Circular Dichroism Spectrometer Research Articles

Rheological properties and structure of myofibrillar protein extracted from Oratosquilla oratoria muscle as affected by ultra-high pressure

ABSTRACT Oratosquilla oratoria rich in myofibrillar protein is often shelled, but traditional methods of shelling have certain limitations. In order to better apply the ultra-high pressure (UHP) technology for shelling, the effects of UHP on rheological properties and structure of myofibrillar protein (MP) from O. oratoria treated with a pressure of 300–400 MPa were determined by rheometer and circular dichroism spectrometer. The results showed that in the frequency ramp tests using rheometer, the complex viscosity and G’ of MP decreased by 59.08% and 73.70%, respectively, when the pressure reached 400 MPa with holding time of 8 min. As frequency increased, the MP exhibited shear thinning; meanwhile, its storage modulus (G’) increased, and solid characteristics became more prominent. In the temperature ramp tests, the MP denatured and its fluidity increased with the increase of temperature. Moreover, the MP from O. oratoria treated with UHP was degraded, and the ionic bond and hydrogen bond were destroyed, while the hydrophobic interaction and disulfide bond increased; the structure of α-helix and β-sheet in the MP converted to β-turn with the increase of pressure and holding time. These findings can provide references for the application of UHP in food processing.

Engineering Clostridium absonum 7α-hydroxysteroid Dehydrogenase for Enhancing Thermostability Based on Flexible Site and ΔΔG Prediction.

Enhancing thermostability of the 7α-Hydroxysteroid dehydrogenases (7α-HSDHs) is beneficial to its industrial application broadly. For protein engineering to enhance thermostability the nonrational strategy, directed evolution, has been applied in obtaining more stable proteins through error-prone PCR or DNA rearrangement generating random mutations. However, the successful application of directed evolution needs to build a large mutant library. Site-directed mutations of CA 7α-HSDH had been performed to probe the relationship between the compactness increasing and thermostability enhancing. Although most of the mutations in β-sheet core predicted by MAESTRO became more stable than wild type, unfortunately, all the mutations suffered dramatic activity loss. The main objective of this study was to verify effects of the mutations in helices selected from the predicting results through MAESTRO on thermostability improving of CA 7α-HSDH. Seven mutants, S22L, P124L, A125L, N171L, A195Q, L197E and Y259E were synthesized and verified through DNA sequencing in Sangon Biotech (Sangon, Shanghai, China). The two mutants, A104P and G105P were prepared by over-lapping PCR. The GST-fusion expression vector, pGEX-6p-1 (GE Healthcare), was used for protein expression with restriction sites BamH I and Not I. Thermostability was measured by circular dichroism (CD) spectrometer (MOS-450, BioLogic Inc). All the enzymes were diluted in PBS (pH 7.3, 10 mM) to OD222 value between 0.8 and 1, and temperature varied from 20°C to 95°C. Activity of enzyme was assayed by measuring the production of NADPH by UV-visible spectrophotometer at 340 nm. The activity assay was performed in 2 mL reaction mixture which contained PBS (pH 7.3, 10 mM), NADP+ (0.5 mM) and taurocholic acid (TCA) at 25°C. Based on unfolding free energy changes (ΔΔG) prediction seven mutations of Clostridium absonum (CA) 7α-HSDH were selected and experimentally verified, and these mutants fitted three-state denaturation model well, among which S22L located in the αA possessed the greatest Tm N→I increase (> 8°C). Mutants P124L, L197E, N171L and Y259E also became more stable than wild type CA 7α-HSDH with different ranges. Meanwhile, thermostability of the two mutants, A104P and G105P (in the coil between βD and αD) resulting from the proline substitution method decreased significantly. Enzyme activity assays indicated that mutant L197E located in αF maximally maintained 28.7% of catalytic efficiency, and activity of the five mutants, P124L, A125L, N171L, A104P and G105P cannot be detected. Although all the mutants' activities decreased, the mutant L197E with the maximum activity retain suggested that the loop structure (residues 194 to 211) may be the favored candidate sites to enhance thermostability. In addition, CA 7α-HSDH may suffer structural destruction resulting from the proline substitution in A104 and G105.

