Native Crystals Research Articles

Ym1 protein crystals promote type 2 immunity

Spontaneous protein crystallization is a rare event, yet protein crystals are frequently found in eosinophil-rich inflammation. In humans, Charcot-Leyden crystals (CLCs) are made from galectin-10 (Gal10) protein, an abundant protein in eosinophils. Although mice do not encode Gal10 in their genome, they do form pseudo-CLCs, made from the chitinase-like proteins Ym1 and/or Ym2, encoded by Chil3 and Chil4 and made by myeloid and epithelial cells respectively. Here, we investigated the biological effects of pseudo-CLCs since their function is currently unknown. We produced recombinant Ym1 crystals which were shown to have identical crystal packing and structure by X-ray crystallography as in vivo native crystals derived from murine lung. When administered to the airways of mice, crystalline but not soluble Ym1 stimulated innate and adaptive immunity and acted as a type 2 immune adjuvant for eosinophilic inflammation via triggering of dendritic cells (DCs). Murine Ym1 protein crystals found at sites of eosinophilic inflammation reinforce type 2 immunity and could serve as a surrogate model for studying the biology of human CLCs.

Ym1 protein crystals promote type 2 immunity.

Spontaneous protein crystallization is a rare event, yet protein crystals are frequently found in eosinophil-rich inflammation. In humans, Charcot-Leyden crystals (CLCs) are made from galectin-10 (Gal10) protein, an abundant protein in eosinophils. Although mice do not encode Gal10 in their genome, they do form pseudo-CLCs, made from the chitinase-like proteins Ym1 and/or Ym2, encoded by Chil3 and Chil4 and made by myeloid and epithelial cells respectively. Here, we investigated the biological effects of pseudo-CLCs since their function is currently unknown. We produced recombinant Ym1 crystals which were shown to have identical crystal packing and structure by X-ray crystallography as in vivo native crystals derived from murine lung. When administered to the airways of mice, crystalline but not soluble Ym1 stimulated innate and adaptive immunity and acted as a type 2 immune adjuvant for eosinophilic inflammation via triggering of dendritic cells (DCs). Murine Ym1 protein crystals found at sites of eosinophilic inflammation reinforce type 2 immunity and could serve as a surrogate model for studying the biology of human CLCs.

Starch ordered structures control starch reassembly behaviors during heat–moisture treatment for modulating its digestibility

This study investigated the effect of starch crystallinity on starch reassembly behaviors during the heat–moisture treatment (HMT) using starches with A-type crystal content of 0.00%–19.03%. The results showed that HMT reduced the native starch crystal content from 19.03% to 15.02% and increased starch thermostability, leading to a decrease in rapidly digestible starch (RDS) content from 86.91% to 76.71%. Moreover, starches containing a crystal content of 2.51%–8.11% exhibited significant reassembly during the HMT, and the resulting modified starches had more crystals and less RDS of 63.43%–69.31%. Interestingly, starches lacked A-type crystals but had some helical structures exhibiting A-type crystalline structures and lower digestibility after HMT. These findings verified that starch could significantly reassemble to form crystalline structures during the HMT. Controlling the crystal content of starch granules, particularly between 2.51% and 8.11%, was a promising approach for promoting starch reassembly during HMT and reducing starch digestibility.

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.

Crystal structures of the molecular class A β-lactamase TEM-171 and its complexes with tazobactam

The resistance of bacteria to β-lactam antibiotics is primarily caused by the production of β-lactamases. Here, novel crystal structures of the native β-lactamase TEM-171 and two complexes with the widely used inhibitor tazobactam are presented, alongside complementary data from UV spectroscopy and fluorescence quenching. The six chemically identical β-lactamase molecules in the crystallographic asymmetric unit displayed different degrees of disorder. The tazobactam intermediate was covalently bound to the catalytic Ser70 in the trans-enamine configuration. While the conformation of tazobactam in the first complex resembled that in published β-lactamase-tazobactam structures, in the second complex, which was obtained after longer soaking of the native crystals in the inhibitor solution, a new and previously unreported tazobactam conformation was observed. It is proposed that the two complexes correspond to different stages along the deacylation path of the acyl-enzyme intermediate. The results provide a novel structural basis for the rational design of new β-lactamase inhibitors.

