Proline Isomerization Research Articles

Sequence and structural determinants of RNAPII CTD phase-separation and phosphorylation by CDK7

The intrinsically disordered carboxy-terminal domain (CTD) of the largest subunit of RNA Polymerase II (RNAPII) consists of multiple tandem repeats of the consensus heptapeptide Y1-S2-P3-T4-S5-P6-S7. The CTD promotes liquid-liquid phase-separation (LLPS) of RNAPII in vivo. However, understanding the role of the conserved heptad residues in LLPS is hampered by the lack of direct biochemical characterization of the CTD. Here, we generated a systematic array of CTD variants to unravel the sequence-encoded molecular grammar underlying the LLPS of the human CTD. Using in vitro experiments and molecular dynamics simulations, we report that the aromaticity of tyrosine and cis-trans isomerization of prolines govern CTD phase-separation. The cis conformation of prolines and β-turns in the SPXX motif contribute to a more compact CTD ensemble, enhancing interactions among CTD residues. We further demonstrate that prolines and tyrosine in the CTD consensus sequence are required for phosphorylation by Cyclin-dependent kinase 7 (CDK7). Under phase-separation conditions, CDK7 associates with the surface of the CTD droplets, drastically accelerating phosphorylation and promoting the release of hyperphosphorylated CTD from the droplets. Our results highlight the importance of conformationally restricted local structures within spacer regions, separating uniformly spaced tyrosine stickers of the CTD heptads, which are required for CTD phase-separation.

Peri active site catalysis of proline isomerisation is the molecular basis of allomorphy in β-phosphoglucomutase

Metabolic regulation occurs through precise control of enzyme activity. Allomorphy is a post-translational fine control mechanism where the catalytic rate is governed by a conformational switch that shifts the enzyme population between forms with different activities. β-Phosphoglucomutase (βPGM) uses allomorphy in the catalysis of isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Herein, we describe structural and biophysical approaches to reveal its allomorphic regulatory mechanism. Binding of the full allomorphic activator β-glucose 1,6-bisphosphate stimulates enzyme closure, progressing through NAC I and NAC III conformers. Prior to phosphoryl transfer, loops positioned on the cap and core domains are brought into close proximity, modulating the environment of a key proline residue. Hence accelerated isomerisation, likely via a twisted anti/C4-endo transition state, leads to the rapid predominance of active cis-P βPGM. In contrast, binding of the partial allomorphic activator fructose 1,6-bisphosphate arrests βPGM at a NAC I conformation and phosphoryl transfer to both cis-P βPGM and trans-P βPGM occurs slowly. Thus, allomorphy allows a rapid response to changes in food supply while not otherwise impacting substantially on levels of important metabolites.

A Simple Generic Model of Elastin-Like Polypeptides with Proline Isomerization.

A generic model of elastin-like polypeptides (ELP) is derived that includes proline isomerization (ProI). As a case study, conformational transition of a -[valine-proline-glycine-valine-glycine]- sequence is investigated in aqueous ethanol mixtures. While the non-bonded interactions are based on the Lennard-Jones (LJ) parameters, the effect of ProI is incorporated by tuning the intramolecular 3- and 4-body interactions known from the underlying all-atom simulations into the generic model. One of the key advantages of such a minimalistic model is that it readily decouples the effects of geometry and the monomer-solvent interactions due to the presence of ProI, thus gives a clearer microscopic picture that is otherwise rather nontrivial within the all-atom setups. These results are consistent with the available all-atom and experimental data. The model derived here may pave the way to investigate large scale self-assembly of ELPs or biomimetic polymers ingeneral.