Cuprous oxide nanostructures tuned by histidine-containing peptides and their photocatalytic activities

Peptides have been used as modifiers and templates in the fabrication of a variety of inorganic nanostructures including noble metals, silica oxide, and metal oxides. In this work, three peptides, including HGGGHGHGGGHG (HG12), HGGGHG (HG6) and RHTDGLRRIAAR (CN225), were used to tune the morphologies of cuprous oxide (Cu2O) nanoparticles. Unlike the morphologies of modified octahedron with polyvinylpyrrolidone (PVP) and truncated cube with cetyltrimethyl ammonium bromide (CTAB), the Cu2O nanoparticles regulated by peptides showed completely different shapes and size. In the presence of HG12, the nanoparticles still showed octahedral morphology with the edge length of 84 nm, but there were distinct defects on the (1 1 1) facets. The Cu2O nanoparticles were sphere-like with the diameter of ∼86 nm under the regulation of HG6. In the presence of CN225, the Cu2O sample was spherical aggregates of smaller nanoparticles, and the diameter of the aggregates was about 128 nm. The interactions between Cu2+ and peptides have been investigated through UV–Vis spectrometer, circular dichroism spectrometer (CD) and isothermal titration calorimetry (ITC). The different coordinating structures of peptide molecules to Cu2+ ions dominated the nucleation and succedent crystal growth. The cuprous oxide synthesized with CN225 showed the best photocatalytic activity among the synthesized Cu2O particles, and about 67% methyl orange could be removed within 2 h under UV–Vis irradiation. The high photocatalytic activity of Cu2O-CN225 was attributed to the relatively small size and the (1 1 1) crystal planes on the primary particles.

12-Deoxyroridin J and 12-Deoxyepiisororidin E from Calcarisporium arbuscular

The culture broth of Calcarisporium arbuscular afforded 12-deoxyroridin J (1) and 12-deoxyepiisororidin E (2). Their structures were elucidated based on NMR and ECD analyses. Both molecules showed negative ECD splitting at 240 nm and a negative Cotton effect at 200 nm. Density functional theory calculations revealed that the former is caused due to the ECD exciton coupling between the C1′–C3′ enone and C7′–C11′ dienone chromophores, whereas the latter corresponds to the longer wavelength fragment of the negative ECD splitting in the vacuum ultraviolet (VUV) region at 190 nm which cannot be observed with conventional circular dichroism spectrometers. Simultaneous isolation of the regular roridins suggested that 1 and 2 are obtained through the same biosynthetic pathway. Since the 12-epoxy functionality is introduced at an early biosynthetic stage, 1 and 2 are assumed to be derived from reductions of the 12,13-epoxide in the corresponding regular roridins at a later biosynthetic stage.

Enhanced Circular Dichroism via Symmetry Breaking in a Chiral Plasmonic Nanoparticle Oligomer

A chiral plasmonic nanoparticle oligomer, consisting of four symmetrically arranged nanodisks of different heights and having different optical absorption responses to left and right-handed circularly polarized light illumination, has been experimentally reported in the literature. The resulting circular dichroism (CD) signal was detectable with state of the art CD spectrometers but was much weaker than those of existing chiral nanostructures, i.e., three-dimensional (3-D) chiral metamaterials. In this letter, via symmetry breaking in such an oligomer, the author demonstrates that the CD can be enhanced up to six times compared to that of a symmetric oligomer, and is in the range of a relevant 3-D chiral metamolecule. Through investigation of geometrical parameters including particle size, asymmetric and symmetric gaps, the CD evolution was reported, which provides a useful guideline for design of two-dimensional chiral oligomers adopted as efficient probes for CD spectroscopic applications.

Molecular weight distribution, rheological property and structural changes of sodium alginate induced by ultrasound

