Protein Aggregation State Research Articles

STORM image denoising and information extraction.

Stochastic optical reconstruction microscopy (STORM) is extensively utilized in the fields of cell and molecular biology as a super-resolution imaging technique for visualizing cells and molecules. Nonetheless, the imaging process of STORM is frequently susceptible to noise, which can significantly impact the subsequent image analysis. Moreover, there is currently a lack of a comprehensive automated processing approach for analyzing protein aggregation states from a large number of STORM images. This paper initially applies our previously proposed denoising algorithm, UNet-Att, in STORM image denoising. This algorithm was constructed based on attention mechanism and multi-scale features, showcasing a remarkably efficient performance in denoising. Subsequently, we propose a collection of automated image processing algorithms for the ultimate feature extractions and data analyses of the STORM images. The information extraction workflow effectively integrates automated methods of image denoising, objective image segmentation and binarization, and object information extraction, and a novel image information clustering algorithm specifically developed for the morphological analysis of the objects in the STORM images. This automated workflow significantly improves the efficiency of the effective data analysis for large-scale original STORM images.

Application of soy protein isolate-naringenin complexes as fat replacers in low-fat cream: Based on protein conformational changes, aggregation states and interfacial adsorption behavior

In this study, changes in the structural and functional properties of soybean protein isolate (SPI)-naringenin (NG) complexes under different amounts of naringenin treatments were explored, elucidating the effect of the complexes as fat replacers at the 15 % substitution level on the properties of low-fat cream. Finally, the correlation between the structure and function of the complex and the properties of low-fat cream was further analyzed. The addition of NG promotes the increase of SPI aggregation and particle size, and reduces the interfacial tension of the complex. Meanwhile, at the mass ratio of 48:3, NG and SPI formed a dendritic network structure suitable for stabilizing cream. The fat properties of cream indicate that low-fat creams stabilized by appropriate proportions of SPI-NG complexes displayed small and dense fat crystal network structures. In addition, low-fat cream stabilized by the SPI-NG complexes have improved whipping time, overrun, firmness, storage stability and rheological properties compared to natural SPI. It is worth noting that the overall quality of the cream stabilized by the SPI-NG complex with a mass ratio of 48:3 was almost close to that of full-fat cream. Therefore, this study promotes the potential applications of protein-polyphenol complexes as fat replacers in the food industry.

Theanine improves the gelation of soy protein isolate by modifying protein conformation and enhancing molecular interaction

This study systematically investigated the dose-effect relationship and regulatory mechanism of theanine on the gelling properties of soy protein isolate (SPI). The results indicated that the strength and water holding capacity of 0.10% theanine-SPI gel increased by 238.80% and 1.13% compared with pure SPI gel, respectively. Hydrogen bonds and van der Waals forces mainly promoted the binding of theanine to β-conglycinin and glycinin. This binding could further change the protein aggregation state (reducing particle size and irregular aggregation), functional groups (exposing more hydrophilic amino acid residues and sulfhydryl groups), and molecular structure (α-helix and β-turn transforming into β-sheet). Furthermore, a more uniform and dense three-dimensional gel network was formed in the composite gels, offering the structural basis for improving the performance of the SPI gel. The results will provide theoretical basis and technical support for the precise regulation of gel properties of the functional amino acid-SPI complex as well as the efficient creation of new SPI-based foods.

Extractability and chromatographic separation of proteins from potato (Solanum tuberosum L.) trimmings

By-products from the potato processing industry, like potato trimmings, are sustainable sources of proteins. Here, a size-exclusion high performance liquid chromatography (SE-HPLC) method was applied to simultaneously determine the extractability and aggregation state of proteins from three batches of potato trimmings of different cultivars. Obtained SE-HPLC profiles allowed distinguishing between the patatin and protease inhibitor fractions of potato proteins. Moreover, only 75% of the crude proteins could be extracted in phosphate buffer containing sodium dodecyl sulfate and a reducing agent, indicating the presence of physical extraction barriers. Ball milling for 5 min significantly increased protein extractability, but prolonged treatment resulted in aggregation of native patatin and a reduced protein extractability. Microwave-dried trimmings had a lower protein extractability than freeze-dried trimmings. In future research, the SE-HPLC method can be used to examine changes in potato protein (fractions) as a result of processing.

