ABSTRACT
Bioconjugation of polymers to proteins is a method to impart improved stability and pharmacokinetic properties to biologic systems. However, the precise effects of polymer architecture on the resulting bioconjugates are not well understood. Particularly, cyclic polymers are known to possess unique features such as a decreased hydrodynamic radius when compared to their linear counterparts of the same molecular weight, but have not yet been studied. Here, we report the first bioconjugation of a cyclic polymer, poly(ethylene glycol) (PEG), to a model protein, T4 lysozyme, containing a single engineered cysteine residue (V131C). We compare the stability and activity of this conjugate with those of a linear PEG-T4 lysozyme analogue of similar molecular weight. Furthermore, we used molecular dynamics (MD) simulations to determine the behavior of the polymer-protein conjugates in solution. We introduce cyclic polymer-protein conjugates as potential candidates for the improvement of biologic therapeutics.
Subject(s)
Molecular Dynamics Simulation , Muramidase , Polyethylene Glycols , Polyethylene Glycols/chemistry , Muramidase/chemistry , Bacteriophage T4/enzymologyABSTRACT
The structural diversity of biological macromolecules in different environments contributes complexity to enzymological processes vital for cellular functions. Fluorescence resonance energy transfer and electron microscopy are used to investigate the enzymatic reaction of T4 DNA ligase catalyzing the ligation of nicked DNA. The data show that both the ligase-AMP complex and the ligase-AMP-DNA complex can have four conformations. This finding suggests the parallel occurrence of four ligation reaction pathways, each characterized by specific conformations of the ligase-AMP complex that persist in the ligase-AMP-DNA complex. Notably, these complexes have DNA bending angles of ≈0°, 20°, 60°, or 100°. The mechanism of parallel reactions challenges the conventional notion of simple sequential reaction steps occurring among multiple conformations. The results provide insights into the dynamic conformational changes and the versatile attributes of T4 DNA ligase and suggest that the parallel multiple reaction pathways may correspond to diverse T4 DNA ligase functions. This mechanism may potentially have evolved as an adaptive strategy across evolutionary history to navigate complex environments.
Subject(s)
DNA Ligases , DNA , DNA Ligases/metabolism , DNA/metabolism , DNA/genetics , DNA/chemistry , DNA Repair , Fluorescence Resonance Energy Transfer/methods , Nucleic Acid Conformation , Bacteriophage T4/enzymology , Bacteriophage T4/genetics , Bacteriophage T4/metabolism , Microscopy, Electron/methodsABSTRACT
T4 polynucleotide kinase (T4 PNK) phosphorylates the 5'-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3'-phosphate group is present. In this study, the T4 PNK-catalyzed conversion of adenosine 3'-monophosphate (3'-AMP) to adenosine-3',5'-bisphosphate was characterized using isothermal titration calorimetry (ITC). Although ITC is typically used to study ligand binding, in this case the instrument was used to evaluate enzyme kinetics by monitoring the heat production due to reaction enthalpy. The reaction was initiated with a single injection of 3'-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30 °C, and Michaelis-Menten analysis was performed on the reaction rates derived from the plot of differential power versus time. The Michaelis-Menten constant, KM, was 13 µM, and the turnover number, kcat, was 8 s-1. The effect of inhibitors was investigated using pyrophosphate (PPi). PPi caused a dose-dependent decrease in the apparent kcat and increase in the apparent KM under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3'-AMP were determined to be -25 kJ/mol and 43 kJ/mol, respectively. ITC is seldom used as a tool to study enzyme kinetics, particularly for technically-challenging enzymes such as kinases. This study demonstrates that quantitative analysis of kinase activity can be amenable to the ITC single injection approach.
Subject(s)
Calorimetry , Polynucleotide 5'-Hydroxyl-Kinase , Kinetics , Calorimetry/methods , Polynucleotide 5'-Hydroxyl-Kinase/metabolism , Polynucleotide 5'-Hydroxyl-Kinase/chemistry , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Thermodynamics , Bacteriophage T4/enzymology , Diphosphates/chemistry , Diphosphates/metabolism , PhosphorylationABSTRACT
The use of computer simulation for binding affinity prediction is growing in drug discovery. However, its wider use is constrained by the accuracy of the free energy calculations. The key sources of error are the force fields used to depict molecular interactions and insufficient sampling of the configurational space. To improve the quality of the force field, we developed a Python-based computational workflow. The workflow described here uses the minimal basis iterative stockholder (MBIS) method to determine atomic charges and Lennard-Jones parameters from the polarized molecular density. This is done by performing electronic structure calculations on various configurations of the ligand when it is both bound and unbound. In addition, we validated a simulation procedure that accounts for the protein and ligand degrees of freedom to precisely calculate binding free energies. This was achieved by comparing the self-adjusted mixture sampling and nonequilibrium thermodynamic integration methods using various protein and ligand conformations. The accuracy of predicting binding affinity is improved by using MBIS-derived force field parameters and a validated simulation procedure. This improvement surpasses the chemical precision for the eight aromatic ligands, reaching a root-mean-square error of 0.7 kcal/mol.