In this study, the effects of ultrasound with different ultrasonic frequencies on the properties of sodium alginate (ALG) were investigated, which were characterized by the means of the multi-angle laser light scattering photometer analysis (GPC-MALLS), rheological analysis, circular dichroism (CD) spectrometer and scanning electron microscope (SEM). It showed that the molecular weight (Mw) and molecular number (Mn) of the untreated ALG was 1.927×105g/mol and 4.852×104g/mol, respectively. The Mw of the ultrasound treated ALG was gradually increased from 3.50×104g/mol to 7.34×104g/mol while the Mn of ALG was increased and then decreased with the increase of the ultrasonic frequency. The maximum value of Mn was 9.988×104g/mol when the ALG was treated by ultrasound at 40kHz. It indicated that ultrasound could induce ALG degradation and rearrangement. The number of the large molecules and small molecules of ALG was changed by ultrasound. The value of dn/dc suggested that the ultrasound could enhance the stability of ALG. Furthermore, it was found that ALG treated by ultrasound at 50kHz tended to be closer to a Newtonian behavior, while the untreated and treated ALG solutions exhibited pseudoplastic behaviours. Moreover, CD spectra demonstrated that ultrasound could be used to improve the strength of the gel by changing the ratio of M/G, which showed that the minimum ratio of M/G of ALG treated at 135kHz was 1.34. The gel-forming capacity of ALG was correlated with the content of G-blocks. It suggested that ALG treated by ultrasound at 135kHz was stiffer in the process of forming gels. The morphology results indicated that ultrasound treatment of ALG at 135kHz increased its hydrophobic interaction and interfacial activity. This study is important to explore the effect of ultrasound on ALG in improving the physical properties of ALG as food additives, enzyme and drug carriers.

The Effect of Shear on the Structural Conformation of rhGH and IgG1 in Free Solution

The effect of hydrodynamic forces on proteins in free solution, also referred to as shear stress in multiple drug substance and drug product processing steps, was investigated by means of in situ and inline biophysical measurements. The use of a quartz Couette cell in combination with a circular dichroism spectrometer allowed simultaneously the creation of simple shear flow and direct measurements of the proteins' secondary and tertiary structure. Recombinant human growth hormone and an IgG1 mAb were chosen as model proteins. Under the exclusion of interfacial effects by the addition of a surfactant, no unfolding was observed due to shearing for 30 min up to the highest possible shear rate under laminar flow (3840 s−1). In another experiment, guanidine hydrochloride was added to a surfactant-protected and sheared sample to lower the thermodynamic and mechanical stability of the proteins. However, even under these destabilizing conditions, the proteins showed no change in their secondary and tertiary structure. We conclude that shear stress in terms of velocity gradients is unlikely to unfold the investigated proteins in free solution up to shear rates of at least 104 s−1.

New Magneto‐Optical Film of Ce,Ga:GIG with High Performance

Ce3+‐doped Gd3Fe5O12 (Ce:GIG) film has a good application prospect in the field of integrated optical device. In this article, Ce:GIG and Ce,Ga:GIG films were deposited onto the quartz glass substrate by using radio‐frequency magnetron sputtering technology. The crystal phase, surface morphology, magnetization, and magnetic circular dichroism properties of films were characterized by using the X‐ray powder diffraction, atomic force microscopy, vibrating sample magnetometer, and circular dichroism spectrometer. The results show that as‐prepared Ce,Ga:GIG films has a good quality and show an excellent magneto‐optical performance, and the doping of Ga3+ ion and the annealing process have significant effect on the magnetism and magneto‐optical performances. It is expected that Ce,Ga:GIG film with a moderate Ga3+‐doping content is a better candidate than Ce:GIG and Ce:YIG films for the next generation of integrated optical isolator and other magneto‐optical equipment.

Photonic spin Hall effect in gap–plasmon metasurfaces for on-chip chiroptical spectroscopy

Chiral structures possessing differential optical responses to light circular polarization are very common in biological and organic compounds. Attaining chiroptical effects is of great biochemical importance, yet requires complicated structures. Circular dichroism (CD) spectrometers measuring the differential absorption between left- (LCP) and right-circular (RCP) polarizations involve complex hardware to switch laser polarization and manage data acquisition sequentially. Here, we present compact and power-efficient metasurface-based chiroptical spectroscopy solutions based on gap–plasmon metasurfaces (GPMSs). First, a minimalistic design of a real-time CD spectrometer is obtained by using the photonic spin Hall effect (PSHE) in a single GPMS, which spatially separates LCP and RCP spectra. It is the smallest CD spectrometer to our knowledge. Another GPMS-based device built with the same approach rotates light polarization by 45° through adding a phase shift between LCP and RCP. Thus, PSHE in GPMS can provide efficient solutions to vital applications including biosensing, DNA structural analysis, and stereochemistry.

Circular Dichroism of Chiral Nematic Films of Cellulose Nanocrystals Loaded with Plasmonic Nanoparticles.

