Scalable Analysis of Dipole Moment Fluctuations for Characterizing Intermolecular Interactions and Structural Stability.

Accurately predicting protein-ligand interactions is essential in computational molecular biochemistry and in silico drug development. Monitoring changes in molecular dipole moments through molecular dynamics simulations provides valuable insights into dipole-dipole interactions, which are critical for understanding protein structure stability and predicting protein-ligand binding affinity. In this study, we propose a novel method to monitor changes in the interangle between dipole vectors of residue molecules within proteins and ligand molecules, aiming to evaluate the strength and consistency of interactions within the complex. Additionally, we extend the concept of positional root-mean-square fluctuation (RMSF), commonly used for protein structure stability analysis, to dipole moments, thus defining dipole moment RMSF. This enables us to analyze the stability of dipole moments for each residue within the protein and compare them across residues and between binding and non-binding complexes. Using the CRBP1-retinoic acid complex as our model system, we observed a significant difference in the interangle change of dipole moments for the key residue at the residue-level between the non-binding and binding complexes. Furthermore, we found that the dipole moment RMSF value of the non-binding complex was substantially larger than that of the binding complex, indicating greater dipole moment instability in the non-binding complex. Leveraging the concept of scalability inherent in the calculation of dipole moment vectors, we systematically expanded the residues within the protein's primary secondary structure. Our dipole moment analysis approach can provide valuable predictive insights into complex candidates, especially in situations where experimental comparisons are challenging.

Efficient Super-Resolution Method for Targets Observed by Satellite SAR.

This study presents an efficient super-resolution (SR) method for targets observed by satellite synthetic aperture radar (SAR). First, a small target image is extracted from a large-scale SAR image and undergoes proper preprocessing. The preprocessing step is adaptively designed depending on the types of movements of targets. Next, the principal scattering centers of targets are extracted using the compressive sensing technique. Subsequently, an impulse response function (IRF) of the satellite SAR system (IRF-S) is generated using a SAR image of a corner reflector located at the calibration site. Then, the spatial resolution of the IRF-S is improved by the spectral estimation technique. Finally, according to the SAR signal model, the super-resolved IRF-S is combined with the extracted scattering centers to generate a super-resolved target image. In our experiments, the SR capabilities for various targets were investigated using quantitative and qualitative analysis. Compared with conventional SAR SR methods, the proposed scheme exhibits greater robustness towards improvement of the spatial resolution of the target image when the degrees of SR are high. Additionally, the proposed scheme has faster computation time (CT) than other SR algorithms, irrespective of the degree of SR. The novelties of this study can be summarized as follows: (1) the practical design of an efficient SAR SR scheme that has robustness at a high SR degree; (2) the application of proper preprocessing considering the types of movements of targets (i.e., stationary, moderate motion, and complex motion) in SAR SR processing; (3) the effective evaluation of SAR SR capability using various metrics such as peak signal-to-noise ratio (PSNR), structural similarity index (SSIM), focus quality parameters, and CT, as well as qualitative analysis.

Protein Repair and Analysis Server: A Web Server to Repair PDB Structures, Add Missing Heavy Atoms and Hydrogen Atoms, and Assign Secondary Structures by Amide Interactions.

The Protein Data Bank (PDB) file format developed at Brookhaven National Laboratory is the most popular file format to store biological data and is widely supported by many software programs for the editing and visualization of macromolecular structures. Unfortunately, many of these structures have variety of issues ranging from missing side chains and/or atoms to alternate locations (rotamers). To fix these flaws, one has to learn a new program, compile modules, and install libraries. To overcome these challenges, we present Protein Repair and Analysis Server (PRAS), an easy-to-use web server to repair protein structures and add missing atoms. The PRAS program flow has two main sections. In the first section, several subroutines are called to deal with sequence microheterogeneity, rotamers, missing side chains, heavy atoms, or hydrogen atoms. A log file is generated detailing irregularities and their fixes as carried out by the program. In the later section, the program uses the error-corrected protein structure to assign secondary structure elements based on amide-amide interactions of the backbone. Plots of four Ramachandran types (general, glycine, proline, and pre-proline) and percentage of secondary structure elements; a log file and the error-corrected PDB file can be downloaded by clicking on the download link generated by the server. The server is freely available and is accessed through its web address at www.protein-science.com. Alternatively, users can download the source code from the server or install the program on their local machines following the instructions at https://pypi.org/project/Pras-Server/ or https://github.com/osita-sunday-nnyigide/Pras_Server. While the present work focuses on the repair of structural data in the PDB format, the server is capable of fixing similar errors in the extensible and newly adopted mmCIF format.