Structure-Based Modeling of the Mechanical Behavior of Cross-Linked Enzyme Crystals

Because of their high volumetric catalytic activity, in addition to their high chemical and thermal resistances, enzymes in the form of protein crystals are an excellent choice for application as immobilized biocatalysts. However, mechanical stability is a requirement for the processability of immobilisates, in addition to the protein crystals retaining their enzymatic activity, and this is closely related to the crystal structure. In this study, the influence of protein engineering on the mechanical stability of cross-linked enzyme crystals (CLECs) was investigated using a genetically modified model protein in which additionally cysteines were introduced on the protein surface for targeted cross-linking. The results showed that the mechanical stability of crystals of the mutant proteins in the native form was decreased compared to native wild-type crystals. However, specific cross-linking of the introduced amino acid residues in the mutant proteins resulted in their increased mechanical stability compared to wild-type CLECs. In order to determine the correlation between the crystal structure and the resulting mechanical properties of CLECs to enable targeted cross-linking, a previously developed model was revised and then used for the two model proteins. This model can explain the mechanically investigated relationships, such as the anisotropic crystal behavior and the influence of a linker or mutation on the micromechanical properties and, hence, can be helpful for the tailor-made production of CLECs.

Control of the supercrystallinity, nanocrystallinity, morphology and magnetism of cobalt nanoparticle assemblies. Towards novel colloidal crystals with high magnetic anisotropy

Starting from a colloidal solution of dodecanoic acid-coated Co nanoparticles (NP) of 8.1 nm in size and of low anisotropy and using a solvent-mediated ligand-ligand interaction strategy, we obtain either colloidal crystals or supercrystalline films. The comparison between the structural and magnetic properties of these two different architectures of supercrystals evidences improved magnetic properties for the colloidal crystals. This is interpreted as a result of both an increase in the mesoscopic coherence length and a decrease in the interparticle distance. In this work we extend this comparative study between the two types of supercrystals after annealing treatment. A common batch of NP is used for the native and the annealed samples in order to optimize the accuracy of the comparison. A complete set of structural characterizations and magnetic measurements shows that upon annealing the NP evolve from soft to hard magnetic NP, with changes of both the anisotropy constant and saturation mangnetization, while the supercrystal structures remain unchanged. Both native and annealed colloidal crystals remain characterized by a smaller interparticle distance compared to the corresponding films, inducing higher dipolar interactions, which is evidenced by an increase in the blocking temperature. In this study, we highlight the first fcc colloidal crystals made of hcp-Co single crystals resulting from the annealing of their native counterparts made of fcc-Co polycrystals. In addition, the comparative magnetic study between both the annealed supercrystalline films and colloidal crystals evidences enhanced magnetic properties for the latter ones.

Stages of Kitaigorodsky Aufbau Principle Detached in the Cocrystals of Cp2MX2 (M = Ti, Zr; X = Cl, Br, I) with σ- and π-Hole Donors

Organometallic halogenides Cp2MX2 (M = Ti, Zr; X = Cl, Br, I) were utilized as σ- and π-hole acceptors in interaction with deficient perfluoroarenes. The chain assembly of Cp2MX2 molecules stabilized by H–X hydrogen bonds, characteristic for their native crystals, recur unchanged in their 1:1 cocrystals with 1,4-DITFB. The arrangement between the Cp2MX2 and 1,4-DITFB chains in the cocrystals clearly depends on the directionality of lone electron pairs of halogen X. These series of cocrystals let us speak about the supramolecular dissolution, which is reverse to the Kitaigorodsky’s Aufbau Principle, used for the analysis and prediction of molecular crystal packing. The latter suggests four stages of aggregation on the way from individual molecules (stage 0) to the 3D crystal lattice (stage 3). Two stages, which are in between, operate with 1D molecular stacks and 2D molecular layers, which are more like representations rather than real entities in the crystal. However, in the process of supramolecular dissolution, these feebly associated molecules, chains, and layers appear as detached, individual, and quite objective entities.

Elemental Depth Profiling of Intact Metal-Organic Framework Single Crystals by Scanning Nuclear Microprobe.