In the search for induced chiral plasmonic activity, cholesteric films formed by cellulose nanocrystals have attracted great interest as potential hosts for plasmonic nanoparticles. Circular dichroism (CD) spectra of the composite films exhibit two peaks, one of which is ascribed to the cholesteric host and the other one to plasmonic chiroptical activity of the plasmonic nanoparticles. Here we report the results of comprehensive studies of extinction and CD properties of composite films formed by different types of cellulose nanocrystals and different types of plasmonic nanoparticles. We show that the second peak in the CD spectra acquired using CD spectrometers appears as the result of the local reduction of the CD signal of the host material, due to excessive absorption by the nanoparticles, and thus it cannot be interpreted as induced plasmonic chiroptical activity. Instead, we propose an alternative way to measure CD spectra of plasmonic cholesteric films by using Mueller matrix transmission ellipsometry. The results of this study are important for ongoing research in the field of chiral plasmonics and for the optical characterization of a broad range of chiral nematic nanostructured materials.

Attractors in Sequence Space: Peptide Morphing by Directed Simulated Evolution.

Certain antimicrobial peptides (AMPs) and mitochondrial targeting peptides (mTPs) share common features such as a positive net charge, helical propensity, and the ability to interact with lipid membranes[1]. While a primary function of AMPs is to disrupt membrane integrity, many mTPs interact with the mitochondrial membranes and the translocator complex, leaving the membranes intact when proteolytically degraded by matrix-located protease(s)[2]. We present a computational approach that may help rationalize the delicate balance of these effects and other sequence-activity relationships. We implemented and applied a directed evolutionary strategy for stepwise peptide morphing of one peptide into another (MoPED, Morphing of Peptides by Evolutionary Design). For the prospective application, we chose cationic peptides as representatives of both peptide classes and converted an AMP (Protonectin, start sequence)[3] into an mTP (target or “attractor” sequence). Novel peptides were generated based on a chemical similarity index (Grantham substitution matrix).[4] The target sequence served as an attractor point for evolutionary exploration of sequence space. We synthesized and tested the individual peptides that were generated during the morphing process. The designed peptides showed systematic loss of membranolytic potential with increasing distance from the start sequence. The results of biophysical and bacterial growth experiments confirmed the applicability of MoPED to designing chemically motivated peptide derivatives. Receiver operating characteristic (ROC) analysis advocates the inducible peptide α-helicity as a semi-quantitative indicator of membranolytic antimicrobial activity.

The S229L Colon Tumor-associated Variant of DNA Polymerase β Induces Cellular Transformation as a Result of Decreased Polymerization Efficiency

DNA polymerase β (Pol β) plays a key role in base excision repair (BER) by filling in small gaps that are generated after base adducts are excised from the DNA. Pol β is mutated in a large number of colorectal tumors, and these mutations may drive carcinogenesis. In the present study, we wished to determine whether the S229L somatic Pol β variant identified in a stage 3 colorectal tumor is a driver of carcinogenesis. We show that S229L does not possess any defects in binding to either DNA or nucleotides compared with the WT enzyme, but exhibits a significant loss of polymerization efficiency, largely due to an 8-fold decrease in the polymerization rate. S229L participates in BER, but due to its lower catalytic rate, does so more slowly than WT. Expression of S229L in mammalian cells induces the accumulation of BER intermediate substrates, chromosomal aberrations, and cellular transformation. Our results are consistent with the interpretation that S229L is a driver of carcinogenesis, likely as a consequence of its slow polymerization activity during BER in vivo.

Modeling, Characterizing, and Accommodating Static Birefringence in Circular and Linear Dichroism Spectroscopy

Nearly all circular dichroism (CD) and linear dichroism (LD) spectrometers use a photoelastic modulator (PEM) in which an optical element is stressed using a high-tension voltage (HT) signal to induce birefringence. The birefringence consequently produces a phase difference between perpendicular polarization states of light passing through the PEM that is appropriate to CD or LD measurements. However, even without external stress (i.e., at zero HT) the PEM exhibits an inherent static birefringence. This article discusses the characterization of the static birefringence inherent to a PEM and its effect on the measurement of CD and LD, as well as the development and implementation of a novel model that accommodates for the presence of static birefringence. The model is validated with CD and LD experiments using purely chiral or linearly structured molecules (camphorsulfonic acid and chrysazin).