Super-Resolution Procedure for Target Responses in KOMPSAT-5 Images.

Recently, target analysis using satellite SAR images has received much attention in the area of satellite SAR remote sensing. Because the spatial resolution of the target response in the satellite SAR image is a main factor that has a large effect on target analysis performances, the improvement of the spatial resolution of target response is required to enhance the target analysis capability. However, the spatial resolution is already determined in the satellite SAR system design process. To solve the above problem, the super-resolution techniques that have been applied to radar images can be utilized. However, the application of the super-resolution techniques to the target response in the satellite SAR image is not simple due to the following reasons. First, the target's motion induces severe blurring of the target response, which impedes the successful improvement of spatial resolution. Next, the zero-region in the frequency spectrum of the target image containing the target response also hinders the generation of the super-resolved image. To successfully improve spatial resolution of the satellite SAR image, the super-resolution techniques should be combined with proper preprocessing steps that can cope with the above two issues. In this paper, the whole super-resolution procedure for target responses in KOMPSAT-5 images is described. To the best of the authors' knowledge, the description of the whole super-resolution procedure for target responses is the first ever attempt in the area of satellite SAR. First, a target image containing the target response is extracted from a large-scale KOMPSAT-5 image. Subsequently, the target image is transformed to be appropriate for the utilization of super-resolution techniques by proper preprocessing steps, considering the direction of super resolution and the motion of the target. Then, some super-resolution techniques are utilized to improve the spatial resolutions and qualities of the target images. The super-resolution performances of the proposed scheme are validated using various target images for point static, extended static, and extended moving targets. The novelties of this paper can be summarized as follows: (1) the practical design of whole super-resolution processing for real satellite SAR images; (2) the performance evaluation of super-resolution techniques on real satellite SAR images. The results show that the proposed scheme can led to noticeable improvements of spatial resolution of the target images for various types of targets with reliable computation times. In addition, the proposed scheme also enhanced PSLR, ISLR, and IC, leading to clearer scattering information of the principal scatterers. Consequently, the proposed method can assist in extracting more precise and meaningful information for targets represented in KOMPSAT-5 images, which means great potential for target recognition.

Combinatorial Effect of Ligand and Ligand-Binding Site Hydrophobicities on Binding Affinity.

Computational methods to study protein-ligand interactions at a molecular level have been successful to a certain extent in predicting the pose, atomic interactions, and so forth, but poor efficiency in estimating a protein-ligand binding affinity is still a crucial problem to be solved. Analyzing the protein-ligand interactions quantitatively is one primary concern for understanding. Qualitative analysis of these interactions may lead to better insights about protein-ligand interactions. To perform such an analysis, the macroscopic molecular properties of the protein and ligand can be studied in detail and should be correlated with the ligand-binding affinity. This detailed study can be helpful in designing the ligands and the ligand-binding site as well. In this study, we attempted to identify the hydrophobic/hydrophilic features of a ligand and ligand-binding site and check their correlation with the experimental affinity of the protein-ligand complexes. This combinatorial analysis of ligand log P and binding site hydrophobicity on data set distribution and binding affinity suggested two critical findings. The hydrophobic ligands bind to hydrophilic and hydrophobic pockets equally, whereas hydrophilic ligands are specific to hydrophilic pockets. The combination of the hydrophobic ligand-hydrophobic pocket prefers high-affinity values compared to other combinations. Although these results cannot be used for atomic-level design of ligands or binding sites, they are expected to be used as a reference for screening the ligands for a given target binding site.

RiSLnet: Rapid identification of smart mutant libraries using protein structure network. Application to thermal stability enhancement.

A key point of protein stability engineering is to identify specific target residues whose mutations can stabilize the protein structure without negatively affecting the function or activity of the protein. Here, we propose a method called RiSLnet (Rapid identification of Smart mutant Library using residue network) to identify such residues by combining network analysis for protein residue interactions, identification of conserved residues, and evaluation of relative solvent accessibility. To validate its performance, the method was applied to four proteins, that is, T4 lysozyme, ribonuclease H, barnase, and cold shock protein B. Our method predicted beneficial mutations in thermal stability with ~62% average accuracy when the thermal stability of the mutants was compared with the ones in the Protherm database. It was further applied to lysine decarboxylase (CadA) to experimentally confirm its accuracy and effectiveness. RiSLnet identified mutations increasing the thermal stability of CadA with the accuracy of ~60% and significantly reduced the number of candidate residues (~99%) for mutation. Finally, combinatorial mutations designed by RiSLnet and in silico saturation mutagenesis yielded a thermally stable triple mutant with the half-life (T 1/2 ) of 114.9 min at 58°C, which is approximately twofold higher than that of the wild-type.