In vitro protein digestibility of RuBisCO-enriched wheat dough: a comparative study with pea and gluten proteins.

Growing demand for sustainable, plant-based protein sources has stimulated interest in new ingredients for food enrichment. This study investigates the nutritional and digestive implications of enriching wheat dough with RuBisCO, in comparison to pea protein-enriched and gluten-enriched doughs. The protein quality and digestibility of these enriched doughs were analysed through dough characterization, in vitro digestion experiments and biochemical analysis of digesta. Our findings indicate that an enrichment at 10% of RuBisCO or pea proteins improves the chemical score and the in vitro PDCAAS (IV-PDCAAS) score of wheat dough as compared to the control dough. Digestibility assays suggest that RuBisCO introduction modifies the protein hydrolysis kinetics: the nitrogen release is lower during gastric digestion but larger during intestinal digestion than other samples. The analysis of the protein composition of the soluble and insoluble parts of digesta, using size-exclusion chromatography, reveals that the protein network in RuBisCO-enriched dough is more resistant to gastric hydrolysis than the ones of other doughs. Indeed, non-covalently bound peptides and disulfide-bound protein aggregates partly composed of RuBisCO subunits remain insoluble at the end of the gastric phase. The digestion of these protein structures is then mostly performed during the intestinal phase. These results are also discussed in relation to the digestive enzymatic cleavage sites, the presence of potential enzyme inhibitors, the protein aggregation state and the secondary structures of the protein network in each dough type.

Investigating the mechanism of antioxidants as egg white powder flavor modifiers.

The uses of egg white powder (EWP) are restricted because of its odor. It is necessary to find a method to improve its flavor. In this paper, three different antioxidants - green tea extract (GTE), sodium ascorbate (SA), and glutathione (GSH) - were selected to modify the flavor. The physicochemical and structural properties of EWP were investigated to study the mechanism of the formation and release of volatile compounds. Antioxidants can modify the overall flavor of EWP significantly, inhibiting the generation or release of nonanal, 3-methylbutanal, heptanal, decanal, geranyl acetone, and 2-pemtylfuran. A SA-EWP combination showed the lowest concentration of 'off' flavor compounds; GTE-EWP and GSH-EWP could reduce several 'off' flavor compounds but increased the formation of geranyl acetone and furans. The changes in the carbonyl content and the amino acid composition confirmed the inhibition of antioxidants with the oxidative degradation of proteins or characteristic amino acids. The results of fluorescence spectroscopy and Fourier transform infrared (FTIR) spectroscopy provided structural information regarding EWP, which showed the release of volatile compounds decreased due to structural changes. For example, the surface hydrophobicity increased and the protein aggregation state changed. Antioxidants reduce the 'off' flavor of EWP in two ways: they inhibit protein oxidation and Maillard reactions (they inhibit formation of 3-methylbutanal and 2-pemtylfuran) and they enhance the binding ability of heat-denatured proteins (reducing the release of nonanal, decanal, and similar compounds). © 2023 Society of Chemical Industry.

Role of the pea protein aggregation state on their interfacial properties

HypothesisPlant protein ingredients from similar sources can vary in functionality not only because of compositional differences, but also because of differences in their structure depending on their processing history. It is essential to understand these distinctions to develop novel food emulsion using plant proteins. It is hypothesized that differing interfacial properties can be attributed to their structures, aggregation, and colloidal states. ExperimentsThe adsorption behavior of a commercial protein isolate, homogenized or non-homogenized, was compared to a mildly extracted isolate to evaluate the effect of aggregation state and structural differences. After characterization of the particle size and protein composition, the interfacial properties were compared. FindingsAtomic force microscopy provided evidence of interfaces packed with protein oligomers regardless of the treatment. Differences in adsorption kinetics and interfacial shear rheology depending on oil polarity suggested different interfacial structures. A polydisperse mixture of protein oligomers resulted in increased rearrangements and protein-protein interactions at the interface. Homogenization of commercial proteins resulted in a lower interfacial tension and less elastic interfaces compared to those of native proteins due to the presence of larger aggregates. This study highlights how the interfacial properties can be related to the protein aggregation state resulting from differences in processing history.

Investigation of refractive index increment with crystallin protein aggregation.

