged, more probable phases could be obtained across a wider range of doses. Therefore, our findings show that it is essential to choose a lower dose than 10 MGy for de novo structure determination by SWSX. In particular, data collection using a dose of 5 MGy proved to be optimal in balancing the amount of signal available while reducing the amount of damage as much as possible.

A photoenhanced oxidation of amino acids and the cross-linking of lysozyme mediated by tetrazolium salts.

Tetrazolium salts (TZs) are pervasively utilized as precursors in the dye industry, colorimetric probes in enzyme assays and for exploring nanomaterial toxicity, but its own toxicity is not investigated enough so far. Using femtosecond transient absorption spectroscopy, nanosecond pulse radiolysis (ns-PRL), western blotting and UV-vis absorption spectroscopy, here we characterized a neutral tetrazolinyl radical (with the same maximum absorption at 420 nm and different lifetimes of 5.0 and 9.0 µs for two selected TZs), the key intermediate of TZs reduction, and noticed TZs-formazan production under UV light irradiation accompanied by 41% increase in the cross-linking of lysozyme (Lyso, model protein) compared to TZs-free sample, which uncovered the photoenhanced oxidation of TZs towards Lyso. The ns-PRL in a reductive atmosphere simulated the electron/proton donors of amino acid residues in Lyso upon photoexcitation and revealed the reduction mechanism of TZs, as that first followed one-electron-transfer and then probably proton-coupled electron transfer. This is the first time to report on the photoenhanced oxidation mechanism of TZs, which would provide new insights into the applications of TZs in cell biology, "click" chemistry and nanotoxicology.

Effect of ultrasound-irradiation combined pretreatment on the foamability of liquid egg white.

This study aims to optimize the ultrasound-irradiation combined pretreatment conditions to enhance the liquid egg white (LEW) foamability and investigate the changing mechanism about the physical and structural properties of LEW during the processing. Results indicated that the foamability of the LEW was increased by ultrasound-irradiation combined pretreatment to the highest value of 92.6% (irradiation dose = 33 kGy, ultrasound time = 6 min, and ultrasound power = 300 W). Three significant proteins in LEW (ovalbumin, ovotransferrin, and lysozyme) were chosen to explore the change law on their physical and structural properties. Results indicated that ultrasound-irradiation combined pretreatment increased the solubility and reduced the pH and particle size of ovalbumin and lysozyme, thus enhancing the foamability of LEW. Furthermore, the fluorescence spectra changes implied the un-folding and destruction of ovalbumin, ovotransferrin, and lysozyme during the ultrasound-irradiation combined pretreatment. Moreover, circular dichroism spectroscopy analysis revealed that the pretreatment decreased α-helix and ß-sheet of the ovalbumin, ovotransferrin, and lysozyme, which bring out the improvement of LEW foamability. The present study indicated that ultrasound-irradiation combined pretreatment had the potential to be implemented to enhance the foamability of LEW.

Interactions of the cis and trans states of an azobenzene photoswitch with lysozyme induced by red and blue light.

Exploring the interaction between an azobenzene-based photoswitch and natural protein can help elucidate how the photo-control of an optical molecule participates in the transmission and delivery of proteins, as well as the effects of azo-switch trans and cis states on protein configurations. In this study, fluorescence analysis, circular dichroism spectroscopy, molecular docking, and molecular dynamics simulations were used to study the interaction among different configurations of tetra-ortho-methoxy substituted azobenzene di-maleimide (toM-ABDM), a red light-induced optical azo-switch, and lysozyme (LYZ). Results showed that toM-ABDM caused the static quenching of LYZ. The cis toM-ABDM had stronger binding affinity than trans toM-ABDM. The noncovalent interaction, hydrogen bonds and van der Waals forces, could not regulate the conformation of LYZ in photo-control. A binding model of toM-ABDM and LYZ in different forms induced by red and blue light was further established by computer simulation.

Ultraviolet irradiation of trypsin, lysozyme and ß-galactosidase: how does UVC affect these enzymes when used as a secondary barrier against adventitious agents?