The growing field of MOF–catalyst composites often relies on postsynthetic modifications for the installation of active sites. In the resulting MOFs, the spatial distribution of the inserted catalysts has far-reaching ramifications for the performance of the system and thus needs to be precisely determined. Herein, we report the application of a scanning nuclear microprobe for accurate and nondestructive depth profiling of individual UiO-66 and UiO-67 (UiO = Universitetet i Oslo) single crystals. Initial optimization work using native UiO-66 crystals yielded a microbeam method which avoided beam damage, while subsequent analysis of Zr/Hf mixed-metal UiO-66 crystals demonstrated the potential of the method to obtain high-resolution depth profiles. The microbeam method was further used to analyze the depth distribution of postsynthetically introduced organic moieties, revealing either core–shell or uniform incorporation can be obtained depending on the size of the introduced molecule, as well as the number of carboxylate binding groups. Finally, the spatial distribution of platinum centers that were postsynthetically installed in the bpy binding pockets of UiO-67-bpy (bpy = 5,5′-dicarboxyy-2,2′-bipyridine) was analyzed by microbeam and contextualized. We expect that the method presented herein will be applicable for characterizing a wide variety of MOFs subjected to postsynthetic modifications and provide information crucial for their optimization as functional materials.

Crystal structure determination of the armadillo repeat domain of Drosophila SARM1 using MIRAS phasing.

The crystal structure determination of the armadillo repeat motif (ARM) domain of Drosophila SARM1 (dSARM1ARM) is described, which required the combination of a number of sources of phase information in order to obtain interpretable electron-density maps. SARM1 is a central executioner of programmed axon degeneration, a common feature of the early phase of many neurodegenerative diseases. SARM1 is held in the inactive state in healthy axons by its N-terminal auto-inhibitory ARM domain, and is activated to cleave NAD upon injury, triggering subsequent axon degeneration. To characterize the molecular mechanism of SARM1 activation, it was sought to determine the crystal structure of the SARM1 ARM domain. Here, the recombinant production and crystallization of dSARM1ARM is described, as well as the unconventional process used for structure determination. Crystals were obtained in the presence of NMN, a precursor of NAD and a potential activator of SARM1, only after in situ proteolysis of the N-terminal 63 residues. After molecular-replacement attempts failed, the crystal structure of dSARM1ARM was determined at 1.65 Å resolution using the MIRAS phasing technique with autoSHARP, combining data from native, selenomethionine-labelled and bromide-soaked crystals. The structure will further the understanding of SARM1 regulation.

The structural landscape of ferrocenyl polychalcogenides

Halogen bond (HaB) assisted co-crystallization strategy was used to explore the structural landscape of ferrocenic polychalcogenides varying from a rather compact ferrocenophane triselenide (1,1’-FcSe3) to bulky diferrocenyl ditelluride (Fc2Te2) and its mixed-valent derivative (FcTeTeI2Fc). Comparison of the supramolecular organization in their native crystals and respective cocrystals with the iconic halogen bond donor, 1,4-diiodotetrafluorobenzene (p-DITFB), demonstrate three different patterns: 1) conservation of the chain structures for FcTeTeI2Fc, 2) transformation of single Se···Se intermolecular interaction to double for FcSe3, and 3) for Fc2Te2, in the absence of specific and directed intermolecular interactions, the cocrystal packing of Fc2Te2 is governed only by p-DITFB HaBs directionality. This allows a rough glimpse of ferrocenic polychalcogenides structural landscape and shows the directions and methods for its further detailization.

Magmatic-hydrothermal origin for Carlin-type Au deposits: Evidences from in-situ S-Pb isotopic compositions of sulfides, the Chang’an Au deposit, southern Ailaoshan tectonic zone