In camera-type eyes, the lens is responsible for focusing light that is necessary for vision. The eye lens is composed of transparent and highly concentrated crystallin proteins. Post-translational modifications in crystallin proteins lead to protein aggregation and cataract formation, the leading cause of blindness worldwide. When light travels through an interface, it bends or refracts. The refractive index increment, dn/dc for proteins can be predicted based on its amino acid sequence. The experimentally measured refractive index increment for wild type βγ crystallin's has previously been shown to significantly deviate from the predicted values. Accurate refractive index increment values are important for data analysis in protein aggregation. This study looks at protein aggregation states and serves to further investigate discrepancies between predicted and measured refractive index increments by looking at a series of βγ crystallin's with differing amounts of deamidation mutations using multi-angle light scattering.

Amide Proton Transfer-Weighted Imaging Detects Hippocampal Proteostasis Disturbance Induced by Sleep Deprivation at 7.0 T MRI.

Sleep deprivation leads to hippocampal injury. Proteostasis disturbance is an important mechanism linking sleep deprivation and hippocampal injury. However, identifying noninvasive imaging biomarkers for hippocampal proteostasis disturbance remains challenging. Amide proton transfer-weighted (APTw) imaging is a chemical exchange saturation transfer technique based on the amide protons in proteins and peptides. We aimed to explore the ability of APTw imaging in detecting sleep deprivation-induced hippocampal proteostasis disturbance and its biological significance, as well as its biological basis. In vitro, the feasibility of APTw imaging in detecting changes of the protein state was evaluated, demonstrating that APTw imaging can detect alterations in the protein concentration, conformation, and aggregation state. In vivo, the hippocampal APTw signal declined with increased sleep deprivation time and was significantly lower in sleep-deprived rats than that in normal rats. This signal was positively correlated with the number of surviving neurons counted in Nissl staining and negatively correlated with the expression of glucose-regulated protein 78 evaluated in immunohistochemistry. Differentially expressed proteins in proteostasis network pathways were identified in the hippocampi of normal rats and sleep-deprived rats via mass spectrometry proteomics analysis, providing the biological basis for the change of the hippocampal APTw signal in sleep-deprived rats. These findings demonstrate that APTw imaging can detect hippocampal proteostasis disturbance induced by sleep deprivation and reflect the extent of neuronal injury and endoplasmic reticulum stress.

Jet cooking dramatically improves the wet strength of soy adhesives

AbstractThe impact of jet cooking on shear strength of soy‐and‐water adhesives was investigated to understand the higher shear strength of commercial soy protein isolates compared to soy flours. Soy flour‐based wood adhesives are appealing because of their bio‐based content, low formaldehyde emission, and low cost, but their commercial application is limited by low wet cohesive strength. Previous researchers proposed that the process of jet cooking (steam injection with high turbulence followed by rapid cooling) was responsible for the high (~3 MPa) wet shear strength of adhesives made with commercially produced soy protein isolate, using the ASTM D 7998 test. In this work, we show that jet cooking did dramatically increase the wet strength of laboratory‐produced, native‐state soy protein isolate from 0.6 to 3 MPa, a strength similar to many commercial isolates. Jet cooking was far less effective at developing wet strength of soy flours, but greatly increased the viscosity of virtually all our soy materials. We hypothesize that the benefits of jet cooking are primarily a result of nonequilibrium protein aggregation states because subsequent wet autoclaving of jet cooked soy proteins dramatically decreased wet strength. The dramatic differences in adhesive properties between commercial soy protein isolates and soy flours suggests that the common practice of using results obtained with commercial isolates to predict the performance of soy flour adhesives is inappropriate.

Neuroprotection and Non-Invasive Brain Stimulation: Facts or Fiction?

Non-Invasive Brain Stimulation (NIBS) techniques, such as transcranial Direct Current Stimulation (tDCS) and repetitive Magnetic Transcranial Stimulation (rTMS), are well-known non-pharmacological approaches to improve both motor and non-motor symptoms in patients with neurodegenerative disorders. Their use is of particular interest especially for the treatment of cognitive impairment in Alzheimer's Disease (AD), as well as axial disturbances in Parkinson's (PD), where conventional pharmacological therapies show very mild and short-lasting effects. However, their ability to interfere with disease progression over time is not well understood; recent evidence suggests that NIBS may have a neuroprotective effect, thus slowing disease progression and modulating the aggregation state of pathological proteins. In this narrative review, we gather current knowledge about neuroprotection and NIBS in neurodegenerative diseases (i.e., PD and AD), just mentioning the few results related to stroke. As further matter of debate, we discuss similarities and differences with Deep Brain Stimulation (DBS)-induced neuroprotective effects, and highlight possible future directions for ongoing clinical studies.