Proline Peptide Bond Isomerization in Ubiquitin Under Folding and Denaturing Conditions by Pressure-Jump NMR

Proline isomerization is widely recognized as a kinetic bottleneck in protein folding, amplified for proteins rich in Pro residues. We introduced repeated hydrostatic pressure jumps between native and pressure-denaturing conditions inside an NMR sample cell to study proline isomerization in the pressure-sensitized L50A ubiquitin mutant. Whereas in two unfolded heptapeptides, X-Pro peptide bonds isomerized ca 1.6-fold faster at 1 bar than at 2.5 kbar, for ubiquitin ca eight-fold faster isomerization was observed for Pro-38 and ca two-fold for Pro-19 and Pro-37 relative to rates measured in the pressure-denatured state. Activation energies for isomerization in pressure-denatured ubiquitin were close to literature values of 20 kcal/mole for denatured polypeptides but showed a substantial drop to 12.7 kcal/mole for Pro-38 at atmospheric pressure. For ubiquitin isomers with a cis E18-P19 peptide bond, the 1-bar NMR spectrum showed sharp resonances with near random coil chemical shifts for the C-terminal half of the protein, characteristic of an unfolded chain, while most of the N-terminal residues were invisible due to exchange broadening, pointing to a metastable partially folded state for this previously recognized ‘folding nucleus’. For cis-P37 isomers, a drop in pressure resulted in the rapid loss of nearly all unfolded-state NMR resonances, while the recovery of native state intensity revealed a slow component attributed to cis → trans isomerization of P37. This result implies that the NMR-invisible cis-P37 isomer adopts a molten globule state that encompasses the entire length of the ubiquitin chain, suggestive of a structure that mostly resembles the folded state.

Proline Isomerization and Molten Globular Property of TgPDCD5 Secreted from Toxoplasma gondii Confers Its Regulation of Heparin Sulfate Binding.

Toxoplasmosis, caused by Toxoplasma gondii, poses risks to vulnerable populations. TgPDCD5, a secreted protein of T. gondii, induces apoptosis through heparan sulfate-mediated endocytosis. The entry mechanism of TgPDCD5 has remained elusive. Here, we present the solution structure of TgPDCD5 as a helical bundle with an extended N-terminal helix, exhibiting molten globule characteristics. NMR perturbation studies reveal heparin/heparan sulfate binding involving the heparan sulfate/heparin proteoglycans-binding motif and the core region, influenced by proline isomerization of P107 residue. The heterogeneous proline recruits a cyclophilin TgCyp18, accelerating interconversion between conformers and regulating heparan/heparin binding. These atomic-level insights elucidate the binary switch's functionality, expose novel heparan sulfate-binding surfaces, and illuminate the unconventional cellular entry of pathogenic TgPDCD5.

Enhanced Sampling of Configuration and Path Space in a Generalized Ensemble by Shooting Point Exchange.

The computer simulation of many molecular processes is complicated by long timescales caused by rare transitions between long-lived states. Here, we propose a new approach to simulate such rare events, which combines transition path sampling with enhanced exploration of configuration space. The method relies on exchange moves between configuration and trajectory space, carried out based on a generalized ensemble. This scheme substantially enhances the efficiency of the transition path sampling simulations, particularly for systems with multiple transition channels, and yields information on thermodynamics, kinetics and reaction coordinates of molecular processes without distorting their dynamics. The method is illustrated using the isomerization of proline in the KPTP tetrapeptide.

Replacement of the native cis prolines by alanine leads to simplification of the complex folding mechanism of a small globular protein

The folding mechanism of MNEI, a single-chain variant of naturally occurring double-chain monellin, is complex, with multiple parallel refolding channels. To determine whether its folding energy landscape could be simplified, the two native cis-prolines, Pro41 and Pro93, were mutated, singly and together, to Ala. The stability of P93A was the same as that of the wild-type protein, pWT; however, P41A and P41AP93A were destabilized by ∼0.9 kcal mol−1. The effects of the mutations on the very fast, fast, slow, and very slow phases of folding were studied. They showed that heterogeneity in the unfolded state arises due to cis to trans isomerization of the Gly92-Pro93 peptide bond. The Pro41 to Ala mutation abolished the very slow phase of folding, whereas surprisingly, the Pro93 to Ala mutation abolished the very fast phase of folding. Double-jump, interrupted folding experiments indicated that two sequential trans to cis proline isomerization steps, of the Gly92-Pro93 peptide bond followed by the Arg40-Pro41 peptide bond, lead to the formation of the native state. They also revealed the accumulation of a late native-like intermediate, N∗, which differs from the native state in the isomeric status of the Arg40-Pro41 bond, as well as in a few tertiary contacts as monitored by near-UV CD measurements. The Pro to Ala mutations not only eliminated the cis to trans Pro isomerization reaction in the unfolded state, but also the two trans to cis Pro isomerization reactions during folding. By doing so, and by differentially affecting the relative stabilities of folding intermediates, the mutations resulted in a simplification of the folding mechanism. The two Pro to Ala mutations together accelerate folding to such an extent that the native state forms more than 1000-fold faster than in the case of pWT.