Synthesis, Chiroptical Properties and Density Functional Theory Calculations of 3,3’‐Biphenyl‐2,2’‐BiTropone

The synthesis of new bitropone derivatives, namely, 3,3'-biphenyl-2,2'-bitropone and 7,7'-biphenyl-2,2'-bitropone, are reported. Isolation of enantiomers arising from restricted rotation around the C-C bond connecting the tropone moieties was attempted by means of chiral high performance liquid chromatography (HPLC). No separation was obtained for 7,7'-biphenyl-2,2'-bitropone. For 3,3'-biphenyl-2,2'-bitropone, difficulties were encountered because of the low separation factor of the peaks and the presence of a rapid racemization process. However, quantitative chiroptical data on the antipodes were obtained by linking a circular dichroism (CD) spectrometer and a UV-vis spectrophotometric detector in series to the HPLC instrument. The analysis of the CD and UV-vis spectra in terms of absolute conformations was done with the help of theoretical calculations performed at the Density Functional Theory (DFT) level. The most stable conformations of the 3,3'-biphenyl-2,2'-bitropone in its ground state were obtained. Starting from these minimum energy conformations, it was possible to compute theoretical CD and UV absorption spectra that fit well with the experimental ones. From this comparison the absolute configuration to the antipodes was assigned. Finally, the effect of the presence of the two lateral phenyl substituents on the structure of the bitropone and hence on the CD spectrum is discussed.

Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

Allosteric regulation often controls key branch points in metabolic processes. Mycobacterium tuberculosis 2-hydroxy-3-oxoadipate synthase (HOAS), a thiamin diphosphate (ThDP)-dependent enzyme, produces 2-hydroxy-3-oxoadipate using 2-ketoglutarate and glyoxylate. The proposed chemical mechanism in analogy with other ThDP-dependent carboligases involves multiple ThDP-bound covalent intermediates. Acetyl coenzyme A is an activator, and GarA, a forkhead association domain-containing protein known to regulate glutamate metabolism, is an allosteric inhibitor of HOAS. Steady state kinetics using assays to study the first half and the full catalytic cycle suggested that the regulators act at different steps in the overall mechanism. To explore the modes of regulation and to test the effects on individual catalytic steps, we performed circular dichroism (CD) studies using a non-decarboxylatable 2-ketoglutarate analog and determined the distribution of ThDP-bound covalent intermediates during the steady state of the HOAS reaction using one-dimensional (1)H gradient carbon heteronuclear single quantum coherence NMR. The results suggest that acetyl coenzyme A acts as a mixed V and K type activator and predominantly affects the predecarboxylation steps. GarA does not inhibit the formation of the predecarboxylation analog and does not affect the accumulation of the postdecarboxylation covalent intermediate derived from 2-ketoglutarate; however, it decreases the abundance of the product ThDP adduct in the HOAS pathway. Thus, the two regulators act on different halves of the catalytic cycle in an unusual regulatory regime.

Automated Circular Dichroism Spectroscopy for Medium-Throughput Analysis of Protein Conformation

Circular dichroism (CD) spectroscopy is a powerful method for monitoring conformational changes of biomolecules. For peptides and proteins, it is highly sensitive to changes in secondary structure, which may be caused by alterations in amino acid composition or solution conditions (e.g., temperature, pH, salts, detergents, denaturants, and excipients), post-translational modifications, self-association, or ligand binding. The assets of CD spectroscopy are that the signal is directly linked to structure, the analyte is measured without labels and in solution, the technique requires low sample amounts, and data analysis is straightforward. However, CD spectroscopy has remained a low-throughput method because it imposes high requirements on the optical quality of sample cells and thus cannot be performed in microplate-reader format. Here, we introduce an automated CD spectrometer equipped with a low-birefringence flow-through cell that is coupled to a three-axis robotic liquid-handling system. This enables unattended CD measurements on up to 384 samples, including sample transfer from 96-well plates into the flow-through cell, data acquisition, and cell cleaning. We show that the accuracy, precision, and reproducibility afforded by the new instrument are excellent and exemplify how the advantages offered by automated CD spectroscopy can be exploited to quantify protein stability by titration with chemical denaturants.