The Chang’an Au deposit is a large Au deposit located in the south of the Ailaoshan-Red River tectonic zone, southwestern margin of the South China block. The deposit exhibit characters similar to Carlin-type Au deposits in Nevada and Dian-Qian-Gui “Golden Triangle”, including tectonic evolution (deformed and rifted cratonic margins), orebody controlled by lithology and structure and invisible Au ionically bound in arsenian pyrite and pyrite. Mineralization is controlled by a strike-slip fault and occurs as barren milky quartz veins, disseminated gold-bearing pyrite and arsenopyrite in the Early Paleozoic sandstone, siltstone and limestone. Gold is invisibly and ionically bound in arsenopyrite and pyrite or locally occurs as isolated native gold crystals (micron scale). Wall rock alteration includes sulfide, silicic, sericite and decarbonized alterations. Three hydrothermal mineralizing stages (I, II, III) can be identified: milky quartz-pyrite (I), quartz-sericite-pyrite-arsenopyrite (II) and dolomite-calcite-quartz (III). Different from those deposit in Dian-Qian-Gui, numerous plutons intruded in and around the mine. The syenite dikes are pyritized, thus, formed prior to the mineralization while lamprophyre dikes are unaltered and crosscut by the syenite dikes. The syenite dikes yielded zircon U − Pb ages of ca. 35.8 Ma and the lamprophyre yielded similar Ar-Ar age of ca. 36.6 Ma, largely constraining the age of Au mineralization. In situ S–Pb isotopic compositions for ore pyrite and arsenopyrite were measured by fs-LA-MC-ICP-MS. Sulfides from Chang’an show a uniform range from –6.4‰ to + 4.6‰, a typical characteristic of magmatic-derived S. Such sulfur isotopic compositions are similar to those of sulfides from Carlin-type Au deposits, but different from those of sulfides from orogenic-type deposit, which commonly exhibit a wide range of δ34S values. Pb isotopic ratios of pyrite and arsenopyrite are similar to those of the intrusive rocks in the deposit with more radiogenic Pb, suggesting that the metal is probably sourced from the coeval intrusive rocks and sedimentary wall rocks. Combined with the low Au concentrations of the sedimentary wall rocks, Au in the Chang’an deposit are probably sourced from the intrusive rocks. Differences between the Chang’an deposit and typical orogenic-type Au deposit include: (1) Orebodies in the Chang’an deposit exhibit transitional contact relationship with the host rocks and boundaries between them are defined by cut-off grade while these boundaries in typical orogenic-type deposits are clearly defined. (2) Sulfidized and decarbonized alterations are quite significant in the Chang’an deposit but commonly absent in typical orogenic-type deposits. (3) The Chang’an deposit have lower CO2 contents of ore-forming fluids and more shallow ore-forming depth relative to typical orogenic-type deposits. Based on the ore deposit geology, dating and isotopic data, the Chang’an mineralization is similar to typical Carlin-type gold deposit, formed by ore fluids originated from a deep magmatic S source in an post-collisional intracontinental setting at ca. 36 Ma.

Redefining Protein Interfaces within Protein Single Crystals with DNA.

Proteins are exquisite nanoscale building blocks: molecularly pure, chemically addressable, and inherently selective for their evolved function. The organization of proteins into single crystals with high positional, orientational, and translational order results in materials where the location of every atom can be known. However, controlling the organization of proteins is challenging due to the myriad interactions that define protein interfaces within native single crystals. Recently, we discovered that introducing a single DNA-DNA interaction between protein surfaces leads to changes in the packing of proteins within single crystals and the protein-protein interactions (PPIs) that arise. However, modifying specific PPIs to effect deliberate changes to protein packing is an unmet challenge. In this work, we hypothesized that disrupting and replacing a highly conserved PPI with a DNA-DNA interaction would enable protein packing to be modulated by exploiting the programmability of the introduced oligonucleotides. Using concanavalin A (ConA) as a model protein, we circumvent potentially deleterious mutagenesis and exploit the selective binding of ConA toward mannose to noncovalently attach DNA to the protein surface. We show that DNA association eliminates the major PPI responsible for crystallization of native ConA, thereby allowing subtle changes to DNA design (length, complementarity, and attachment position) to program distinct changes to ConA packing, including the realization of three novel crystal structures and the deliberate expansion of ConA packing along a single crystallographic axis. These findings significantly enhance our understanding of how DNA can supersede native PPIs to program protein packing within ordered materials.

