Calcium phosphate controls nucleation and growth of calcium oxalate crystal phases in kidney stones.

Kidney stone disease is a serious disease due to the severe pain it causes, high morbidity, and high recurrence rate. Notably, calcium oxalate stones are the most common type of kidney stone. Calcium oxalate appears in two forms in kidney stones: the stable phase, monohydrate (COM), and the metastable phase, dihydrate (COD). Particularly, COM stones with concentric structures are hard and difficult to treat. However, the factor determining the growth of either COM or COD crystals in the urine, which is supersaturated for both phases, remains unclear. This study shows that calcium phosphate ingredients preferentially induce COM crystal nucleation and growth, by observing and analyzing kidney stones containing both COM and COD crystals. The forms of calcium phosphate are not limited to Randall's plaques (1-2 mm size aggregates, which contain calcium phosphate nanoparticles and proteins, and form in the renal papilla). For example, aggregates of strip-shaped calcium phosphate crystals and fields of dispersed calcium phosphate microcrystals (nano to micrometer order) also promote the growth of concentric COM structures. This suggests that patients who excrete urine with a higher quantity of calcium phosphate crystals may be more prone to forming hard and troublesome COM stones.

Utilization of low-stability variants in protein evolutionary engineering.

Evolutionary engineering involves repeated mutations and screening and is widely used to modify protein functions. However, it is important to diversify evolutionary pathways to eliminate the bias and limitations of the variants by using traditionally unselected variants. In this study, we focused on low-stability variants that are commonly excluded from evolutionary processes and tested a method that included an additional restabilization step. The esterase from the thermophilic bacterium Alicyclobacillus acidocaldarius was used as a model protein, and its activity at its optimum temperature of 65 °C was improved by evolutionary experiments using random mutations by error-prone PCR. After restabilization using low-stability variants with low-temperature (37 °C) activity, several re-stabilizing variants were obtained from a large number of variant libraries. Some of the restabilized variants achieved by removing the destabilizing mutations showed higher activity than that of the wild-type protein. This implies that low-stability variants with low-temperature activity can be re-evolved for future use. This method will enable further diversification of evolutionary pathways.

The impact of crystal phase transition on the hardness and structure of kidney stones.

Calcium oxalate kidney stones, the most prevalent type of kidney stones, undergo a multi-step process of crystal nucleation, growth, aggregation, and secondary transition. The secondary transition has been rather overlooked, and thus, the effects on the disease and the underlying mechanism remain unclear. Here, we show, by periodic micro-CT images of human kidney stones in an ex vivo incubation experiment, that the growth of porous aggregates of calcium oxalate dihydrate (COD) crystals triggers the hardening of the kidney stones that causes difficulty in lithotripsy of kidney stone disease in the secondary transition. This hardening was caused by the internal nucleation and growth of precise calcium oxalate monohydrate (COM) crystals from isolated urine in which the calcium oxalate concentrations decreased by the growth of COD in closed grain boundaries of COD aggregate kidney stones. Reducing the calcium oxalate concentrations in urine is regarded as a typical approach for avoiding the recurrence. However, our results revealed that the decrease of the concentrations in closed microenvironments conversely promotes the transition of the COD aggregates into hard COM aggregates. We anticipate that the suppression of the secondary transition has the potential to manage the deterioration of kidney stone disease.

Induced Circular Dichroism Analysis of Thermally Induced Conformational Changes on Protein Binding Sites Under a Crowding Environment.

Protein-ligand interactions in crowded cellular environments play a crucial role in biological functions. The crowded environment can perturb the overall protein structure and local conformation, thereby influencing the binding pathway of protein-ligand reactions within the cellular milieu. Therefore, a detailed understanding of the local conformation is crucial for elucidating the intricacies of protein-ligand interactions in crowded cellular environments. In this study, we investigated the feasibility of induced circular dichroism (ICD) using 8-anilinonaphthalene-1-sulfonic acid (ANS) for local conformational analysis at the binding site in a crowding environment. Bovine serum albumin (BSA) concentration-dependent measurements were performed to assess the feasibility of ANS-ICD for analyzing protein interior binding sites. The results showed distinct changes in the ANS-ICD spectra of BSA solutions, indicating their potential for analyzing the internal conformation of proteins. Moreover, temperature-dependent measurements were performed in dilute and crowding environments, revealing distinct denaturation pathways of BSA binding sites. Principal component analysis of ANS-ICD spectral changes revealed lower temperature pre-denaturation in the crowded solution than that in the diluted solution, suggesting destabilization of binding sites owing to self-crowding repulsive interactions. The established ANS-ICD method can provide valuable conformational insights into protein-ligand interactions in crowded cellular environments.