Separation efficiency of free-solution conjugated electrophoresis with drag-tags incorporating a synthetic amino acid.

DNA sequencing or separation by conventional capillary electrophoresis with a polymer matrix has some inherent drawbacks, such as the expense of polymer matrix and limitations in sequencing read length. As DNA fragments have a linear charge-to-friction ratio in free solution, DNA fragments cannot be separated by size. However, size-based separation of DNA is possible in free-solution conjugate electrophoresis (FSCE) if a "drag-tag" is attached to DNA fragments because the tag breaks the linear charge-to-friction scaling. Although several previous studies have demonstrated the feasibility of DNA separation by free-solution conjugated electrophoresis, generation of a monodisperse drag-tag and identification of a strong, site-specific conjugation method between a DNA fragment and a drag-tag are challenges that still remain. In this study, we demonstrate an efficient FSCE method by conjugating a biologically synthesized elastin-like polypeptide (ELP) and green fluorescent protein (GFP) to DNA fragments. In addition, to produce strong and site-specific conjugation, a methionine residue in drag-tags is replaced with homopropargylglycine (Hpg), which can be conjugated specifically to a DNA fragment with an azide site.

Structural and sequence features of two residue turns in beta-hairpins.

Beta-turns in beta-hairpins have been implicated as important sites in protein folding. In particular, two residue ß-turns, the most abundant connecting elements in beta-hairpins, have been a major target for engineering protein stability and folding. In this study, we attempted to investigate and update the structural and sequence properties of two residue turns in beta-hairpins with a large data set. For this, 3977 beta-turns were extracted from 2394 nonhomologous protein chains and analyzed. First, the distribution, dihedral angles and twists of two residue turn types were determined, and compared with previous data. The trend of turn type occurrence and most structural features of the turn types were similar to previous results, but for the first time Type II turns in beta-hairpins were identified. Second, sequence motifs for the turn types were devised based on amino acid positional potentials of two-residue turns, and their distributions were examined. From this study, we could identify code-like sequence motifs for the two residue beta-turn types. Finally, structural and sequence properties of beta-strands in the beta-hairpins were analyzed, which revealed that the beta-strands showed no specific sequence and structural patterns for turn types. The analytical results in this study are expected to be a reference in the engineering or design of beta-hairpin turn structures and sequences.

Redesigning the type II' ß-turn in green fluorescent protein to type I': implications for folding kinetics and stability.

Both Type I' and Type II' ß-turns have the same sense of the ß-turn twist that is compatible with the ß-sheet twist. They occur predominantly in two residue ß-hairpins, but the occurrence of Type I' ß-turns is two times higher than Type II' ß-turns. This suggests that Type I' ß-turns may be more stable than Type II' ß-turns, and Type I' ß-turn sequence and structure can be more favorable for protein folding than Type II' ß-turns. Here, we redesigned the native Type II' ß-turn in GFP to Type I' ß-turn, and investigated its effect on protein folding and stability. The Type I' ß-turns were designed based on the statistical analysis of residues in natural Type I' ß-turns. The substitution of the native "GD" sequence of i+1 and i+2 residues with Type I' preferred "(N/D)G" sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' ß-turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' ß-turns in natural ß-hairpins can be further optimized by converting the sequence to Type I'.

A variant of green fluorescent protein exclusively deposited to active intracellular inclusion bodies.

BACKGROUND: Inclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Recently, some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science and nanotechnology. RESULTS: In this study, we characterized the IBs of DL4, a deletion variant of green fluorescent protein which forms active intracellular aggregates. The DL4 proteins expressed in Escherichia coli were exclusively deposited to IBs, and the IBs were estimated to be mostly composed of active proteins. The spectral properties and quantum yield of the DL4 variant in the active IBs were almost same with those of its native protein. Refolding and stability studies revealed that the deletion mutation in DL4 didn't affect the folding efficiency of the protein, but destabilized its structure. Analyses specific for amyloid-like structures informed that the inner architecture of DL4 IBs might be amorphous rather than well-organized. The diameter of fluorescent DL4 IBs could be decreased up to 100-200 nm by reducing the expression time of the protein in vivo. CONCLUSIONS: To our knowledge, DL4 is the first GFP variant that folds correctly but aggregates exclusively in vivo without any self-aggregating/assembling tags. The fluorescent DL4 IBs have potentials to be used as fluorescent biomaterials. This study also suggests that biologically active IBs can be achieved through engineering a target protein itself.