Experimental NOE, Chemical Shift, and Proline Isomerization Data Provide Detailed Insights into Amelotin Oligomerization.

Amelotin is an intrinsically disordered protein (IDP) rich in Pro residues and is involved in hydroxyapatite mineralization. It rapidly oligomerizes under physiological conditions of pH and pressure but reverts to its monomeric IDP state at elevated pressure. We identified a 105-residue segment of the protein that becomes ordered upon oligomerization, and we used pressure-jump NMR spectroscopy to measure long-range NOE contacts that exist exclusively in the oligomeric NMR-invisible state. The kinetics of oligomerization and dissociation were probed at the residue-specific level, revealing that the oligomerization process is initiated in the C-terminal half of the segment. Using pressure-jump NMR, the degree of order in the oligomer at the sites of Pro residues was probed by monitoring changes in cis/trans equilibria relative to the IDP state after long-term equilibration under oligomerizing conditions. Whereas most Pro residues revert to trans in the oligomeric state, Pro-49 favors a cis configuration and three Pro residues retain an unchanged cis fraction, pointing to their local lack of order in the oligomeric state. NOE contacts and secondary 13C chemical shifts in the oligomeric state indicate the presence of an 11-residue α-helix, preceded by a small intramolecular antiparallel β-sheet, with slower formation of long-range intermolecular interactions to N-terminal residues. Although none of the models generated by AlphaFold2 for the amelotin monomer was consistent with experimental data, subunits of a hexamer generated by AlphaFold-Multimer satisfied intramolecular NOE and chemical shift data and may provide a starting point for developing atomic models for the oligomeric state.

Proline Isomerization: From the Chemistry and Biology to Therapeutic Opportunities.

Proline isomerization, the process of interconversion between the cis- and trans-forms of proline, is an important and unique post-translational modification that can affect protein folding and conformations, and ultimately regulate protein functions and biological pathways. Although impactful, the importance and prevalence of proline isomerization as a regulation mechanism in biological systems have not been fully understood or recognized. Aiming to fill gaps and bring new awareness, we attempt to provide a wholistic review on proline isomerization that firstly covers what proline isomerization is and the basic chemistry behind it. In this section, we vividly show that the cause of the unique ability of proline to adopt both cis- and trans-conformations in significant abundance is rooted from the steric hindrance of these two forms being similar, which is different from that in linear residues. We then discuss how proline isomerization was discovered historically followed by an introduction to all three types of proline isomerases and how proline isomerization plays a role in various cellular responses, such as cell cycle regulation, DNA damage repair, T-cell activation, and ion channel gating. We then explore various human diseases that have been linked to the dysregulation of proline isomerization. Finally, we wrap up with the current stage of various inhibitors developed to target proline isomerases as a strategy for therapeutic development.

Not Only Expansion: Proline Content and Density Also Induce Disordered Protein Conformation Compaction

Intrinsically disordered proteins (IDPs) adopt a wide array of different conformations that can be constrained by the presence of proline residues, which are frequently found in IDPs. To assess the effects of proline, we designed a series of peptides that differ with respect to the number of prolines in the sequence and their organization. Using high-resolution atomistic molecular dynamics simulations, we found that accounting for whether the proline residues are clustered or isolated contributed significantly to explaining deviations in the experimentally-determined gyration radii of IDPs from the values expected based on the Flory scaling-law. By contrast, total proline content makes smaller contribution to explaining the effect of prolines on IDP conformation. Proline residues exhibit opposing effects depending on their organizational pattern in the IDP sequence. Clustered prolines (i.e., prolines with ≤2 intervening non-proline residues) result in expanded peptide conformations whereas isolated prolines (i.e., prolines with >2 intervening non-proline residues) impose compacted conformations. Clustered prolines were estimated to induce an expansion of ∼20% in IDP dimension (via formation of PPII structural elements) whereas isolated prolines were estimated to induce a compaction of ∼10% in IDP dimension (via the formation of backbone turns). This dual role of prolines provides a mechanism for conformational switching that does not rely on the kinetically much slower isomerization of cis proline to the trans form. Bioinformatic analysis demonstrates high populations of both isolated and clustered prolines and implementing them in coarse-grained molecular dynamics models illustrates that they improve the characterization of the conformational ensembles of IDPs.