Quantitative analysis of calcium oxalate monohydrate and dihydrate for elucidating the formation mechanism of calcium oxalate kidney stones.

We sought to identify and quantitatively analyze calcium oxalate (CaOx) kidney stones on the order of micrometers, with a focus on the quantitative identification of calcium oxalate monohydrate (COM) and dihydrate (COD). We performed Fourier transform infrared (FTIR) spectroscopy, powder X-ray diffraction (PXRD), and microfocus X-ray computed tomography measurements (microfocus X-ray CT) and compared their results. An extended analysis of the FTIR spectrum focusing on the 780 cm-1 peak made it possible to achieve a reliable analysis of the COM/COD ratio. We succeeded in the quantitative analysis of COM/COD in 50-µm2 areas by applying microscopic FTIR for thin sections of kidney stones, and by applying microfocus X-ray CT system for bulk samples. The analysis results based on the PXRD measurements with micro-sampling, the microscopic FTIR analysis of thin sections, and the microfocus X-ray CT system observation of a bulk kidney stone sample showed roughly consistent results, indicating that all three methods can be used complementarily. This quantitative analysis method evaluates the detailed CaOx composition on the preserved stone surface and provides information on the stone formation processes. This information clarifies where and which crystal phase nucleates, how the crystals grow, and how the transition from the metastable phase to the stable phase proceeds. The phase transition affects the growth rate and hardness of kidney stones and thus provides crucial clues to the kidney stone formation process.

Real-time monitoring of polyacrylamide gel electrophoresis by the shadowgraph technique.

Polyacrylamide gel electrophoresis (PAGE) with sodium dodecyl sulphate (SDS) and Coomassie brilliant blue (CBB) staining is widely used in protein research and requires time for electrophoresis, staining and destaining. Because the protein bands electrophoresed in the gel are invisible in most cases, the results cannot be observed until the whole process is complete. In this study, shadowgraph was applied to detect biomolecules such as proteins during electrophoresis. A simple optical system and camera-enabled real-time monitoring of migration and separation of individual protein bands in polyacrylamide gels without staining. The visibility was high enough that it was possible to visualize substances other than proteins, such as DNA. This method provides protein profiles instantly in the early stage of electrophoresis. The elimination of the staining and destaining steps will help save researchers' time. The method is also environmentally friendly and will help reduce the generation of waste solutions containing synthetic dyes.

Dispersion Effect of Molecular Crowding on Ligand-Protein Surface Binding Sites of Escherichia coli RNase HI.

The molecular crowding effect on ligand-protein interactions, which plays several crucial roles in life processes, has been investigated using various models by adding crowding agents to mimic the intracellular environment. Several studies evaluating this effect have focused on the ligand-protein binding reaction of well-structured binding sites with rigid conformations. However, the crowding effect on flexible binding sites is not well-understood, especially in terms of the conformations. In this work, to elucidate the detailed molecular mechanism underlying the ligand-protein interactions with flexible binding sites on a protein surface, we studied the interaction between the basic protrusion of Escherichia coli ribonuclease HI (RNase HI) and 8-anilinonaphthalene-1-sulfonic acid (ANS). The RNase HI concentration-dependent measurement of ANS fluorescence combined with the multivariate analysis and the fluorescence vibronic structure analysis revealed an increase in the heterogeneous species with an increase in the protein concentration, which is a different behavior from that of proteins with rigid binding sites. This result indicates that ANS molecules bind to the additional binding sites because of the destabilization of the main sites by the excluded volume effect in a crowded environment. The fluorescence vibronic structure analysis yields a detailed molecular picture, indicating that the main species of ANS can have a distorted structure. On the other hand, some ANS molecules move to the minor binding sites of a different microenvironment to secure a stabilized structure. These spectroscopic analyses may show a hypothesis, suggesting that the decrease in the ΔG difference between the main and minor sites due to destabilization of the main binding site could lower the potential barrier between them, inducing the dispersion of binding pathways.

Heterologous Expression and Catalytic Properties of Codon-Optimized Small-Sized Bromelain from MD2 Pineapple.

