ABSTRACT
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.
Subject(s)
Molecular Dynamics Simulation , Protein Binding , Protein Stability , Proteins , Ligands , Proteins/chemistry , Protein ConformationABSTRACT
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.
Subject(s)
Data Compression , Radar , Algorithms , Signal-To-Noise RatioABSTRACT
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.
Subject(s)
Retinol-Binding Proteins , Tretinoin , Retinol-Binding Proteins, Cellular/metabolism , Tretinoin/metabolism , Retinol-Binding Proteins/metabolism , Molecular Dynamics Simulation , Vitamin A/metabolism , Ligands , Retinoids/metabolismABSTRACT
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.
Subject(s)
COVID-19 , Molecular Dynamics Simulation , Humans , RNA, Viral , SARS-CoV-2 , Proteins/chemistry , Protein BindingABSTRACT
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.
Subject(s)
Immunoglobulin Fc Fragments , Molecular Dynamics Simulation , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/metabolism , MutationABSTRACT
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.
Subject(s)
MotionABSTRACT
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.
Subject(s)
Chlorella , Microalgae , Biofuels , Cell Wall , Lipids , BiomassABSTRACT
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.
Subject(s)
Amides , Hydrogen , Databases, Protein , Internet , Proline , Protein Conformation , Protein Structure, Secondary , Proteins/chemistry , SoftwareABSTRACT
Molecular docking approach has been extensively used to predict the ligand's binding conformation in the binding pocket of protein. However, its prediction accuracy is still limited and highly dependent on target protein-ligand complexes. In this study, we investigated the effects of ligand torsion number, ligand hydrophobicity, and binding-site hydrophobicity on the prediction accuracy of Autodock, a popular molecular docking tool, combinatorially as well as respectively. A clear understanding of how these properties affect the prediction accuracy was observed when these properties were studied combinatorially rather than individually. The combination of low ligand torsion number-hydrophilic ligand-hydrophobic binding site provided the best prediction accuracy while the high ligand torsion number-hydrophilic ligand-hydrophobic binding pocket combination showed the least prediction accuracy. This study allowed us to determine the molecular properties of complex, showing relatively higher or low prediction accuracy and can be employed as a reference in the molecular docking studies using Autodock.
Subject(s)
Proteins , Binding Sites , Ligands , Molecular Conformation , Molecular Docking Simulation , Protein Binding , Proteins/metabolismABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
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.
Subject(s)
Proteins , Binding Sites , Hydrophobic and Hydrophilic Interactions , Ligands , Models, Molecular , Protein Binding , Proteins/metabolismABSTRACT
The binding interactions of the surfactants: anionic sodium dodecyl sulphate (SDS), cationic cetyltrimethylammonium bromide (CTAB), non-ionic octyl glucoside (OG), and zwitterionic 3-[Hexadecyl(dimethyl)ammonio]-1-propanesulfonate (HPS), with bovine serum albumin (BSA) were investigated by computer simulation. The results disclosed that the surfactants bound stably between hydrophobic subdomain IIA and IIIA where tryptophan-213 residue, an important intrinsic fluorophore in BSA is housed. The interactions of the surfactants with the BSA were electrostatic and hydrophobic interactions. The head-groups of SDS, HPS and OG formed hydrogen bonds with the BSA, while that of CTAB was shielded from intermolecular hydrogen-bonding due to intervening methyl groups. Subsequently, molecular dynamics (MD) simulation of the protein-surfactant complexes revealed that hydrogen bonds formed by OG were stronger than those of SDS and HPS. However, the decomposed force-field energies showed that OG had the least interaction energy with the BSA. In addition to MD simulation, it was found by density functional theory (DFT) that the differences in the coulomb interaction energies can be attributed to charge distribution in the surfactants. Overall, free energies calculated by linear interaction energy (LIE) proved that the binding of each surfactant was dominated by differences between van der Waals interactions in bound and free states.