Trypsin is one of the essential raw materials used in the manufacturing of biopharmaceutical products. As an animal derived product, it can potentially carry a serious risk of contamination with adventitious agents that can result in production shut down and lost product. To mitigate these risks, several methods are currently being used in the industry to remove contamination including physical and chemical methods. Ultraviolet-C (UVC) light is known to inactivate adventitious agents that are resistant to physical and chemical methods and could be a secondary barrier strategy. In this study, we investigated the effect of UVC irradiation on the activity and structure of trypsin. Extreme doses of UVC light were applied to trypsin using a collimated beam apparatus. The effect of UVC light on trypsin enzymatic activity was measured using a colorimetric activity assay and the effect on structure was analyzed by spectrophotometry, gel electrophoresis, and mass spectrometry. To broaden the scope, the effect of UVC light on the activity of two additional enzymes, lysozyme and ß-galactosidase, was also examined. At high doses of UVC light, changes to protein structure and protein fragmentation resulted in decreased trypsin activity. However, minimal damage was observed at doses applicable to inactivating adventitious agents, making UVC a feasible treatment for viral inactivation of trypsin products.

Effect of Technically Relevant X-Ray Doses on the Structure and Function of Alcohol Dehydrogenase and Hen Egg-White Lysozyme.

PURPOSE: The effect of different irradiation doses on the structure and activity of lyophilized powders of Hen Egg-White Lysozyme (HEWL) and alcohol dehydrogenase (ADH) was investigated using these substances as models for robust and sensitive proteins, respectively. Three doses were selected to cover the ranges of radio-sterilization (25kGy), treatment of blood products (25Gy) and annual background radiation dose (approximately 2mGy). The results offer an initial screening of different irradiation doses and support the development of X-ray imaging methods as non-destructive process analytical technology (PAT) tools for detecting the visible particulate matters in such products. METHODS: HEWL and ADH were exposed to X-rays in the solid state. The effect of irradiation was determined directly after irradiation and after storage. Structural changes and degradation were investigated using SAXS, SDS-PAGE and HPLC-MS. Protein functionality was assessed via activity assays. RESULTS: Lower irradiation doses of 25Gy and 2mGy had no significant impact on the structure and enzyme activity. The dose of 25kGy caused a significant decrease in the enzyme activity and structural changes immediately after irradiation of ADH and after storage of irradiated HEWL at -20°C. CONCLUSION: The results emphasize the importance of careful selection of radiation doses for development of X-ray imaging methods as PAT tools inspection of solid biopharmaceutical products.

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry.

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (∼100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day-1 . Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs.

Frequency Dependent Non- Thermal Effects of Oscillating Electric Fields in the Microwave Region on the Properties of a Solvated Lysozyme System: A Molecular Dynamics Study.

The use of microwaves in every day's applications raises issues regarding the non thermal biological effects of microwaves. In this work we employ molecular dynamics simulations to advance further the dielectric studies of protein solutions in the case of lysozyme, taking into consideration possible frequency dependent changes in the structural and dynamic properties of the system upon application of electric field in the microwave region. The obtained dielectric spectra are identical with those derived in our previous work using the Fröhlich-Kirkwood approach in the framework of the linear response theory. Noticeable structural changes in the protein have been observed only at frequencies near its absorption maximum. Concerning Cα position fluctuations, different frequencies affected different regions of the protein sequence. Furthermore, the influence of the field on the kinetics of protein-water as well as on the water-water hydrogen bonds in the first hydration shell has been studied; an extension of the Luzar-Chandler kinetic model was deemed necessary for a better fit of the applied field results and for the estimation of more accurate hydrogen bond lifetime values.

Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt.

X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltd in situ micro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ∼100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. High-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.

Hard X-ray-induced optical luminescence via biomolecule-directed metal clusters.

Here, we demonstrate that biomolecule-directed metal clusters are applicable in the study of hard X-ray excited optical luminescence, promising a new direction in the development of novel X-ray-activated imaging probes.

To scavenge or not to scavenge, that is STILL the question.

An extensive radiation chemistry literature would suggest that the addition of certain radical scavengers might mitigate the effects of radiation damage during protein crystallography diffraction data collection. However, attempts to demonstrate and quantify such an amelioration and its dose dependence have not yielded consistent results, either at room temperature (RT) or 100 K. Here the information thus far available is summarized and reasons for this lack of quantitative success are identified. Firstly, several different metrics have been used to monitor and quantify the rate of damage, and, as shown here, these can give results which are in conflict regarding scavenger efficacy. In addition, significant variation in results from data collected from crystals treated in nominally the same way has been observed. Secondly, typical crystallization conditions contain substantial concentrations of chemical species which already interact strongly with some of the X-ray-induced radicals that the added scavengers are intended to intercept. These interactions are probed here by the complementary technique of on-line microspectrophotometry carried out on solutions and crystals held both at 100 K and RT, the latter enabled by the use of a beamline-mounted humidifying device. With the help of computational chemistry, attempts are made to assign some of the characteristic spectral features observed experimentally. A further source of uncertainty undoubtedly lies in the challenge of reliably measuring the parameters necessary for the accurate calculation of the absorbed dose (e.g. crystal size and shape, beam profile) and its distribution within the volume of the crystal (an issue addressed in detail in another article in this issue). While microspectrophotometry reveals that the production of various species can be quenched by the addition of scavengers, it is less clear that this observation can be translated into a significant gain in crystal dose tolerance for macromolecular crystallographers.