Bromelain is a unique enzyme-based bioactive complex containing a mixture of cysteine proteases specifically found in the stems and fruits of pineapple (Ananas comosus) with a wide range of applications. MD2 pineapple harbors a gene encoding a small bromelain cysteine protease with the size of about 19 kDa, which might possess unique properties compared to the other cysteine protease bromelain. This study aims to determine the expressibility and catalytic properties of small-sized (19 kDa) bromelain from MD2 pineapple (MD2-SBro). Accordingly, the gene encoding MD2-SBro was firstly optimized in its codon profile, synthesized, and inserted into the pGS-21a vector. The insolubly expressed MD2-SBro was then resolubilized and refolded using urea treatment, followed by purification by glutathione S-transferase (GST) affinity chromatography, yielding 14 mg of pure MD2-SBro from 1 L of culture. The specific activity and catalytic efficiency (kcat/Km) of MD2-SBro were 3.56 ± 0.08 U mg-1 and 4.75 ± 0.23 × 10-3 µM-1 s-1, respectively, where optimally active at 50 °C and pH 8.0, and modulated by divalent ions. The MD2-SBro also exhibited the ability to scavenge the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) with an IC50 of 0.022 mg mL-1. Altogether, this study provides the production feasibility of active and functional MD2-Bro as a bioactive compound.

Multicolor imaging of calcium-binding proteins in human kidney stones for elucidating the effects of proteins on crystal growth.

The pathogenesis of kidney stone formation includes multi-step processes involving complex interactions between mineral components and protein matrix. Calcium-binding proteins in kidney stones have great influences on the stone formation. The spatial distributions of these proteins in kidney stones are essential for evaluating the in vivo effects of proteins on the stone formation, although the actual distribution of these proteins is still unclear. We reveal micro-scale distributions of three different proteins, namely osteopontin (OPN), renal prothrombin fragment 1 (RPTF-1), and calgranulin A (Cal-A), in human kidney stones retaining original mineral phases and textures: calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD). OPN and RPTF-1 were distributed inside of both COM and COD crystals, whereas Cal-A was distributed outside of crystals. OPN and RPTF-1 showed homogeneous distributions in COM crystals with mosaic texture, and periodically distributions parallel to specific crystal faces in COD crystals. The unique distributions of these proteins enable us to interpret the different in vivo effects of each protein on CaOx crystal growth based on their physico-chemical properties and the complex physical environment changes of each protein. This method will further allow us to elucidate in vivo effects of different proteins on kidney stone formation.

Revisiting the Rate-Limiting Step of the ANS-Protein Binding at the Protein Surface and Inside the Hydrophobic Cavity.

8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The difference in fluorescence can not only be caused by the microenvironment but can also be affected by the binding affinity, which is represented by the binding constant (K). However, the overall binding process considering the binding constant is not fully understood, which requires the ANS fluorescence binding mechanism to be examined. In this study, to reveal the rate-limiting step of the ANS-protein binding process, protein concentration-dependent measurements of the ANS fluorescence of lysozyme and bovine serum albumin were performed, and the binding constants were analyzed. The results suggest that the main factor of the binding process is the microenvironment at the binding site, which restricts the attached ANS molecule, rather than the attractive diffusion-limited association. The molecular mechanism of ANS-protein binding will help us to interpret the molecular motions of ANS molecules at the binding site in detail, especially with respect to an equilibrium perspective.

Insertion loop-mediated folding propagation governs efficient maturation of hyperthermophilic Tk-subtilisin at high temperatures.

The serine protease Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1-IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk-subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk-subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur. Alanine substitution of the aspartate residue in IS3 disturbed the intraloop hydrogen-bonding network, as evidenced by crystallographic analysis, resulting in compromised folding at high temperatures. Taking into account the high conservation of IS3 across hyperthermophilic homologues, we propose that the presence of IS3 is important for folding of hyperthermophilic subtilisins in high-temperature environments.

Exploring mutable conserved sites and fatal non-conserved sites by random mutation of esterase from Sulfolobus tokodaii and subtilisin from Thermococcus kodakarensis.

