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.

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.

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.

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.

Spectroscopic Analysis of Protein-Crowded Environments Using the Charge-Transfer Fluorescence Probe 8-Anilino-1-Naphthalenesulfonic Acid.

The molecular behaviors of proteins under crowding conditions are crucial for understanding the protein actions in intracellular environments. Under a crowded environment, the distance between protein molecules is almost the same size as the molecular level, thus, both the excluded volume effect and short ranged soft chemical interaction on protein surface could induce the complicated influence on the protein behavior cooperatively. Recently, various kinds of analytical approaches from macroscopic to microscopic aspects have been made to evaluate the crowding effect. The method, however, has not been established to evaluate the surface specific interactions on protein surface. In this study, the analytical method to evaluate the crowding effect has been suggested by using a charge-transfer fluorescence probe, ANS. By employing the unique property of ANS attaching to charged residues on the surface of lysozyme, the crowding effect was focused, while the case was compared as a reference, in which ANS is confined in hydrophobic pockets of BSA. Consequently, the surface specific changes of fluorescence spectra were readily observed under the crowded environment, whereas the fluorescence spectra of ANS in protein inside did not change. This result suggests the fluorescence spectra of ANS binding to protein surface have the capability to estimate the crowding effect of proteins.

Protein Evolution is Potentially Governed by Protein Stability: Directed Evolution of an Esterase from the Hyperthermophilic Archaeon Sulfolobus tokodaii.

The study of evolution is important to understand biological phenomena. During evolutionary processes, genetic changes confer amino acid substitutions in proteins, resulting in new or improved functions. Unfortunately, most mutations destabilize proteins. Thus, protein stability is a significant factor in evolution; however, its role remains unclear. Here, we simply and directly explored the association between protein activity and stability in random mutant libraries to elucidate the role of protein stability in evolutionary processes. In the first random mutation of an esterase from Sulfolobus tokodaii, approximately 20% of the variants displayed higher activity than wild-type protein (i.e., 20% evolvability). During evolutionary processes, the evolvability depended on the stability of template proteins, indicating that protein evolution is potentially governed by protein stability. Furthermore, decreased activity could be recovered during evolution by maintaining the stability of variants. The results suggest that protein sequence space for its evolution is able to expand during nearly neutral evolution where mutations are slightly deleterious for activity but rarely fatal for stability. Molecular evolution is a crucial phenomenon that has continued since the birth of life on earth, and mechanism underlying it is simple; therefore, this could be demonstrated by our simple experiments. These findings also can be applied to protein engineering.

Structural Basis for the Serratia marcescens Lipase Secretion System: Crystal Structures of the Membrane Fusion Protein and Nucleotide-Binding Domain.

Serratia marcescens secretes a lipase, LipA, through a type I secretion system (T1SS). The T1SS for LipA, the Lip system, is composed of an inner membrane ABC transporter with its nucleotide-binding domains (NBD), LipB, a membrane fusion protein, LipC, and an outer membrane channel protein, LipD. Passenger protein secreted by this system has been functionally and structurally characterized well, but relatively little information about the transporter complex is available. Here, we report the crystallographic studies of LipC without the membrane anchor region, LipC-, and the NBD of LipB (LipB-NBD). LipC- crystallographic analysis has led to the determination of the structure of the long α-helical and lipoyl domains, but not the area where it interacts with LipB, suggesting that the region is flexible without LipB. The long α-helical domain has three α-helices, which interacts with LipD in the periplasm. LipB-NBD has the common overall architecture and ATP hydrolysis activity of ABC transporter NBDs. Using the predicted models of full-length LipB and LipD, the overall structural insight into the Lip system is discussed.

Behavior of Bovine Serum Albumin Molecules in Molecular Crowding Environments Investigated by Raman Spectroscopy.

The behavior of proteins in crowded environments is dominated by protein-crowder interactions (the entropic/excluded volume effect) and protein-protein interactions (the soft chemical effect). The details of these interactions, however, are not fully understood. In this study, the behavior of bovine serum albumin (BSA) in crowded environments, including high protein concentrations and in the presence of another protein, was investigated by Raman spectroscopy. A detailed analysis of the water, Tyr, and Phe Raman bands revealed that the excluded volume effect with an increase in the protein concentration changed the local environment of hydrophobic residues. In contrast, no specific changes to the secondary structure were observed from the analysis of the concentration dependence of the amide I band. BSA was experimentally shown to adopt a more compact state in the presence of the crowding agent. Moreover, H-D exchange experiments of the amide I band revealed that the intramolecular hydrogen bonds of BSA were strengthened in the presence of the protein crowder. Thus, the Raman spectroscopy results have revealed the molecular behavior of proteins in crowded environments by extracting information about the excluded volume effect, soft chemical interactions, and the hydration effect.

Laser ablation for protein crystal nucleation and seeding.

With the recent development in pulsed lasers with ultrashort pulse widths or wavelengths, spatially precise, low-damage processing by femtosecond or deep-UV laser ablation has shown promise for the production of protein single crystals suitable for X-ray crystallography. Femtosecond laser processing of supersaturated solutions can shorten the protein nucleation period or can induce nucleation at low supersaturation, which improves the crystal quality of various proteins including membrane proteins and supra-complexes. In addition to nucleation, processing of protein crystals by femtosecond or deep-UV laser ablation can produce single crystalline micro- or macro-seeds without deterioration of crystal quality. This tutorial review gives an overview of the successful application of laser ablation techniques to nucleation and seeding for the production of protein single crystals, and also describes the advantages from a physico-chemical perspective.