Subject(s)
Cetrimonium/chemistry , Glucosides/chemistry , Molecular Dynamics Simulation , Serum Albumin, Bovine/chemistry , Sodium Dodecyl Sulfate/chemistry , Surface-Active Agents/chemistry , Amino Acids/chemistry , Animals , Binding Sites , Cattle , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Kinetics , Ligands , Protein Domains , Static ElectricityABSTRACT
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.
Subject(s)
Computational Biology/methods , Genetic Testing/methods , Hot Temperature , Mutant Proteins/chemistry , Protein Stability , Mutant Proteins/genetics , Time FactorsABSTRACT
Macroscopic descriptors have become valuable as coarse-grained features of complex proteins and are complementary to microscopic descriptors. Proteins macroscopic geometric features provide effective clues in the quantification of distant similarity and close dissimilarity searches for structural comparisons. In this study, we performed a systematic comparison of ß-barrels, one of the important classes of protein folds in various transmembrane (TM) proteins against cytoplasmic barrels to estimate the conformational features using a joint-based descriptor. The approach uses joint coordinates and dihedral angles (ß and γ) based on the ß-strand joints and loops to determine the arrangements and propensities at the local and global levels. We then confirmed that there is a clear preference in the overall ß and γ distribution, arrangements of ß-strands and loops, signature patterns, and the number of strand effects between TM and cytoplasmic ß-barrel geometries. As a robust and simple approach, we determine that the joint-based descriptor could provide a reliable static structural comparison aimed at macroscopic level between complex protein conformations.
Subject(s)
Cytoplasm/chemistry , Membrane Proteins/chemistry , Databases, Protein , Protein Conformation, beta-Strand , Protein Structure, SecondaryABSTRACT
The interactions of bovine serum albumin (BSA) with urea/water were investigated by computer simulation. It was revealed that the BSA-hydrophobic residues in urea solutions favored contact with urea more than with water. Energy decomposition analysis showed that van der Waals energy was the dominant driving force behind urea affinity for hydrophobic residues, whereas coulombic attraction was largely responsible for water affinity for these residues. Meanwhile, urea-BSA hydrogen bond energies were found to be weaker than water-BSA hydrogen bond energies. The greater strength of water-BSA hydrogen bonds than urea-BSA hydrogen bonds, and the opposing preferential interaction between the BSA and urea suggest that disruption of hydrophobic interaction predominates urea-protein denaturation. In pure water, hydrophobic residues showed aggregation tendencies at 323 K, suggesting an increase in hydrophobicity, while at 353 K the residues were partly denatured due to loss of hydrogen bonds; thus, disruption of hydrophobic interactions appeared to contribute less to thermal denaturation.
Subject(s)
Serum Albumin, Bovine/chemistry , Urea/chemistry , Computer Simulation , Entropy , Hot Temperature , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Protein Denaturation , Solvents/chemistry , Water/chemistryABSTRACT
3-Hydroxypropionic acid (3-HP) can be produced via the biological route involving two enzymatic reactions: dehydration of glycerol to 3-hydroxypropanal (3-HPA) and then oxidation to 3-HP. However, commercial production of 3-HP using recombinant microorganisms has been hampered with several problems, some of which are associated with the toxicity of 3-HPA and the efficiency of NAD+ regeneration. We engineered α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense for the second reaction to address these issues. The residues in the binding sites for the substrates, 3-HPA and NAD+, were randomized, and the resulting libraries were screened for higher activity. Isolated KGSADH variants had significantly lower Km values for both the substrates. The enzymes also showed higher substrate specificities for aldehyde and NAD+, less inhibition by NADH, and greater resistance to inactivation by 3-HPA than the wild-type enzyme. A recombinant Pseudomonas denitrificans strain with one of the engineered KGSADH variants exhibited less accumulation of 3-HPA, decreased levels of inactivation of the enzymes, and higher cell growth than that with the wild-type KGSADH. The flask culture of the P. denitrificans strain with the mutant KGSADH resulted in about 40% increase of 3-HP titer (53 mM) compared with that using the wild-type enzyme (37 mM).
