Existence of twisting in dislocation-free protein single crystals.

The growth of high-quality protein crystals is a prerequisite for the structure analysis of proteins by X-ray diffraction. However, dislocation-free perfect crystals such as silicon and diamond have been so far limited to only two kinds of protein crystals, such as glucose isomerase and ferritin crystals. It is expected that many other high-quality or dislocation-free protein crystals still exhibit some imperfection. The clarification of the cause of imperfection is essential for the improvement of crystallinity. Here, we explore twisting as a cause of the imperfection in high-quality protein crystals of hen egg-white lysozyme crystals with polymorphisms (different crystal forms) by digital X-ray topography with synchrotron radiation. The magnitude of the observed twisting is 10−6 to 10−5°/µm which is more than two orders smaller than 10−3 to 104°/µm in other twisted crystals owing to technique limitations with optical and electron microscopy. Twisting is clearly observed in small crystals or in the initial stage of crystal growth. It is uniformly relaxed with crystal growth and becomes smaller in larger crystals. Twisting is one of main residual defects in high-quality crystals and determines the crystal perfection. Furthermore, it is presumed that the handedness of twisting can be ascribed to the anisotropic interaction of chiral protein molecules associated with asymmetric units in the crystal forms. This mechanism of twisting may correspond to the geometric frustration proposed as a primary mechanism of twisting in molecular crystals. Our finding provides insights for the understanding of growth mechanism and the growth control of high-quality crystals.

Analysis of oscillatory rocking curve by dynamical diffraction in protein crystals.

High-quality protein crystals meant for structural analysis by X-ray diffraction have been grown by various methods. The observation of dynamical diffraction in protein crystals is an interesting topic because dynamical diffraction generally occurs in perfect crystals such as Si crystals. However, to our knowledge, there is no report yet on protein crystals showing clear dynamical diffraction. We wonder whether the perfection of protein crystals might still be low compared with that of high-quality Si crystals. Here, we present observations of the oscillatory profile of rocking curves for protein crystals such as glucose isomerase crystals. The oscillatory profiles are in good agreement with those predicted by the dynamical theory of diffraction. We demonstrate that dynamical diffraction occurs even in protein crystals. This suggests the possibility of the use of dynamical diffraction for the determination of the structure and charge density of proteins.

Unraveling Polymorphism and Twisting in Near-Perfect Protein Crystals.

Crystals ideally have well-formed shapes and periodic arrangements of constituent components, such as atoms and molecules. Twisting, an unconventional crystal morphology, presents itself as a puzzling and natural phenomenon. The coexistence of a continuous twisting structure and crystalline order poses a paradox. Numerous mechanisms to explain twisting have been proposed, and the elucidation of the underlying causes of spontaneous nonlong-range translational order twisting in crystals has been desired. Here, we demonstrate twisting and perfect crystals controlled by the crystal polymorphs of macromolecular crystals. We establish that the presence of either a perfectly periodic crystalline arrangement or twisting is linked to anisotropic interactions arising from salt bridges among protein molecules. Employing the dynamical theory of X-ray diffraction, we discern that twisting serves as an imperfection that cannot be attributed to conventional crystal defects within crystals. These insights suggest the origin of crystal twisting and methods for controlling crystal perfection.

Formation of Biogenic Manganese Oxide Nodules on Hyphae of a New Fungal Isolate of Periconia That Immobilizes Aqueous Copper.