Nonenzymatic Posttranslational Modifications and Peptide Cleavages Observed in Peptide Epimers.

Posttranslational modifications (PTMs) play vital roles in cellular homeostasis and are implicated in various pathological conditions. This work uses two ion mobility spectrometry-mass spectrometry (IMS-MS) modalities, drift-tube IMS (DT-IMS) and trapped IMS (TIMS), to characterize three important nonenzymatic PTMs that induce no mass loss: l/d isomerization, aspartate/isoaspartate isomerization, and cis/trans proline isomerization. These PTMs are assessed in a single peptide system, the recently discovered pleurin peptides, Plrn2, from Aplysia californica. We determine that the DT-IMS-MS/MS can capture and locate asparagine deamidation into aspartate and its subsequent isomerization to isoaspartate, a key biomarker for age-related diseases. Additionally, nonenzymatic peptide cleavage via in-source fragmentation is evaluated for differences in the intensities and patterns of fragment peaks between these PTMs. Peptide fragments resulting from in-source fragmentation, preceded by peptide denaturation by liquid chromatography (LC) mobile phase, exhibited cis/trans proline isomerization. Finally, the effects of differing the fragmentation voltage at the source and solution-based denaturation conditions on in-source fragmentation profiles are evaluated, confirming that LC denaturation and in-source fragmentation profoundly impact N-terminal peptide bond cleavages of Plrn2 and the structures of their fragment ions. With that, LC-IMS-MS/MS coupled with in-source fragmentation could be a robust method to identify three important posttranslational modifications: l/d isomerization, Asn-deamidation leading to Asp/IsoAsp isomerization, and cis/trans proline isomerization.

Probing Peptidylprolyl Bond cis/trans Status Using Distal 19 F NMR Reporters.

A method for measuring peptidylprolyl bond cis-trans conformational status in peptide models is described, using 4-fluorophenylalanine (4FPhe) as a distal reporter for 19 F NMR. The %cis-Pro population was measured for peptides of the general structure Ac-X-Pro-Z-Ala-Ala-4FPhe (X and Z are proteinogenic amino acids) at pH 7.4, and provided conformational populations consistent with literature values obtained by more complex methods. This approach was applied to probe the prolyl bond status in pentapeptide models of the intrinsically disordered C-terminal region of α-synuclein, which mirrored the preferences in the Ac-X-Pro-Z-Ala-4FPhe models. Advantageously, the 19 F reporter group does not need to be adjacent to or attached to proline to provide quantifiable signals and distal 4-fluorophenylalanines can be placed so as not to influence prolyl bond conformation. Finally, we demonstrated that the prolyl bond status is not significantly affected by pH when there are ionisable amino acid residues at the carboxyl side of proline, which makes 19 F NMR an invaluable tool with which to study proline isomerism at a range of pHs and in different solvents and buffers.

Proline isomerization compensates for the effect of phosphorylation in conformations of the disordered RNA polymerase II CTD.

RNA Polymerase II transcribes mRNA. The largest subunit of this complex contains a C-terminal domain (CTD) that is an intrinsically disordered protein with a repetitive amino acid heptad sequence making the domain very difficult to study. Kinases and Phosphatases regulate CTD through post-translational modifications. Small angle x-ray scattering data shows very little change in CTD compaction with phosphorylation, despite the repulsion between the negatively charged phosphate groups. Data also shows an increase in Pro6 isomerization when Ser5 is phosphorylated. Prior work shows that a variation of the consensus sequence where Ser7 is replaced with Asn increases the population of the proline cis conformation. We use Gaussian accelerated molecular dynamics simulations to determine how the CTD domain conformation and dynamics are impacted by phosphorylation. Simulations were run on a 21-residue construct consisting of three heptads, with the middle heptad containing Asn7. Results show that due to proline isomerization, the domain can move reversibly between a compact and extended structure. The phosphorylated CTD domain samples more prolines in cis, which compensates for the effect of phosphorylation, resulting in a similar radius of gyration to the unphosphorylated domain. Future work includes running simulations with the consensus sequence for all three heptads of our construct to determine why having asparagine in position 7 increases Pro6 isomerization compared to serine.