Changes in the Secondary Structure and Assembly of Proteins on Fluoride Ceramic (CeF3) Nanoparticle Surfaces.

Fluoride nanoparticles (NPs) are materials utilized in the biomedical field for applications including imaging of the brain. Their interactions with biological systems and molecules are being investigated, but the mechanism underlying these interactions remains unclear. We focused on possible changes in the secondary structure and aggregation state of proteins on the surface of NPs and investigated the principle underlying the changes using the amyloid β peptide (Aβ16–20) based on infrared spectrometry. CeF3 NPs (diameter 80 nm) were synthesized via thermal decomposition. Infrared spectrometry showed that the presence of CeF3 NPs promotes the formation of the β-sheet structure of Aβ16–20. This phenomenon was attributed to the hydrophobic interaction between NPs and Aβ peptides in aqueous environments, which causes the Aβ peptides to approach each other on the NP surface and form ordered hydrogen bonds. Because of the coexisting salts on the secondary structure and assembly of Aβ peptides, the formation of the β-sheet structure of Aβ peptides on the NP surface was suppressed in the presence of NH4+ and NO3– ions, suggesting the possibility that Aβ peptides were adsorbed and bound to the NP surface. The formation of the β-sheet structure of Aβ peptides was promoted in the presence of NH4+, whereas it was suppressed in the presence of NO3– because of the electrostatic interaction between the lysine residue of the Aβ peptide and the ions. Our findings will contribute to comparative studies on the effect of different NPs with different physicochemical properties on the molecular state of proteins.

Protein aggregation and Ca2+-induced gelation of soymilk after heat treatment under slightly alkaline conditions

Alkaline heat treatment of soymilk is widely used in the production of soy products. This study aimed to clarify the effect of the thermal aggregation state of proteins on the coagulation characteristics of soymilk. Soymilk with different pH levels (6.6–8.0) was preheated, and the gelation processes induced by CaSO4·2H2O and the gel properties were investigated. Results showed that the aggregation and binding pattern of proteins changed after slightly alkaline heat treatment. The ability of 7S and the acidic (A) subunit of 11S to bind to the basic (B) subunit of 11S weakened. These subunits converted into non-particulate proteins, which decreased particulate protein content and particle size. The number of sulfhydryl groups on the protein surface reduced by 45%–62%, and more disulfide bonds formed in the protein subunits during recombination. During gelation, the soymilk after alkaline heat treatment exhibited a slow intermolecular binding in the “pre-aggregation” stage, which led to late gelation onset and a uniform gel network. With the increase in non-gelable protein content in the gel, the water holding capacity (WHC) enhanced and but the hardness decreased. When the pH was further increased (pH ≥ 7.8) to the isoelectric point of the B subunit, the B subunit excessively aggregated and formed large and dense aggregates, which increased gel hardness and produced a rough and uneven network.

Optimization of Membrane Protein TmrA Purification Procedure Guided by Analytical Ultracentrifugation.

Membrane proteins are involved in various cellular processes. However, purification of membrane proteins has long been a challenging task, as membrane protein stability in detergent is the bottleneck for purification and subsequent analyses. Therefore, the optimization of detergent conditions is critical for the preparation of membrane proteins. Here, we utilize analytical ultracentrifugation (AUC) to examine the effects of different detergents (OG, Triton X-100, DDM), detergent concentrations, and detergent supplementation on the behavior of membrane protein TmrA. Our results suggest that DDM is more suitable for the purification of TmrA compared with OG and TritonX-100; a high concentration of DDM yields a more homogeneous protein aggregation state; supplementing TmrA purified with a low DDM concentration with DDM maintains the protein homogeneity and aggregation state, and may serve as a practical and cost-effective strategy for membrane protein purification.

Synuclein Family Members Prevent Membrane Damage by Counteracting α-Synuclein Aggregation.