Mn(II)-oxidizing microorganisms are considered to play significant roles in the natural geochemical cycles of Mn and other heavy metals because the insoluble biogenic Mn oxides (BMOs) that are produced by these microorganisms adsorb other dissolved heavy metals and immobilize them as precipitates. In the present study, a new Mn(II)-oxidizing fungal strain belonging to the ascomycete genus Periconia, a well-studied plant-associating fungal genus with Mn(II)-oxidizing activity that has not yet been exami-ned in detail, was isolated from natural groundwater outflow sediment. This isolate, named strain TS-2, was confirmed to oxidize dissolved Mn(II) and produce insoluble BMOs that formed characteristic, separately-located nodules on their hyphae while leaving major areas of the hyphae free from encrustation. These BMO nodules also adsorbed and immobilized dissolved Cu(II), a model analyte of heavy metals, as evidenced by elemental mapping ana-lyses of fungal hyphae-BMO assemblages using a scanning electron microscope with energy-dispersive X-ray spectroscopy (SEM-EDX). Analyses of functional genes within the whole genome of strain TS-2 further revealed the presence of multiple genes predicted to encode laccases/multicopper oxidases that were potentially responsible for Mn(II) oxidation by this strain. The formation of BMO nodules may have functioned to prevent the complete encrustation of fungal hyphae, thereby enabling the control of heavy metal concentrations in their local microenvironments while maintaining hyphal functionality. The present results will expand our knowledge of the physiological and morphological traits of Mn(II)-oxidizing Periconia, which may affect the natural cycle of heavy metals through their immobilization.

Unique Mechanical Properties of Gel-Incorporating Protein Crystals.

Crystalline materials that are grown in gel media exhibit reinforced mechanical characteristics. Studies on the mechanical properties of protein crystals are limited in numbers because of the difficulty in growing high-quality large crystals. This study shows the demonstration of the unique macroscopic mechanical properties by compression tests of large protein crystals grown in both solution and agarose gel. Particularly, the gel-incorporating protein crystals exhibit larger elastic limits and a higher fracture stress compared with the native protein crystals without gel. Conversely, the change in the Young's modulus corresponding to if the crystals incorporate the gel network is negligible. This suggests that gel networks affect only the fracture phenomenon. Thus, reinforced mechanical characteristics that cannot be obtained by the gel or the protein crystal alone can be developed. By combining the gel media and protein crystals, the gel-incorporating protein crystals show the potential to toughen without sacrificing other mechanical properties.

Diffusion Coefficient of Intracrystalline Water in Intrinsic Hen Egg-White Lysozyme Crystals Determined by Confocal Raman Spectroscopy.

Protein crystals composed of protein molecules are expected as a novel porous material. They have high porosity, and the knowledge of the diffusion of intracrystalline water is important. In this study, the diffusion coefficient of intracrystalline water in intrinsic hen egg-white lysozyme (HEWL) crystals was determined by a method that combines confocal Raman spectroscopy and air convection with controlled relative humidity. Similar to common porous materials, the drying process of the protein crystals includes three periods: constant-rate drying, falling-rate drying, and equilibrium state. During the falling-rate drying period, the drying rate depends on the diffusion of intracrystalline water in the protein crystal. The gradient of the water content was measured using confocal Raman spectroscopy. The diffusion coefficient of the intrinsic HEWL crystals was determined as 3.1 × 10-7 cm2/s with a water content of 36.3 vol %. The estimated diffusion coefficients of the intrinsic HEWL crystals without cross-linking were in close agreement with those of the cross-linked protein crystals. This study is timely as the knowledge of the intrinsic diffusion coefficient is useful not only for understanding the mechanism of hydration of proteins but also in practical applications such as porous materials, drug binding, and cryoprotectant soaks.

Radiation-induced defects in protein crystals observed by X-ray topography.

The characterization of crystal defects induced by irradiation, such as X-rays, charged particles and neutrons, is important for understanding radiation damage and the associated generation of defects. Radiation damage to protein crystals has been measured using various methods. Until now, these methods have focused on decreased diffraction intensity, volume expansion of unit cells and specific damage to side chains. Here, the direct observation of specific crystal defects, such as dislocations, induced by X-ray irradiation of protein crystals at room temperature is reported. Dislocations are induced even by low absorbed doses of X-ray irradiation. This study revealed that for the same total absorbed dose, the formation of defects appears to critically depend on the dose rate. The relationship between dislocation energy and dose energy was analyzed based on dislocation theory associated with elasticity theory for crystalline materials. This demonstration of the crystal defects induced by X-ray irradiation could help to understand the underlying mechanisms of X-ray-induced radiation damage.