Automated Circular Dichroism Spectroscopy for Medium-Throughput Quantification of Protein Conformation

Circular dichroism (CD) spectroscopy is a powerful method for monitoring conformational changes of biomolecules. For proteins, it is highly sensitive to changes in secondary structure, which, in turn, are influenced by amino acid composition, posttranslational modifications, solution conditions (e.g., temperature, pH, salts, detergents, denaturants, excipients, etc.), and ligand binding. The CD signal is directly linked to protein structure, the analyte is in solution and label-free, the technique requires low sample amounts, and data analysis is straightforward. However, CD spectroscopy has remained a low-throughput method because it imposes high requirements on the optical quality of sample cells and thus cannot be performed in microplate-reader format. Here, we introduce an automated CD spectrometer that is equipped with a flow-through cell and coupled to a 3-axis robotic liquid handler. This enables completely unattended CD measurements on up to 384 samples, including sample transfer from 96-well plates into the flow-through cell, data acquisition, and cell cleaning. We demonstrate that the accuracy, precision, and data quality of the automated CD spectrometer are as good as those of a conventional, manually operated instrument and exemplify how the advantages offered by automated CD spectroscopy can be exploited in protein-unfolding experiments using chemical denaturants.

Chiral Glutathione-protected Ag Triangular Nanoplates Synthesized by Protectant-substitution Reaction: Chiroptical and Surface Structure Analysis

Abstract Chiral Ag:SG (glutathione) triangular nanoplates were synthesized by substitution reaction of achiral Ag:poly(vinylpyrrolidone) (PVP) nanoplates with glutathione free GSH molecules. Analysis of the chiroptical properties of the synthesized nanoplates using a circular dichroism spectrometer revealed characteristic Cotton effects with wavelengths that corresponded well to those of plasmon resonances for Ag nanoplates. X-ray photoelectron spectrometry analysis of their chemical compositions revealed that complete substitution of PVP molecules with GSH molecules proceeds on the nanoplates.

A sensitive method based on fluorescence-detected circular dichroism for protein local structure analysis

We report an improved fluorescence-detected circular dichroism (FDCD)-based analytical method that is useful for probing protein three-dimensional structures. The method uses a novel FDCD device with an ellipsoidal mirror that functions on a standard circular dichroism (CD) spectrometer and eliminates all artifacts. Our experiments demonstrated three important findings. First, the method is applicable to any proteins either by using intrinsic fluorescence derived from tryptophan residues or by introducing a fluorescent label onto nonfluorescent proteins. Second, by using intrinsic fluorescence, FDCD spectroscopy can detect a structural change in the tertiary structure of metmyoglobin due to stepwise denaturation on a change in pH. Such changes could not be detected by conventional CD spectroscopy. Third, based on the typical advantages of fluorescence-based analyses, FDCD measurements enable observation of only the target proteins in a solution even in the presence of other peptides. Using our ellipsoidal mirror FDCD device, we could observe structural changes of fluorescently labeled calmodulin on binding with Ca2+ and/or interacting with binding peptides. Because FDCD appears to reflect the protein’s local structure around the fluorophore, it may provide a useful means for “pinpoint analysis” of protein structures.

Colon Cancer-associated DNA Polymerase β Variant Induces Genomic Instability and Cellular Transformation

Rapidly advancing technology has resulted in the generation of the genomic sequences of several human tumors. We have identified several mutations of the DNA polymerase β (pol β) gene in human colorectal cancer. We have demonstrated that the expression of the pol β G231D variant increased chromosomal aberrations and induced cellular transformation. The transformed phenotype persisted in the cells even once the expression of G231D was extinguished, suggesting that it resulted as a consequence of genomic instability. Biochemical analysis revealed that its catalytic rate was 140-fold slower than WT pol β, and this was a result of the decreased binding affinity of nucleotides by G231D. Residue 231 of pol β lies in close proximity to the template strand of the DNA. Molecular modeling demonstrated that the change from a small and nonpolar glycine to a negatively charged aspartate resulted in a repulsion between the template and residue 231 leading to the distortion of the dNTP binding pocket. In addition, expression of G231D was insufficient to rescue pol β-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation. More importantly, this suggests that the G231D variant has impaired base excision repair. Together, these data indicate that the G231D variant plays a role in driving cancer.