On the anomalous shapes of native copper crystals from the Michigan Copper Country

Abstract. For over a century, the anomalous shapes of Michigan copper crystals from the Michigan Copper Country have been acknowledged. They are well known by mineral collectors and curated in museums from all around the world; still, their particular habits remain enigmatic. These natural crystals do not seem to follow crystal shape theories, based on the internal three-dimensional crystal structure. In this article, we offer a unique perspective on the formation of Michigan copper crystals. Firstly, we review the most common theories of crystal shapes. Then, taking into account the surface reconstructions induced by adsorbed oxygen, detected by ultra-high vacuum techniques, we present evidence of a strong correlation between these oxygen-induced surface reconstructions and the anomalous shapes. Finally, in order to understand why these shapes are not found in copper at other localities, oxygen dosing was performed using NanoSIMS on different natural copper crystals as a preliminary investigation. The higher oxygen content found in the Michigan copper crystal studied compared to others supports the influence of adsorbed oxygen on the anomalous crystal shapes. This result shows which mechanisms could modify crystal shapes and allow the development of strategies to monitor them, due to the presence of oxygen impurities. This new find is of great importance in shape-dependent catalysis, sensor characteristics, or other properties of material such as nanocrystals.

Expression, purification and crystallization of the N-terminal Solanaceae domain of the Sw-5b NLR immune receptor.

Plant nucleotide-binding domain and leucine-rich repeat receptors (NLRs) play crucial roles in recognizing pathogen effectors and activating plant immunity. The tomato NLR Sw-5b is a coiled-coil NLR (CC-NLR) immune receptor that confers resistance against tospoviruses, which cause serious economic losses in agronomic crops worldwide. Compared with other CC-NLRs, Sw-5b possesses an extended N-terminal Solanaceae domain (SD). The SD of Sw-5b is critical for recognition of the tospovirus viral movement protein NSm. An SD is also frequently detected in many NLRs from Solanaceae plants. However, no sequences homologous to the SD have been detected in animals or in plants other than Solanaceae. The properties of the SD protein are largely unknown, and thus 3D structural information is vital in order to better understand its role in pathogen perception and the activation of immune receptors. Here, the expression, purification and crystallization of Sw-5b SD (amino acids 1-245) are reported. Native and selenomethionine-substituted crystals of the SD protein belonged to space group P3112, with unit-cell parameters a = 81.53, b=81.53, c=98.44 Å and a = 81.63, b = 81.63, c = 98.80 Å, respectively. This is the first report of a structural study of the noncanonical SD domain of the NLR proteins from Solanaceae plants.

Copper-Containing Agates of the Avacha Bay (Eastern Kamchatka, Russia)

The copper-containing agates of the Avacha Bay (Eastern Kamchatka, Russia) have been investigated in this study. Optical microscopy, scanning electron microscopy, electron microprobe analysis, X-ray powder diffraction, Raman spectroscopy, and fluid inclusions were used to investigate the samples. It was found that copper mineralization in agates is represented by native copper, copper sulphides (chalcocite, djurleite, digenite, anilite, yarrowite, rarely chalcopyrite) and cuprite. In addition to copper minerals, sphalerite and native silver were also found in the agates. Native copper is localized in a siliceous matrix in the form of inclusions usually less than 100 microns in size—rarely up to 1 mm—forming dendrites and crystals of a cubic system. Copper sulphides are found in the interstices of chalcedony often cementing the marginal parts of spherule aggregates of silica. In addition, they fill the micro veins, which occupy a cross-cutting position with respect to the concentric bands of chalcedony. The idiomorphic appearance of native copper crystals and clear boundaries with the silica matrix suggest their simultaneous crystallization. Copper sulphides, cuprite, and barite micro veins indicate a later deposition. Raman spectroscopy and X-ray powder diffraction results demonstrated that the Avacha Bay agates contained cristobalite in addition to quartz and moganite. The fluid inclusions study shows that the crystalline quartz in the center of the nodule in agates was formed with the participation of solutions containing a very low salt concentration (<0.3 wt.% NaCl equivalent) at the temperature range 110–50 °C and below. The main salt components were CaCl2 and NaCl, with a probable admixture of MgCl2. The copper mineralization in the agates of the Avacha Bay established in the volcanic strata can serve as a direct sign of their metallogenic specialization.

Halogen bonding in crystals of free 1,2-diiodo-ethene (C2H2I2) and its π-complex [CpMn(CO)2](π-C2H2I2)

Abstract 1,2-trans-diiodo-ethene (C2H2I2) – is an overlooked halogen bond donor, which demonstrate the distinct similarity of the geometry and directionality of I···I halogen bonds around the iodine atoms in its native and CpMn(CO)2(C2H2I2) π-complex crystals. Distortion of the planar geometry of C2H2I2 upon the π-coordination result the distortion of the native planar layered geometry of C2H2I2, so that [CpMn(CO)2](π-C2H2I2) features more complex I···I XB assisted 3D network. Unusual structural parallels between the native C2H2I2 crystals and solid iodine are discussed.