Recrystallization solvent-dependent elastic/plastic flexibility of an n-dodecyl-substituted tetrachlorophthalimide.

A long alkyl-chained organic molecule, 4,5,6,7-tetrachloro-2-dodecylisoindoline-1,3-dione (1), was crystallized into needle-like crystals in dichloromethane (1DCM) or plate-like ones in tetrahydrofuran (1THF) depending on the recrystallisation solvent. X-ray crystallography analyses revealed the alkyl chains of the molecules, in which they were assembled differently, with the former responding flexibly bendable and elastic deformation, and the later being a permanent plastic one by external mechanical stress. The elastic modulus (E) and hardness (H) indicating both compliant and soft nature, reflecting their weak interaction in crystals, were quantified from the nano-indentation test.

Control of strain in subgrains of protein crystals by the introduction of grown-in dislocations.

It is important to reveal the exact cause of poor diffractivity in protein crystals in order to determine the accurate structure of protein molecules. It is shown that there is a large amount of local strain in subgrains of glucose isomerase crystals even though the overall crystal quality is rather high, as shown by clear equal-thickness fringes in X-ray topography. Thus, a large stress is exerted on the subgrains of protein crystals, which could significantly lower the resistance of the crystals to radiation damage. It is also demonstrated that this local strain can be reduced through the introduction of dislocations in the crystal. This suggests that the introduction of dislocations in protein crystals can be effective in enhancing the crystal quality of subgrains of protein crystals. By exploiting this effect, the radiation damage in subgrains could be decreased, leading to the collection of X-ray diffraction data sets with high diffractivity.

Evaluation of crystal quality of thin protein crystals based on the dynamical theory of X-ray diffraction.

Knowledge of X-ray diffraction in macromolecular crystals is important for not only structural analysis of proteins but also diffraction physics. Dynamical diffraction provides evidence of perfect crystals. Until now, clear dynamical diffraction in protein crystals has only been observed in glucose isomerase crystals. We wondered whether there were other protein crystals with high quality that exhibit dynamical diffraction. Here we report the observation of dynamical diffraction in thin ferritin crystals by rocking-curve measurement and imaging techniques such as X-ray topography. It is generally known that in the case of thin crystals it is difficult to distinguish whether dynamical diffraction occurs from only rocking-curve profiles. Therefore, our results clarified that dynamical diffraction occurs in thin protein crystals because fringe contrasts similar to Pendellösung fringes were clearly observed in the X-ray topographic images. For macromolecular crystallography, it is hard to obtain large crystals because they are difficult to crystallize. For thin crystals, dynamical diffraction can be demonstrated by analysis of the equal-thickness fringes observed by X-ray topography.

Ultrafast synthesis of carbon quantum dots from fenugreek seeds using microwave plasma enhanced decomposition: application of C-QDs to grow fluorescent protein crystals.

Herein, we present the rapid synthesis of mono-dispersed carbon quantum dots (C-QDs) via a single-step microwave plasma-enhanced decomposition (MPED) process. Highly-crystalline C-QDs were synthesized in a matter of 5 min using the fenugreek seeds as a sustainable carbon source. It is the first report, to the best of our knowledge, where C-QDs were synthesized using MPED via natural carbon precursor. Synthesis of C-QDs requires no external temperature other than hydrogen (H2) plasma. Plasma containing the high-energy electrons and activated hydrogen ions predominantly provide the required energy directly into the reaction volume, thus maximizing the atom economy. C-QDs shows excellent Photoluminescence (PL) activity along with the dual-mode of excitation-dependent PL emission (blue and redshift). We investigate the reason behind the dual-mode of excitation-dependent PL. To prove the efficacy of the MPED process, C-QDs were also derived from fenugreek seeds using the traditional synthesis process, highlighting their respective size-distribution, crystallinity, quantum yield, and PL. Notably, C-QDs synthesis via MPED was 97.2% faster than the traditional thermal decomposition process. To the best of our knowledge, the present methodology to synthesize C-QDs via natural source employing MPED is three times faster and far more energy-efficient than reported so far. Additionally, the application of C-QDs to produce the florescent lysozyme protein crystals "hybrid bio-nano crystals" is also discussed. Such a guest-host strategy can be exploited to develop diverse and complex "bio-nano systems". The florescent lysozyme protein crystals could provide a platform for the development of novel next-generation polychrome luminescent crystals.