Effect of mid-infrared free-electron laser irradiation on refolding of amyloid-like fibrils of lysozyme into native form.

Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610-1,630 cm(-1), characteristic of a ß-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a ß-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620 cm(-1) (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70-90 % of the native form. Both irradiations at 1,535 cm(-1)(amide II band) and 1,240 cm(-1) (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000 cm(-1) afforded only about 60 % recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.

High-resolution protein structure determination by serial femtosecond crystallography.

Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

Modeling the influence of histone proteins on the sensitivity of DNA to ionizing radiation.

The DNA-binding proteins that are present in chromatin significantly affect the sensitivity of cells to ionizing radiation and to the radiation chemistry of DNA damage. The interaction between protein and DNA modifies the radiation chemistry of the latter. To model these processes, we have examined the effects of ionizing radiation on the minichromosome form of SV40 (which contains histone proteins arranged in nucleosomes) and also on plasmid DNA in the presence of lysozyme. Although high concentrations of lysozyme can bring about an extensive radioprotection by condensation of the plasmid, at lower levels it still produces significant radioprotective effects under conditions where this associative phase separation does not take place. The presence of histones or of lysozyme decreases the yield of modified guanines produced by ionizing radiation. Comparison with previous observations made with oligopeptides suggests that the mechanism responsible is electron donation to guanyl radicals in the DNA by tryptophan and tyrosine residues in the proteins. However, there was no evidence for DNA-protein crosslink formation.

The protective effect of salicylic acid on lysozyme against riboflavin-mediated photooxidation.

As a metabolite of aspirin in vivo, salicylic acid was proved to protect lysozyme from riboflavin-mediated photooxidation in this study. The antioxidative properties of salicylic acid were further studied by using time-resolved laser flash photolysis of 355 nm. It can quench the triplet state of riboflavin via electron transfer from salicylic acid to the triplet state of riboflavin with a reaction constant of 2.25×10(9) M(-1) s(-1). Mechanism of antioxidant activities of salicylic acid on lysozyme oxidation was discussed. Salicylic acid can serve as a potential antioxidant to quench the triplet state of riboflavin and reduce oxidative pressure.

Small-angle solution scattering using the mixed-mode pixel array detector.

Solution small-angle X-ray scattering (SAXS) measurements were obtained using a 128 × 128 pixel X-ray mixed-mode pixel array detector (MMPAD) with an 860 µs readout time. The MMPAD offers advantages for SAXS experiments: a pixel full-well of >2 × 10(7) 10 keV X-rays, a maximum flux rate of 10(8) X-rays pixel(-1) s(-1), and a sub-pixel point-spread function. Data from the MMPAD were quantitatively compared with data from a charge-coupled device (CCD) fiber-optically coupled to a phosphor screen. MMPAD solution SAXS data from lysozyme solutions were of equal or better quality than data captured by the CCD. The read-noise (normalized by pixel area) of the MMPAD was less than that of the CCD by an average factor of 3.0. Short sample-to-detector distances were required owing to the small MMPAD area (19.2 mm × 19.2 mm), and were revealed to be advantageous with respect to detector read-noise. As predicted by the Shannon sampling theory and confirmed by the acquisition of lysozyme solution SAXS curves, the MMPAD at short distances is capable of sufficiently sampling a solution SAXS curve for protein shape analysis. The readout speed of the MMPAD was demonstrated by continuously monitoring lysozyme sample evolution as radiation damage accumulated. These experiments prove that a small suitably configured MMPAD is appropriate for time-resolved solution scattering measurements.

Integrity of proteins in human saliva after sterilization by gamma irradiation.