High-throughput screening for transglutaminase activities using recombinant fluorescent proteins.

Since detailed evaluation of specific transglutaminases (TGs) from various species requires identification of their substrate specificities, rapid substrate screening method by measurement of their relative activities is in great demand. Here, a novel evaluation method of TG activity was developed using two recombinant fluorescent proteins (FPs), that is, eYFP and DsRed, tagged with TG substrate peptides. By cross-linking the two FPs based on the tagged target peptide sequences at their C-terminus, the expression of co-transformed TG allows quenching of the yellow fluorescence intensities. It was shown that the degree of in vivo fluorescent quenching by the TG activity agrees well with its in vitro reaction data, suggesting that this system can be used to identify relative substrate specificity of TGs for target peptide sequences. Using this method, the lysine substrates of TGs from Bacillus species (BTG) were evaluated, and the newly selected pentapeptide, KTKTN showed almost the same reactivity with the well-known hexa-lysine (K6) substrate for BTG reaction.

Glutamine (Q)-peptide screening for transglutaminase reaction using mRNA display.

Information on subsite specificity of the transglutaminase (TG) is important to design any specific peptides for TG's applications and inhibitor studies. Here, mRNA display was introduced for identifying the subsite specificity of TG from Streptomyces mobaraensis (STG). Functionally active peptides expressed from mRNA display library were differentially conjugated to hexa lysine (K6-beads according to their relative activities for STG. The active peptide substrates for STG were enriched through six rounds of screening, and its corresponding cDNA/mRNA sequences were identified by DNA sequencing. The results showed that tripeptides such as LQQ and TQP do not show any activity for STG, but the minimum size of the peptide displaying STG activity is pentapeptide. One such predicted peptide sequence, that is, RLQQP (TQ1), showed higher reactivity (ca. 182% conjugation yield) to STG than that of the highly active sequence, that is, control-Q (PQPQLPYPQPQLPY), well-known previously for mammalian TG2. Furthermore, when recombinant DsRed was tagged with TQ1 sequence at its C-terminal, DsRed-TQ1 underwent efficient covalent-immobilization onto alginate-gelatin bead by STG reaction, showing a Q-peptide application as a useful tagging molecule.

Analysis of spatial and seasonal distributions of MODIS aerosol optical properties and ground-based measurements of mass concentrations in the Yellow Sea region in 2009.

Satellite-retrieved data on aerosol optical depth (AOD) and Ångström exponent (AE) using a moderate resolution imaging spectrometer (MODIS) were used to analyze large-scale distributions of atmospheric aerosols in East Asia. AOD was relatively high in March (0.44 ± 0.25) and low in September (0.24 ± 0.21) in the East Asian region in 2009. Sandstorms originating from the deserts and dry areas in northern China and Mongolia were transported on a massive scale during the springtime, thus contributing to the high AOD in East Asia. However, whereas PM10 with diameters ≤10 µm was the highest in February at Anmyon, Cheongwon, and Ulleung, located leeward about halfway through the Korean Peninsula, AOD rose to its highest in May. The growth of hygroscopic aerosols attendant on increases in relative humidity prior to the Asian monsoon season contributed to a high AOD level in May. AE typically appears at high levels (1.30 ± 0.37) in August due to anthropogenic aerosols originating from the industrial areas in eastern China, while AOD stays low in summer due to the removal process caused by rainfall. The linear correlation coefficients of the MODIS AOD and ground-based mass concentrations of PM10 at Anmyon, Cheongwon, and Ulleung were measured at 0.4~0.6. Four cases (6 days) of mineral dustfall from sandstorms and six cases (12 days) of anthropogenically polluted particles were observed in the central area of the Korean Peninsula in 2009. PM10 mass concentrations increased at both Anmyon and Cheongwon in the cases of mineral dustfall and anthropogenically polluted particles. Cases of dustfall from sandstorms and anthropogenic polluted particles, with increasing PM10 mass concentrations, showed higher AOD values in the Yellow Sea region.