FK506-Binding Protein 2 Participates in Proinsulin Folding.

Apart from chaperoning, disulfide bond formation, and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94 and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high-molecular-weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic β-cells increased apoptosis without detectable up-regulation of ER stress response genes. Interestingly, FKBP2 mRNA was overexpressed in β-cells from pancreatic islets of T2D patients. Based on molecular modeling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B-chain (P28) is the substrate of FKBP2's isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

Peptidyl Prolyl Isomerase A Modulates the Liquid-Liquid Phase Separation of Proline-Rich IDPs.

Liquid–liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) and the action of molecular chaperones are tightly connected. An important class of molecular chaperones are peptidyl prolyl isomerases, which enhance the cis/trans-isomerization of proline. However, little is known about the impact of peptidyl prolyl isomerases on the LLPS of IDPs, which often contain many prolines. Here, we demonstrate that the most ubiquitous peptidyl prolyl isomerase, peptidyl prolyl isomerase A (PPIA), concentrates inside liquid-like droplets formed by the Alzheimer’s disease-associated protein tau, as well as inside RNA-induced coacervates of a proline–arginine dipeptide repeat protein. We further show that the recruitment of PPIA into the IDP droplets triggers their dissolution and return to a single mixed phase. NMR-based binding and proline isomerization studies provide insights into the mechanism of LLPS modulation. Together, the results establish a regulatory role of proline isomerases on the liquid–liquid phase separation of proline-rich IDPs.

Prolyl Isomerase Pin1 Impacts on Metabolism in Muscle and Adipocytes

Prolyl isomerase Pin1 is associated with various substrates and controls their functions through the isomerization of proline. Interestingly, a high fat diet increases Pin1 levels in adipose tissues. Accordingly, we investigated how Pin1 regulates energy metabolism in adipose tissues. Currently, adipocytes are divided into three types with distinct features. White adipocytes (WATs) store energy as triglycerides under fed conditions and release non-esterified fatty acids and glycerol through lipolysis while fasting. Brown and beige adipocytes, which exist in subcutaneous fat (scWAT), promote energy consumption through thermogenesis. We found that Pin1 impacts both thermogenesis and lipolysis upon interaction with distinct substrates. When mice were exposed to cold stress, both brown adipose tissues (BAT) and scWAT from adipocyte-specific Pin1 knockout (KO) mice showed higher expression levels of thermogenic genes in comparison to those from wild-type mice. Furthermore, we observed that Pin1 binds to the PRDM16 transcriptional cofactor, a major contributor to thermogenic programs, and promotes its degradation. Therefore, Pin1 suppresses thermogenesis. Meanwhile, in white adipocytes, Pin1 is involved in lipolysis. Pin1 deficiency enhanced lipolysis activity in epididymal WAT (epiWAT), but not in scWAT and BAT. In epiWAT, Pin1 interacts with adipose triglyceride lipase (ATGL) which is a rate-limiting enzyme for lipolysis, and downregulates ATGL levels. Finally, adipocyte-specific Pin1 KO mice have less body weight and better glucose metabolism under high fat diet conditions. These observations indicate that Pin1 is involved in the function of adipocytes through its association with PRDM16 and ATGL. Pin1 inhibitors could have therapeutic applications in the treatment of obesity.

Residual Structure in the Denatured State of the Fast-Folding UBA(1) Domain from the Human DNA Excision Repair Protein HHR23A.