Homologous proteins differ in their amino acid sequences at several positions. Generally, conserved sites are recognized as not suitable for amino acid substitution, and thus in evolutionary protein engineering, non-conserved sites are often selected as mutation sites. However, there have also been reports of possible mutations in conserved sites. In this study, we explored mutable conserved sites and immutable non-conserved sites by testing random mutations of two thermostable proteins, an esterase from Sulfolobus tokodaii (Sto-Est) and a subtilisin from Thermococcus kodakarensis (Tko-Sub). The subtilisin domain of Tko-Sub needs Ca2+ ions and the propeptide domain for stability, folding and maturation. The results from the two proteins showed that about one-third of the mutable sites were detected in conserved sites and some non-conserved sites lost enzymatic activity at high temperatures due to mutation. Of the conserved sites in Sto-Est, the sites on the loop, on the surface, and far from the active site are more resistant to mutation. In Tko-Sub, the sites flanking Ca2+-binding sites and propeptide were undesirable for mutation. The results presented here serve as an index for selecting mutation sites and contribute to the expansion of available sequence range by introducing mutations at conserved sites.

Spectroscopic Evidence of the Salt-Induced Conformational Change around the Localized Electric Charges on the Protein Surface of Fibronectin Type III.

The effect of salt on the electrostatic interaction of a protein is an important issue, because addition of salt affects protein stability and association/aggregation. Although adding salt is a generally recognized strategy to improve protein stability, this improvement does not necessarily occur. The lack of an effect upon the addition of salt was previously confirmed for the tenth fibronectin type III domain from human fibronectin (FN3) by thermal stability analysis. However, the detailed molecular mechanism is unknown. In the present study, by employing the negatively charged carboxyl triad on the surface of FN3 as a case study, the molecular mechanism of the inefficient NaCl effect on protein stability was experimentally addressed using spectroscopic methods. Complementary analysis using Raman spectroscopy and 8-anilino-1-naphthalenesulfonic acid fluorescence revealed the three-phase behavior of the salt-protein interaction between NaCl and FN3 over a wide salt concentration range from 100 mM to 4.0 M, suggesting that the Na+-specific binding to the negatively charged carboxyl triad causes a local conformational change around the binding site with an accompanying structural change in the overall protein, which contributes to the protein's structural destabilization. This spectroscopic evidence clarifies the molecular understanding of the inefficiency of salt to improve protein stability. The findings will inform the optimization of formulation conditions.

Microflow system promotes acetaminophen crystal nucleation.

It is known that interfaces have various impacts on crystallization from a solution. Here, we describe crystallization of acetaminophen using a microflow channel, in which two liquids meet and form a liquid-liquid interface due to laminar flow, resulting in uniform mixing of solvents on the molecular scale. In the anti-solvent method, the microflow mixing promoted the crystallization more than bulk mixing. Furthermore, increased flow rate encouraged crystal formation, and a metastable form appeared under a certain flow condition. This means that interface management by the microchannel could be a beneficial tool for crystallization and polymorph control.

Highly active enzymes produced by directed evolution with stability-based selection.

In a directed evolution aimed at improving enzymatic activity, a situation occurs where highly active variants can no longer be obtained from a template protein because the template is already located at a peak (local maximum) in the fitness landscape of activity for the sequence space. To overcome this situation, the template needs to descend the mountain (lose activity) once and climb another higher mountain. However, there is no solid guideline of how the template should go down. Here, we propose a stability index. Previous studies have shown that protein evolution is potentially governed by stability, and that proteins with low activity but high stability are more favorable templates for producing highly active variants. In our earlier works on conventional directed evolution by random mutagenesis of an esterase from Sulfolobus tokodaii, we identified variants with 3-fold higher activity than the wild-type as the highest activity variants. In this work, as a first step, stability-keeping variants were selected by five rounds of random mutagenesis and screening based on halo formation assay using the substrate tributyrin at 70 °C after heat treatment for 30 min at 90 °C. These variants are likely to be scattered at the feet of various mountains in the fitness landscape. Next, these variants were pooled and used as parental proteins for a conventional experiment with activity-based selection, where the activity of variants was assayed using their cell-free extracts on the substrate p-nitrophenyl butyrate at 75 °C. After two rounds of random mutagenesis, we successfully obtained a variant with 9-fold higher activity than the wild-type. These results indicate that the two-step selection by stability and activity enables us more easily to produce markedly activity-improving variants.

Crystal structure of a GH1 ß-glucosidase from Hamamotoa singularis.

A GH1 ß-glucosidase from the fungus Hamamotoa singularis (HsBglA) has high transgalactosylation activity and efficiently converts lactose to galactooligosaccharides. Consequently, HsBglA is among the most widely used enzymes for industrial galactooligosaccharide production. Here, we present the first crystal structures of HsBglA with and without 4'-galactosyllactose, a tri-galactooligosaccharide, at 3.0 and 2.1 Å resolutions, respectively. These structures reveal details of the structural elements that define the catalytic activity and substrate binding of HsBglA, and provide a possible interpretation for its high catalytic potency for transgalactosylation reaction.