Analysis of protein-protein interface with incorporating low-frequency molecular interactions in molecular dynamics simulation.

Protein-protein interactions are vital for various biological processes such as immune reaction, signal transduction, and viral infection. Molecular Dynamics (MD) simulation is a powerful tool for analyzing non-covalent interactions between two protein molecules. In general, MD simulation studies on the protein-protein interface have focused on the analysis of major and frequent molecular interactions. In this study, we demonstrate that minor interactions with low-frequency need to be incorporated to analyze the molecular interactions in the protein-protein interface more efficiently using the complex of SARS-CoV2-RBD and ACE2 receptor as a model system. It was observed that the dominance of interactions in the MD-simulated structures didn't directly correlate with the interactions in the experimentally determined structure. The interactions from the experimentally determined structure could be reproduced better in the ensemble of MD simulated structures by including the less frequent interactions compared to the norm of choosing only highly frequent interactions. Residue Interaction Networks (RINs) analysis also showed that the critical residues in the protein-protein interface could be more efficiently identified by incorporating low-frequency interactions in MD simulation. It is expected that the approach proposed in this study can be a new way of studying protein-protein interaction through MD simulation.

Comparative molecular dynamics study on the features of binding and non-binding modes of retinoic acid in cellular retinol-binding protein (I).

Retinoids play crucial roles in various biological processes by interacting with their carrier proteins such as cellular retinol-binding protein (CRBP). Understanding the molecular interactions between retinoids and CRBP enables their pharmacological and biomedical applications. Experimentally, CRBP(I) does not bind to retinoic acid, but when arginine is introduced into 108th residue instead of glutamine (Q108R), it binds to retinoic acid. Here, molecular dynamics simulations were performed to understand the differences in the microscopic and dynamic behaviors of the non-binding wild-type CRBP(I)-retinoic acid and binding Q108R variant-retinoic acid complexes. The ligand RMSD and RMSF, the binding poses of binding motif amino acids, and the number of hydrogen bonds and salt-bridges revealed the relative instability of the non-binding complex. In particular, the ligand's terminal group showed very different dynamics and interactions. So far, most studies have focused on the binding characteristics of retinoids, but the features of their non-binding modes have not been studied well. This study provides some structural insights into the non-binding modes of a retinoid in CRBP, which may be applicable in retinoid-based drug discovery and protein engineering through computational modeling.

Residue interaction network and molecular dynamics simulation study on the binding of S239D/I332E Fc variant with enhanced affinity to FcγRIIIa receptor.

Engineering of Fc has been adapted as an efficient method for enhanced or reduced affinity towards Fc receptors in the development of therapeutic antibodies. S239D/I332E mutation of Fc induces approximately two logs greater affinity to the FcγRIIIa receptor and has been extensively employed in various Fc engineering studies. It is known that the mutation gives rise to the formation of salt bridges between the mutated residues of Fc and FcγRIIIa, but the overall effect of the mutation in the binding interface of the Fc-FcγRIIIa complex is still unclear. In this study, the molecular interactions in the binding interface of mutant Fc and FcγRIIIa were analyzed and compared with those of wild-type Fc binding through residue interaction network (RIN) analysis and molecular dynamics (MD) simulation. RIN analysis identified specific molecular interactions and Hub residues in the interfaces, and their numbers were increased by introducing the mutation, with maintaining most of the molecular interactions in the wild-type complex. MD simulation study revealed that the numbers of stable electrostatic interactions and stable Hub residues in the mutant complex were higher than those in the wild-type complex. The introduced mutations were shown to form further charge-charge attractive interactions in addition to the identified salt bridges without generating any repulsive interactions. These results are expected to provide further structural insight into Fc variants' design based on the S239D/I332E mutation.

Characteristics of aerosol types during large-scale transport of air pollution over the Yellow Sea region and at Cheongwon, Korea, in 2008.