The 140 amino acid protein α-synuclein (αS) is an intrinsically disordered protein (IDP) with various roles and locations in healthy neurons that plays a key role in Parkinson’s disease (PD). Contact with biomembranes can lead to α-helical conformations, but can also act as s seeding event for aggregation and a predominant β-sheet conformation. In PD patients, αS is found to aggregate in various fibrillary structures, and the shift in aggregation and localization is associated with disease progression. Besides full-length αS, several related polypeptides are present in neurons. The role of many αS-related proteins in the aggregation of αS itself is not fully understood Two of these potential aggregation modifiers are the αS splicing variant αS Δexon3 (Δ3) and the paralog β-synuclein (βS). Here, polarized ATR-FTIR spectroscopy was used to study the membrane interaction of these proteins individually and in various combinations. The method allowed a continuous monitoring of both the lipid structure of biomimetic membranes and the aggregation state of αS and related proteins. The use of polarized light also revealed the orientation of secondary structure elements. While αS led to a destruction of the lipid membrane upon membrane-catalyzed aggregation, βS and Δ3 aggregated significantly less, and they did not harm the membrane. Moreover, the latter proteins reduced the membrane damage triggered by αS. There were no major differences in the membrane interaction for the different synuclein variants. In combination, these observations suggest that the formation of particular protein aggregates is the major driving force for αS-driven membrane damage. The misbalance of αS, βS, and Δ3 might therefore play a crucial role in neurodegenerative disease.

Exploring interactions between lipids and amyloid-forming proteins: A review on applying fluorescence and NMR techniques

A hallmark of Alzheimer’s, Parkinson’s, and other amyloid diseases is the assembly of amyloid proteins into amyloid aggregates or fibrils. In many cases, the formation and cytotoxicity of amyloid assemblies are associated with their interaction with cell membranes. Despite studied for many years, the characterization of the interaction is challenged for reasons on the multiple aggregation states of amyloid-forming proteins, transient and weak interactions in the complex system. Although several strategies such as computation biology, spectroscopy, and imaging methods have been performed, there is an urgent need to detail the molecular mechanism in different time scales and high resolutions. This review highlighted the recent applications of fluorescence, solution and solid-state NMR in exploring the interactions between amyloid protein and membranes attributing to their advantages of high sensitivity and atomic resolution.

Acid‐induced aggregation and gelation of heat‐treated chia proteins

SummaryThis work studied for the first time the acid‐induced aggregation and gelation of heat‐treated chia protein isolates obtained by extraction at pH 10 or 12 (CPI10 and CPI12, respectively). The aggregation state of proteins was modified during acidification. The size of the aggregates was reduced for both samples when the pH decreased but below pH 4.5 further protein aggregation took place for CPI12. Gelation of CPI12 was completed after about 30 min of acidification with glucone‐δ‐lactone. By contrast, this period was not enough to reach a constant value in G′ for CPI10. When gelation was ensured, confocal laser scanning micrographs from those gels revealed a coarse and irregular structure with large pores (median size of diameters: 30 μm). Instead, micrographs from CPI12 cold gels showed a more regular and interconnected network, with smaller pores (median size of diameters: 9 μm). These differences are consistent with a higher elastic behaviour ( = 13.6 ± 0.1 Pa).

The Interaction of Bovine β-Lactoglobulin with Caffeic Acid: From Binding Mechanisms to Functional Complexes.

In this study, the interaction of native and transglutaminase (Tgase) cross-linked β-lactoglobulin (β-LG) with caffeic acid (CA) was examined, aiming to obtain functional composites. Knowledge on the binding affinity and interaction mechanism was provided by performing fluorescence spectroscopy measurements, after heating the native and cross-linked protein at temperatures ranging from 25 to 95 °C. Regardless of the protein aggregation state, a static quenching mechanism of intrinsic fluorescence of β-LG by CA was established. The decrease of the Stern–Volmer constants with the temperature increase indicating the facile dissociation of the weakly bound complexes. The thermodynamic analysis suggested the existence of multiple contact types, such as Van der Waals’ force and hydrogen bonds, between β-LG and CA. Further molecular docking tests indicated the existence of various CA binding sites on the β-LG surface heat-treated at different temperatures. Anyway, regardless of the simulated temperature, the CA-β-LG assemblies appeared to be unstable. Compared to native protein, the CA-β-LG and CA-β-LGTgase complexes (ratio 1:1) exhibited significantly higher antioxidant activity and inhibitory effects on α-glucosidase, α-amylase, and pancreatic lipase, enzymes associated with metabolic syndrome. These findings might help the knowledge-based development of novel food ingredients with valuable biological properties.