Proteolysis of abnormal prion protein with a thermostable protease from Thermococcus kodakarensis KOD1.

The abnormal prion protein (scrapie-associated prion protein, PrP(Sc)) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Since PrP(Sc) is highly resistant to normal sterilization procedures, the decontamination of PrP(Sc) is a significant public health issue. In the present study, a hyperthermostable protease, Tk-subtilisin, was used to degrade PrP(Sc). Although PrP(Sc) is known to be resistant toward proteolytic enzymes, Tk-subtilisin was able to degrade PrP(Sc) under extreme conditions. The level of PrP(Sc) in brain homogenates was found to decrease significantly in vitro following Tk-subtilisin treatment at 100 °C, whereas some protease-resistant fractions remain after proteinase K treatment. Rather small amounts of Tk-subtilisin (0.3 U) were required to degrade PrP(Sc) at 100 °C and pH 8.0. In addition, Tk-subtilisin was observed to degrade PrP(Sc) in the presence of sodium dodecyl sulfate or other industrial surfactants. Although several proteases degrading PrP(Sc) have been reported, practical decontamination procedures using enzymes are not available. This report aims to provide basic information for the practical use of a proteolytic enzyme for PrP(Sc) degradation.

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.

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.

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.

Evolvability of thermophilic proteins from archaea and bacteria.

Proteins from thermophiles possess high thermostability. The stabilization mechanisms differ between archaeal and bacterial proteins, whereby archaeal proteins are mainly stabilized via hydrophobic interactions and bacterial proteins by ion pairs. High stability is an important factor in promoting protein evolution, but the precise means by which different stabilization mechanisms affect the evolution process remain unclear. In this study, we investigated a random mutational drift of esterases from thermophilic archaea and bacteria at high temperatures. Our results indicate that mutations in archaeal proteins lead to improved function with no loss of stability, while mutant bacterial proteins are largely destabilized with decreased activity at high temperatures. On the basis of these findings, we suggest that archaeal proteins possess higher "evolvability" than bacterial proteins under temperature selection and are additionally able to evolve into eukaryotic proteins.

Investigating the structural dependence of protein stabilization by amino acid substitution.

A goal of protein engineering technology is developing methods to increase protein stability. However, rational design of stable proteins is difficult because the stabilization mechanism of proteins is not fully understood. In this study, we examined the structural dependence of protein stabilization by introducing single amino acid substitution into ribonuclease H1 from the psychotropic bacterium Shewanella oneidensis MR-1 (So-RNase H1), which was our model protein. We performed saturation mutagenesis at various sites. Mutations that stabilized So-RNase H1 were screened using an RNase H-dependent temperature-sensitive Escherchia coli strain. Stabilizing mutations were identified by the suppressor mutagenesis method. This method yielded 39 stabilized mutants from 513 mutations at 27 positions. This suggested that more than 90% of mutations caused destabilization even in a psychotropic protein. However, 17 positions had stabilizing mutations, indicating that the stabilization factors were dispersed over many positions. Interestingly, the identified mutations were distributed mainly at exposed or nonconserved sites. These results provide a novel strategy for protein stabilization.

Enzymatic activity of a subtilisin homolog, Tk-SP, from Thermococcus kodakarensis in detergents and its ability to degrade the abnormal prion protein.

BACKGROUND: Tk-SP is a member of subtilisin-like serine proteases from a hyperthermophilic archaeon Thermococcus kodakarensis. It has been known that the hyper-stable protease, Tk-SP, could exhibit enzymatic activity even at high temperature and in the presence of chemical denaturants. In this work, the enzymatic activity of Tk-SP was measured in the presence of detergents and EDTA. In addition, we focused to demonstrate that Tk-SP could degrade the abnormal prion protein (PrPSc), a protease-resistant isoform of normal prion protein (PrPC). RESULTS: Tk-SP was observed to maintain its proteolytic activity with nonionic surfactants and EDTA at 80°C. We optimized the condition in which Tk-SP functions efficiently, and demonstrated that the enzyme is highly stable in the presence of 0.05% (w/v) nonionic surfactants and 0.01% (w/v) EDTA, retaining up to 80% of its activity. Additionally, we also found that Tk-SP can degrade PrPSc to a level undetectable by western-blot analysis. CONCLUSIONS: Our results indicate that Tk-SP has a great potential for technological applications, such as thermo-stable detergent additives. In addition, it is also suggested that Tk-SP-containing detergents can be developed to decrease the secondary infection risks of transmissible spongiform encephalopathies (TSE).

Expression, purification, crystallization and preliminary crystallographic analysis of spermidine acetyltransferase from Escherichia coli.

The spermidine acetyltransferase (SAT) from Escherichia coli catalyses the transfer of acetyl groups from acetyl-CoA to spermidine. SAT has been expressed and purified from E. coli. SAT was crystallized by the sitting-drop vapour-diffusion method to obtain a more detailed insight into the molecular mechanism. Preliminary X-ray diffraction studies revealed that the crystals diffracted to 2.5 Å resolution and belonged to the cubic space group P23, with unit-cell parameters a = b = c = 148.7 Å. They contained four molecules per asymmetric unit.

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.

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.