Stress Responses of Shade-Treated Tea Leaves to High Light Exposure after Removal of Shading.

High-quality green tea is produced from buds and young leaves grown by the covering-culture method, which employs shading treatment for tea plants (Camellia sinensis L.). Shading treatment improves the quality of tea, but shaded tea plants undergo sudden exposures to high light (HL) at the end of the treatment by shade removal. In this study, the stress response of shaded tea plants to HL illumination was examined in field condition. Chl a/b ratio was lower in shaded plants than nonshaded control, but it increased due to exposure to HL after 14 days. Rapid decline in Fv/Fm values and increases in carbonylated protein level were induced by HL illumination in the shaded leaves on the first day, and they recovered thereafter between a period of one and two weeks. These results revealed that shaded tea plants temporarily suffered from oxidative damages caused by HL exposure, but they could also recover from these damages in 2 weeks. The activities of antioxidant enzymes, total ascorbate level, and ascorbate/dehydroascorbate ratio were decreased and increased in response to low light and HL conditions, respectively, suggesting that the upregulation of antioxidant defense systems plays a role in the protection of the shaded tea plants from HL stress.

Affinity shift of ATP upon glycerol binding to a glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1.

Glycerol kinase (GK) is a key enzyme of glycerol metabolism. It participates in glycolysis and lipid membrane biosynthesis. A hexamer of GK from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1(Tk-GK) was identified as a substrate-binding form of the enzyme. Here, the X-ray crystal structure analysis and the biochemical analysis was done and the relationships between its unique oligomer structure and substrate binding affinity were investigated. Wild type GK and mutant K271E GK, which disrupts the hexamer formation interface, were crystallized with and without their substrates and analyzed at 2.19-3.05 Å resolution. In the absence of glycerol, Tk-GK was a dimer in solution. In the presence of its glycerol substrate, however, it became a hexamer consisting of three symmetrical dimers about the threefold axis. Through glycerol binding, all Tk-GK molecules in the hexamer were in closed form as a result of domain-motion. The closed form of Tk-GK had tenfold higher ATP affinity than the open form of Tk-GK. The hexamer structure stabilized the closed conformation and enhanced ATP binding affinity when the GK was bound to glycerol. This molecular mechanism is quite simple activity regulation mechanism among known GKs.

Spectroscopic Signature of the Steric Strains in an Escherichia coli RNase HI Cavity-Filling Destabilized Mutant Protein.

A cavity-filling mutation at a hydrophobic cavity is a useful method for increasing protein stability. This method, however, sometimes destabilizes the protein because of the accompanying structural changes by the steric hindrance around the cavity. Thus, detailed knowledge of unfavorable structural changes is important for a comprehensive understanding of the cavity-filling mutation. In the present study, by employing the cavity-filling mutant of Escherichia coli RNase HI as a case study, the structural change induced by the substitution of Phe for Ala52 (Ala52Phe) was analyzed in detail using Raman spectroscopy. In previous studies, the thermodynamic result apparently indicated a small decrease in ΔG (destabilization) by the mutation. In the present study, Raman differential spectra show a clear structural difference between wild-type E. coli RNase HI and Ala52Phe. Consequently, the direct signature of the conformational strains around the protein cavity is readily acquired, leading to further understanding of the trade-off relationship between the cavity-filling and incidental steric hindrance.

Activity-stability trade-off in random mutant proteins.

In our previous study, we investigated the relationship between protein evolution and stability through the random mutational drift of an esterase from hyperthermophilic archaeon Sulfolobus tokodaii. The results revealed that evolvability, which is the appearance frequency of variants with higher activity than the parent protein, correlates with parental stability. This suggests that protein evolution that does not take stability into account does not make sense. Here, we used those data to further evaluate the relationship between activity and stability in random mutations, revealing that the maximum increase in activity due to mutation conflicts with parental stability. That is, many activated variants are produced when parental stability is high, whereas lower stability offers a few excellent variants with much higher activity. Moreover, we used the random mutant library to compute a novel criterion, robustizability (stabilizability), which is the appearance frequency of variants with a higher stability than the parent protein. Robustizability correlates positively with parental activity and negatively with parental stability. The results indicated that the principle of activity-stability trade-off dominates, in even random mutations. We propose its application in protein engineering via directed evolution by stability selection.