Glycoprotein Profile Assessed by 1H-NMR as a Global Inflammation Marker in Patients with HIV Infection. A Prospective Study.

Plasma glycoproteins are a composite biomarker of inflammation and can be detected by 1H-NMR. The aim of this study was to prospectively appraise the clinical value of plasma glycoproteins assessed by 1H-NMR in people living with HIV (PLWH). A total of 221 patients with HIV infection were recruited and studied at baseline and at 48 and 144 weeks. Patients were distributed into two groups according to baseline CD4+ T-cell number below or above 200 cells/µL. Patients with fewer than 200 cells/µL were distributed into the responders and nonresponders according to antiretroviral therapy (ART) response at 144 weeks. Glycoprotein concentrations were determined by 1H-NMR arising from the protein bond N-acetylglucosamine and N-acetylgalactosamine signals (GlycA); and N-acetylneuraminic acid signal (GlycB) associated with the sugar–protein bond concentration and aggregation state (shapes (height/width)). Basal glycoprotein concentrations were higher in patients with < 200 CD4+ T-cell/μL (Glyc A: 1040(917.9–1199.1) vs. 950.4(845.5–1050.9), p < 0.001, and Glyc B: 521(440.3–610.3) vs. 468.6(417.9–507.0) μ mol/L, p < 0.001) being reduced by ART. The height/width (H/W) ratio was the parameter showing a better association with this clinical status. Baseline glycoproteins predict the condition of responder/nonresponder. In this study, 1H-NMR glycoproteins provide novel insights to assess inflammation status and have prognostic value in PLWH.

Heterologous expression, purification and biochemical characterization of a glutamate racemase (MurI) from Streptococcus mutans UA159.

BackgroundGlutamate racemase (MurI) is a cofactor-independent enzyme that is essential to the bacterial peptidoglycan biosynthesis pathway and has therefore been considered an attractive target for the development of antimicrobial drugs. While in our previous study the essentiality of the murI gene was shown in Streptococcus mutans, the primary aetiologic agent of human dental caries, studies on S. mutans MurI have not yet provided definitive results. This study aimed to produce and characterize the biochemical properties of the MurI from the S. mutans UA159 genome.MethodsStructure characterization prediction and multiple sequence alignment were performed by bioinformatic analysis. Recombinant His6-tagged S. mutans MurI was overexpressed in the expression vector pColdII and further purified using a Ni2+ affinity chromatography method. Protein solubility, purity and aggregation state were analyzed by SDS–PAGE, Western blotting, native PAGE and SEC-HPLC. Kinetic parameters were assessed by a circular dichroism (CD) assay. Kinetic constants were calculated based on the curve fit for the Michaelis–Menten equation. The effects of temperature and pH on enzymatic activity were determined by a series of coupled enzyme reaction mixtures.ResultsThe glutamate racemase gene from S. mutans UA159 was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3). The 264-amino-acid protein, as a mixture of dimeric and monomeric enzymes, was purified to electrophoretic homogeneity. In the CD assay, S. mutans MurI displayed unique kinetic parameters (Km, d-Glu→l-Glu = 0.3631 ± 0.3205 mM, Vmax, d-Glu→l-Glu = 0.1963 ± 0.0361 mM min−1, kcat, d-Glu→l-Glu = 0.0306 ± 0.0065 s−1, kcat/Km, d-Glu→l-Glu = 0.0844 ± 0.0128 s−1 mM−1, with d-glutamate as substrate; Km, l-Glu→d-Glu = 0.8077 ± 0.5081 mM, Vmax, l-Glu→d-Glu = 0.2421 ± 0.0418 mM min−1, kcat, l-Glu→d-Glu = 0.0378 ± 0.0056 s−1, kcat/Km, l-Glu→d-Glu = 0.0468 ± 0.0176 s−1 mM−1, with l-glutamate as substrate). S. mutans MurI possessed an assay temperature optimum of 37.5 °C and its optimum pH was 8.0.ConclusionThe findings of this study provide insight into the structure and biochemical traits of the glutamate racemase in S. mutans and supply a conceivable guideline for employing glutamate racemase in anti-caries drug design.