Microbial contamination of whole human saliva is unwanted for certain in vitro applications, e.g., when utilizing it as a growth substratum for biofilm experiments. The aim of this investigation was to test gamma irradiation for its suitability to sterilize saliva and to investigate the treatment's influence on the composition and integrity of salivary proteins in comparison to filter sterilization. For inhibition of bacterial growth by gamma irradiation, a sterility assurance level of 10(-6) was determined to be reached at a dose of 3.5 kGy. At this dose, the integrity of proteins, as measured by fluorescence, circular dichroism, and gel electrophoretic banding pattern, and the enzymatic activities of salivary amylase and lysozyme were virtually unchanged. Filtration reduced the total protein concentration to about half of its original value and decreased lysozyme activity to about 10%. It can be concluded that irradiation is suitable for sterilizing whole saliva in its native form.

Effect of flash-heat treatment on antimicrobial activity of breastmilk.

BACKGROUND AND OBJECTIVES: The World Health Organization recommends human immunodeficiency virus (HIV)-positive mothers in resource-poor regions heat-treat expressed breastmilk during periods of increased maternal-to-child transmission risk. Flash-heat, a "low tech" pasteurization method, inactivates HIV, but effects on milk protein bioactivity are unknown. The objectives were to measure flash-heat's effect on antimicrobial properties of lactoferrin, lysozyme, and whole milk and on the digestive resistance of lactoferrin and lysozyme. METHODS: Flash-heated and unheated breastmilk aliquots from HIV-positive mothers in South Africa were "spiked" with Staphylococcus aureus and Escherichia coli and then cultured for 0, 3, and 6 hours. Lysozyme and lactoferrin activities were determined by lysis of Micrococcus luteus cells and inhibition of enteropathogenic E. coli, respectively, measured spectrophotometrically. Percentages of proteins surviving in vitro digestion, lactoferrin and lysozyme activity, and bacteriostatic activity of whole milk in heated versus unheated samples were compared. RESULTS: There was no difference in rate of growth of E. coli or S. aureus in flash-heated versus unheated whole milk (p = 0.61 and p = 0.96, respectively). Mean (95% confidence interval) antibacterial activity of lactoferrin was diminished 11.1% (7.8%, 14.3%) and that of lysozyme by up to 56.6% (47.1%, 64.5%) by flash-heat. Digestion of lysozyme was unaffected (p = 0.12), but 25.4% less lactoferrin survived digestion (p < 0.0001). CONCLUSIONS: In summary, flash-heat resulted in minimally decreased lactoferrin and moderately decreased lysozyme bioactivity, but bacteriostatic activity of whole milk against representative bacteria was unaffected. This suggests flash-heated breastmilk likely has a similar profile of resistance to bacterial contamination as that of unheated milk. Clinical significance of the decreased bioactivity should be tested in clinical trials.

Raman-assisted crystallography suggests a mechanism of X-ray-induced disulfide radical formation and reparation.

X-ray-induced chemistry modifies biological macromolecules structurally and functionally, even at cryotemperatures. The mechanisms of x-radiation damage in colored or redox proteins have often been investigated by combining X-ray crystallography with in crystallo-ultraviolet-visible spectroscopy. Here, we used Raman microspectrophotometry to follow the onset of damage in crystalline lysozyme, notably that of disulfide bond breakage. The dose-dependent Raman spectra are consistent with a kinetic model for the rupture of disulfide bonds suggesting the rapid build up of an anionic radical intermediate. This intermediate may either revert back to the oxidized state or evolve toward protonated radical species or cleaved products. The data strongly suggest that back conversion of the anionic radical is significantly accelerated by X-rays, revealing an X-ray-induced "repair" mechanism. The possibility of X-ray-induced chemical repair is an important feature to take into account when assessing radiation damage in macromolecules.

A requirement for bid for induction of apoptosis by photodynamic therapy with a lysosome- but not a mitochondrion-targeted photosensitizer.

Photodynamic therapy (PDT) with lysosome-targeted photosensitizers induces the intrinsic pathway of apoptosis via the cleavage and activation of the BH3-only protein Bid by proteolytic enzymes released from photodisrupted lysosomes. To investigate the role of Bid in apoptosis induction and the role of damaged lysosomes on cell killing by lysosome-targeted PDT, we compared the responses of wild type and Bid-knock-out murine embryonic fibroblasts toward a mitochondrion/endoplasmic reticulum-binding photosensitizer, Pc 4, and a lysosome-targeted sensitizer, Pc 181. Whereas apoptosis and overall cell killing were induced equally well by Pc 4-PDT in both cell lines, Bid(-/-) cells were relatively resistant to induction of apoptosis and to overall killing following PDT with Pc 181, particularly at low PDT doses. Thus, Bid is critical for the induction of apoptosis caused by PDT with the lysosome-specific sensitizers, but dispensable for PDT targeted to other membranes.