GlycoTorch Vina: Docking Designed and Tested for Glycosaminoglycans.

Glycosaminoglycans (GAGs) are a family of anionic carbohydrates that play an essential role in the physiology and pathology of all eukaryotic life forms. Experimental determination of GAG-protein complexes is challenging due to their difficult isolation from biological sources, natural heterogeneity, and conformational flexibility-including possible ring puckering of sulfated iduronic acid from 1C4 to 2SO conformation. To overcome these challenges, we present GlycoTorch Vina (GTV), a molecular docking tool based on the carbohydrate docking program VinaCarb (VC). Our program is unique in that it contains parameters to model 2SO sugars while also supporting glycosidic linkages specific to GAGs. We discuss how crystallographic models of carbohydrates can be biased by the choice of refinement software and structural dictionaries. To overcome these variations, we carefully curated 12 of the best available GAG and GAG-like crystal structures (ranging from tetra- to octasaccharides or longer) obtained from the PDB-REDO server and refined using the same protocol. Both GTV and VC produced pose predictions with a mean root-mean-square deviation (RMSD) of 3.1 Å from the native crystal structure-a statistically significant improvement when compared to AutoDock Vina (4.5 Å) and the commercial software Glide (5.9 Å). Examples of how real-space correlation coefficients can be used to better assess the accuracy of docking pose predictions are given. Comparisons between statistical distributions of empirical "salt bridge" interactions, relevant to GAGs, were compared to density functional theory (DFT) studies of model salt bridges, and water-mediated salt bridges; however, there was generally a poor agreement between these data. Water bridges appear to play an important, yet poorly understood, role in the structures of GAG-protein complexes. To aid in the rapid prototyping of future pose scoring functions, we include a module that allows users to include their own torsional and nonbonded parameters.

Ultra-wide spectral range (0.4–8 μm) transparent conductive ZnO bulk single crystals: a leading runner for mid-infrared optoelectronics

Transparent conductive oxides have been widely applied to solar cells, liquid crystal displays, and other optoelectronic devices because of their excellent transparent conductive properties in the visible-near-infrared (Vis-NIR) region. At present, the transparent conductive oxide films (TCOFs) such as indium tin oxide (ITO), fluorine-doped tin oxide (FTO), and aluminum-doped zinc oxide (AZO) are widely noticed and used. However, a high carrier concentration (≥1020 cm−3) of these TCOFs leads to a result that their transparency will be affected by the plasmons effect, and thus their transparent range will generally end around 2–3 μm. As such, they are more applicable in the Vis-NIR region but unable to meet the requirements of mid-infrared (MIR) optoelectronic devices. Faced with this situation, herein, we reported the native ZnO bulk single crystals with low background carrier concentration (1015–1016 cm−3) and high carrier mobility (μ > 300 cm2 V−1 s−1). These single crystals boast excellent transparent conductive properties (IR cutoff edge: 6–8 μm, Rsheet: 3.7–28.5 Ω/sq) with an advantage that they can be massively produced by a hydrothermal method at a low cost. Based on these merits, the native ZnO bulk single crystals bring a new possibility of breaking the bottleneck of MIR transparent conductivity.

In Situ Absorption and Fluorescence Microspectroscopy Investigation of the Molecular Incorporation Process into Single Nanoporous Protein Crystals.

Protein crystals exhibit distinct three-dimensional structures, which contain well-ordered nanoporous solvent channels, providing a chemically heterogeneous environment. In this paper, the incorporation of various molecules into the solvent channels of native hen egg-white lysozyme crystals was demonstrated using fluorescent dyes, including acridine yellow G, rhodamine 6G, and eosin Y. The process was evaluated on the basis of absorption and fluorescence microspectroscopy at a single-crystal level. The molecular loading process was clearly visualized as a function of time, and it was determined that the protein crystals could act as nanoporous materials. It was found that the incorporation process is strongly dependent on the molecular charge, leading to heterogeneous molecular aggregation, which suggests host–guest interaction of protein crystals from the viewpoint of nanoporous materials.