Synthesis and characterization of Mono-disperse Carbon Quantum Dots from Fennel Seeds: Photoluminescence analysis using Machine Learning.

Herein, we present the synthesis of mono-dispersed C-QDs via single-step thermal decomposition process using the fennel seeds (Foeniculum vulgare). As synthesized C-QDs have excellent colloidal, photo-stability, environmental stability (pH) and do not require any additional surface passivation step to improve the fluorescence. The C-QDs show excellent PL activity and excitation-independent emission. Synthesis of excitation-independent C-QDs, to the best of our knowledge, using natural carbon source via pyrolysis process has never been achieved before. The effect of reaction time and temperature on pyrolysis provides insight into the synthesis of C-QDs. We used Machine-learning techniques (ML) such as PCA, MCR-ALS, and NMF-ARD-SO in order to provide a plausible explanation for the origin of the PL mechanism of as-synthesized C-QDs. ML techniques are capable of handling and analyzing the large PL data-set, and institutively recommend the best excitation wavelength for PL analysis. Mono-disperse C-QDs are highly desirable and have a range of potential applications in bio-sensing, cellular imaging, LED, solar cell, supercapacitor, printing, and sensors.

Correction of the equilibrium temperature caused by slight evaporation of water in protein crystal growth cells during long-term space experiments at International Space Station.

The normal growth rates of the {110} faces of tetragonal hen egg-white lysozyme crystals, R, were measured as a function of the supersaturation σ parameter using a reflection type interferometer under µG at the International Space Station (NanoStep Project). Since water slightly evaporated from in situ observation cells during a long-term space station experiment for several months, equilibrium temperature T(e) changed, and the actual σ, however, significantly increased mainly due to the increase in salt concentration C(s). To correct σ, the actual C(s) and protein concentration C(p), which correctly represent the measured T(e) value in space, were first calculated. Second, a new solubility curve with the corrected C(s) was plotted. Finally, the revised σ was obtained from the new solubility curve. This correction method successfully revealed that the 2.8% water was evaporated from the solution, leading to 2.8% increase in the C(s) and C(p) of the solution.

Growth rate measurements of lysozyme crystals under microgravity conditions by laser interferometry.

The growth rate vs. supersaturation of a lysozyme crystal was successfully measured in situ together with the crystal surface observation and the concentration measurements onboard the International Space Station. A Michelson-type interferometer and a Mach-Zehnder interferometer were, respectively, employed for real-time growth rate measurements and concentration field measurements. The hardware development, sample preparation, operation, and analysis methods are described.

Label-free biomedical imaging of hydrodynamics in single human cells.

Intracellular hydrodynamics is of considerable importance to regulate cellular functions. Using Raman microspectroscopy and imaging, we visualized the time-dependent changes of water concentration in single human acute promyelocytic leukemia HL-60 cells, and also showed the labelfree biomedical imaging of water molecules, lipids and proteins for analyzing molecular distribution. Taken together, our results demonstrated that we can distinguish between intracellular water and other major cell components, and that when the physiological status of cells changes with time, the Raman spectral intensities for intracellular water concentration also changes. Therefore, this biospectroscopic analysis of intracellular hydrodynamics can provide biomarkers to examine activities in dynamic living systems, and has significant potential in determining cell specificity for novel therapeutic approaches.