The structure of the first ubiquitin-associated domain from HHR23A, UBA(1), was determined by X-ray crystallography at a 1.60 Å resolution, and its stability, folding kinetics, and residual structure under denaturing conditions have been investigated. The concentration dependence of thermal denaturation and size-exclusion chromatography indicate that UBA(1) is monomeric. Guanidine hydrochloride (GdnHCl) denaturation experiments reveal that the unfolding free energy, ΔGu°'(H2O), of UBA(1) is 2.4 kcal mol-1. Stopped-flow folding kinetics indicates sub-millisecond folding with only proline isomerization phases detectable at 25 °C. The full folding kinetics are observable at 4 °C, yielding a folding rate constant, kf, in the absence of a denaturant of 13,000 s-1 and a Tanford β-value of 0.80, consistent with a compact transition state. Evaluation of the secondary structure via circular dichroism shows that the residual helical structure in the denatured state is replaced by polyproline II structure as the GdnHCl concentration increases. Analysis of NMR secondary chemical shifts for backbone 15NH, 13CO, and 13Cα atoms between 4 and 7 M GdnHCl shows three islands of residual helical secondary structure that align in sequence with the three native-state helices. Extrapolation of the NMR data to 0 M GdnHCl demonstrates that helical structure would populate to 17-33% in the denatured state under folding conditions. Comparison with NMR data for a peptide corresponding to helix 1 indicates that this helix is stabilized by transient tertiary interactions in the denatured state of UBA(1). The high helical content in the denatured state, which is enhanced by transient tertiary interactions, suggests a diffusion-collision folding mechanism.

The Effects of Proline Isomerization on the Solvation Behavior of Elastin-Like Polypeptides in Water-Ethanol Mixtures.

Elastin-like polypeptides (ELPs) are well-known proline-rich stimulus-responsive polymers. They have broad applications ranging from drug delivery to green chemistry. Recently, the authors have shown that the cis/trans proline isomerization can be used to regulate their conformational behavior while keeping the lower critical solution temperature (LCST) unchanged in pure water. In aqueous ethanol mixtures, ELPs typically exhibit an expanded-collapsed-expanded transition known as the co-non-solvency phenomenon. Since it is unclear how proline isomerization affects the solvation behavior of ELPs in aqueous ethanol mixtures, an all-atom insight on single ELPs has been provided to address this question. It is found that if all proline residues are in the cis state, the peptides only experience a collapsed-expanded transition as ethanol concentration increases, i.e., the initial collapse vanishes because cis isomers prefer the compact structures in pure water. The data from the authors also suggest that proline isomerization does not change the shift in solvation free energy of an ELP with given sequence, but it varies the affinity of the peptide to both the solvent and cosolventmolecules.

Proline isomerization in molecular dynamics simulations of a proline-rich signaling peptide

Disordered proline-rich motifs are common across the proteomes of many species and are often involved in protein-protein interactions. Proline is a unique amino acid due to the covalent bond between the backbone nitrogen and the proline side chain. The resulting five-membered ring allows proline to sample the cis state about its peptide bond, which other residues cannot do as readily. Because proline-rich disordered sequences exist as ensembles that likely include structures with the proline peptide bond in cis, a robust methodology to accurately account for these conformations in the overall ensemble is crucial.

An electrochemical chiral sensor based on competitive host-guest interaction for the discrimination of electroinactive amino acids.

A novel electrochemical chiral sensor has been designed based on the principle of competitive host-guest interaction and utilized for the discrimination of electroinactive proline (Pro) isomers. Electroactive methylene blue (MB) was used as the signal probe, which was combined with multi-walled carbon nanotubes (MWCNTs)-decorated β-cyclodextrin (β-CD), via host-guest interaction, where the oxidation peak currents of MB decreased after isomers of Pro were combined with the MWCNTs-β-CD via a competitive host-guest interaction. Due to the steric configuration of L-Pro matching the cavity of β-CD, more L-Pro than D-Pro was combined with MWCNTs-β-CD, resulting in a more pronounced decrease of MB peak currents. Therefore, the isomers of Pro could be discriminated. Besides Pro, the isomers of electroinactive histidine (His) could also be discriminated with the chiral sensor. In addition, the contents of L-Pro in non-racemic mixtures could be detected with the developed chiral sensor.