Episodes of large-scale transport of airborne dust and anthropogenic pollutant particles from different sources in the East Asian continent in 2008 were identified by National Oceanic and Atmospheric Administration satellite RGB (red, green, and blue)-composite images and the mass concentrations of ground level particulate matter. These particles were divided into dust, sea salt, smoke plume, and sulfate by an aerosol classification algorithm. To analyze the aerosol size distribution during large-scale transport of atmospheric aerosols, aerosol optical depth (AOD) and fine aerosol weighting (FW) of moderate imaging spectroradiometer aerosol products were used over the East Asian region. Six episodes of massive airborne dust particles, originating from sandstorms in northern China, Mongolia, and the Loess Plateau of China, were observed at Cheongwon. Classified dust aerosol types were distributed on a large-scale over the Yellow Sea region. The average PM10 and PM2.5 ratio to the total mass concentration TSP were 70% and 15%, respectively. However, the mass concentration of PM2.5 among TSP increased to as high as 23% in an episode where dust traveled in by way of an industrial area in eastern China. In the other five episodes of anthropogenic pollutant particles that flowed into the Korean Peninsula from eastern China, the anthropogenic pollutant particles were largely detected in the form of smoke over the Yellow Sea region. The average PM10 and PM2.5 ratios to TSP were 82% and 65%, respectively. The ratio of PM2.5 mass concentrations among TSP varied significantly depending on the origin and pathway of the airborne dust particles. The average AOD for the large-scale transport of anthropogenic pollutant particles in the East Asian region was measured to be 0.42 ± 0.17, which is higher in terms of the rate against atmospheric aerosols as compared with the AOD (0.36 ± 0.13) for airborne dust particles with sandstorms. In particular, the region ranging from eastern China, the Yellow Sea, and the Korean Peninsula to the Korea East Sea was characterized by high AOD distributions. In the episode of anthropogenic polluted aerosols, FW averaged 0.63 ± 0.16, a value higher than that in the episode of airborne dust particles (0.52 ± 0.13) with sandstorms, showing that fine anthropogenic pollutant particles contribute greatly to atmospheric aerosols in East Asia.

Cell disruption and lipid extraction from Chlorella species for biorefinery applications: Recent advances.

Chlorella is a promising microalga for CO2-neutral biorefinery that co-produces drop-in biofuels and multiple biochemicals. Cell disruption and selective lipid extraction steps are major technical bottlenecks in biorefinement because of the inherent robustness and complexity of algal cell walls. This review focuses on the state-of-the-art achievements in cell disruption and lipid extraction methods for Chlorella species within the last five years. Various chemical, physical, and biological approaches have been detailed theoretically, compared, and discussed in terms of the degree of cell wall disruption, lipid extractability, chemical toxicity, cost-effectiveness, energy use, scalability, customer preferences, environment friendliness, and synergistic combinations of different methods. Future challenges and prospects of environmental-friendly and efficient extraction technologies are also outlined for practical applications in sustainable Chlorella biorefineries. Given the diverse industrial applications of Chlorella, this review may provide useful information for downstream processing of the advanced biorefineries of other algae genera.

A synthetic gene-metabolic oscillator.

Autonomous oscillations found in gene expression and metabolic, cardiac and neuronal systems have attracted significant attention both because of their obvious biological roles and their intriguing dynamics. In addition, de novo designed oscillators have been demonstrated, using components that are not part of the natural oscillators. Such oscillators are useful in testing the design principles and in exploring potential applications not limited by natural cellular behaviour. To achieve transcriptional and metabolic integration characteristic of natural oscillators, here we designed and constructed a synthetic circuit in Escherichia coli K12, using glycolytic flux to generate oscillation through the signalling metabolite acetyl phosphate. If two metabolite pools are interconverted by two enzymes that are placed under the transcriptional control of acetyl phosphate, the system oscillates when the glycolytic rate exceeds a critical value. We used bifurcation analysis to identify the boundaries of oscillation, and verified these experimentally. This work demonstrates the possibility of using metabolic flux as a control factor in system-wide oscillation, as well as the predictability of a de novo gene-metabolic circuit designed using nonlinear dynamic analysis.

Biosynthetic substitution of tyrosine in green fluorescent protein with its surrogate fluorotyrosine in Escherichia coli.

Introduction of a fluorine moiety into green fluorescent protein offers an interesting novel spectral variant. The calculated binding energy of fluorotyrosine (F-Tyr) (-8.42 kcal/mol) for tyrosyl tRNA synthetase was moderately higher than that of tyrosine (Tyr) (-8.36 kcal/mol). This result directly correlated with the expression level of F-Tyr containing GFP (38 mg/l), which was comparably higher than that of the parent GFP expression level (34 mg/l). Finally, we generated a model structure for GFP to assess possible interaction in the chromophore of the protein structure, which plays an important role in determining the spectral and folding behaviors of the F-Tyr incorporated GFP variant.