Subject(s)
Aldehyde Oxidoreductases/metabolism , Azospirillum brasilense/enzymology , Bacterial Proteins/metabolism , Glyceraldehyde/analogs & derivatives , Lactic Acid/analogs & derivatives , NAD/metabolism , Propane/metabolism , Protein Engineering/methods , Aldehyde Oxidoreductases/chemistry , Glyceraldehyde/metabolism , Glycerol/metabolism , Lactic Acid/metabolism , Protein Conformation , Substrate SpecificityABSTRACT
Joint-based descriptor is a new level of macroscopic descriptor for protein structure using joints of secondary structures as a basic element. Here, we propose how the joint-based descriptor can be applied to examine the conformational distances or differences of transmembrane (TM) proteins. Specifically, we performed three independent studies that measured the global and conformational distances between GPCR A family and its related structures. First, the conformational distances of GPCR A family and other 7TM proteins were evaluated. This provided the information on the distant and close families or superfamilies to GPCR A family and permitted the identification of conserved local conformations. Second, computational models of GPCR A family proteins were validated, which enabled us to estimate how much they reproduce the native conformation of GPCR A proteins at global and local conformational level. Finally, the conformational distances between active and inactive states of GPCR proteins were estimated, which identified the difference of local conformation. The proposed macroscopic joint-based approach is expected to allow us to investigate structural features, evolutionary relationships, computational models and conformational changes of TM proteins in a more simplistic manner.
Subject(s)
Membrane Proteins/chemistry , Protein Structure, Secondary , Receptors, G-Protein-Coupled/chemistry , Computational Biology , Evolution, Molecular , Membrane Proteins/classification , Models, Molecular , Receptors, G-Protein-Coupled/classificationABSTRACT
Cadaverine (1,5-diaminopentane) is a major source of many industrial polyamides such as nylon and chelating agents. Currently, cadaverine is produced by the microbial fermentation of glucose to lysine, which is then decarboxylated by lysine decarboxylase (CadA). However, utilizing CadA for cadaverine production causes enzyme instability. In order to stabilize the CadA homo-decamer structure for in vitro decarboxylation reaction, mutants are designed. Of the four disulfide bond mutants in the multimeric interfacial region, B1 (F14C/K44C) showed a 216-folds increase in the half-life of CadA at 60 °C. On top of B1, another round of mutant screening is performed around F14C and K44C to generate B1/L7M/N8G, which is then examined for cadaverine production (2M lysine and 10% v/v of cell-extract at 50 °C). The reaction pH increased from 4.9 to 8.3, and the final titer of the mutant is 157 g L-1 , that is, 76.7% conversion yield in 9.5 h, whereas the wild-type gave 119 g L-1 , that is, 58.2% conversion yield in 9.5 h.
Subject(s)
Cadaverine/metabolism , Carboxy-Lyases/chemistry , Carboxy-Lyases/metabolism , Lysine/metabolism , Metabolic Engineering/methods , Cadaverine/analysis , Carboxy-Lyases/genetics , Cloning, Molecular , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Models, Molecular , Mutagenesis, Site-DirectedABSTRACT
A macroscopic description of a protein structure allows an understanding of the protein conformations in a more simplistic manner. Here, a new macroscopic approach that utilizes the joints of the protein secondary structures as a basic descriptor for the protein structure is proposed and applied to study the arrangement of secondary structures in helical membrane proteins. Two types of dihedral angle, Ω and λ, were defined based on the joint points of the transmembrane (TM) helices and loops, and employed to analyze 103 non-homologous membrane proteins with 3 to 14 TM helices. The Ω-λ plot, which is a distribution plot of the dihedral angles of the joint points, identified the allowed and disallowed regions of helical arrangement. Analyses of consecutive dihedral angle patterns indicated that there are preferred patterns in the helical alignment and extension of TM proteins, and helical extension pattern in TM proteins is varied as the size of TM proteins increases. Finally, we could identify some symmetric protein pairs in TM proteins under the joint-based coordinate and 3-dimensional coordinates. The joint-based approach is expected to help better understand and model the overall conformational features of complicated large-scale proteins, such as membrane proteins.
Subject(s)
Membrane Proteins/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , Models, MolecularABSTRACT
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.