Subject(s)
Muramidase , Protein Binding , Thermodynamics , Muramidase/chemistry , Muramidase/metabolism , Ligands , Electrons , Bacteriophage T4/enzymology , Mutation , Protein Conformation , Molecular Dynamics Simulation , Models, MolecularABSTRACT
The mechanisms by which viruses hijack the genetic machinery of the cells they infect are of current interest. When bacteriophage T4 infects Escherichia coli, it uses three different adenosine diphosphate (ADP)-ribosyltransferases (ARTs) to reprogram the transcriptional and translational apparatus of the host by ADP-ribosylation using nicotinamide adenine dinucleotide (NAD) as a substrate1,2. NAD has previously been identified as a 5' modification of cellular RNAs3-5. Here we report that the T4 ART ModB accepts not only NAD but also NAD-capped RNA (NAD-RNA) as a substrate and attaches entire RNA chains to acceptor proteins in an 'RNAylation' reaction. ModB specifically RNAylates the ribosomal proteins rS1 and rL2 at defined Arg residues, and selected E. coli and T4 phage RNAs are linked to rS1 in vivo. T4 phages that express an inactive mutant of ModB have a decreased burst size and slowed lysis of E. coli. Our findings reveal a distinct biological role for NAD-RNA, namely the activation of the RNA for enzymatic transfer to proteins. The attachment of specific RNAs to ribosomal proteins might provide a strategy for the phage to modulate the host's translation machinery. This work reveals a direct connection between RNA modification and post-translational protein modification. ARTs have important roles far beyond viral infections6, so RNAylation may have far-reaching implications.
Subject(s)
ADP Ribose Transferases , Bacteriophage T4 , Escherichia coli Proteins , Escherichia coli , NAD , RNA , Viral Proteins , ADP Ribose Transferases/metabolism , Bacteriophage T4/enzymology , Bacteriophage T4/genetics , Bacteriophage T4/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/virology , NAD/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Viral Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , RNA/chemistry , RNA/genetics , RNA/metabolism , Protein Biosynthesis , Gene Expression Regulation, Bacterial , Protein Processing, Post-TranslationalABSTRACT
BACKGROUND: Recombinase (uvsY and uvsX) from bacteriophage T4 is a key enzyme for recombinase polymerase amplification (RPA) that amplifies a target DNA sequence at a constant temperature with a single-stranded DNA-binding protein and a strand-displacing polymerase. The present study was conducted to examine the effects of the N- and C-terminal tags of uvsY on its function in RPA to detect SARS-CoV-2 DNA. METHODS: Untagged uvsY (uvsY-Δhis), N-terminal tagged uvsY (uvsY-Nhis), C-terminal tagged uvsY (uvsY-Chis), and N- and C-terminal tagged uvsY (uvsY-NChis) were expressed in Escherichia coli and purified. RPA reaction was carried out with the in vitro synthesized standard DNA at 41 °C. The amplified products were separated on agarose gels. RESULTS: The minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 105, 60, 600, and 600 copies for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The minimal reaction time at which the amplified products were observed were 20, 20, 30, and 20 min for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The RPA with uvsY-Nhis exhibited clearer bands than that with either of other three uvsYs. CONCLUSIONS: The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA.
Subject(s)
Bacteriophage T4/enzymology , DNA, Viral/chemistry , DNA-Binding Proteins/chemistry , Membrane Proteins/chemistry , Nucleic Acid Amplification Techniques , SARS-CoV-2/chemistry , Viral Proteins/chemistry , DNA, Viral/genetics , SARS-CoV-2/geneticsABSTRACT
Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing RNA circles as PKR inhibitors. Here, we report that RNA circles synthesized by permuted self-splicing thymidylate synthase (td) introns from T4 bacteriophage or by Anabaena pre-tRNA group I intron could induce an immune response. Autocatalytic splicing introduces â¼74 nt td or â¼186 nt Anabaena extraneous fragments that can distort the folding status of original circular RNAs or form structures themselves to provoke innate immune responses. In contrast, synthesized RNA circles produced by T4 RNA ligase without extraneous fragments exhibit minimized immunogenicity. Importantly, directly ligated circular RNAs that form short dsRNA regions efficiently suppress PKR activation 103- to 106-fold higher than reported chemical compounds C16 and 2-AP, highlighting the future use of circular RNAs as potent inhibitors for diseases related to PKR overreaction.
Subject(s)
Protein Kinase Inhibitors/pharmacology , RNA, Circular/pharmacology , eIF-2 Kinase/antagonists & inhibitors , A549 Cells , Bacteriophage T4/enzymology , Bacteriophage T4/genetics , HEK293 Cells , HeLa Cells , Humans , Immunity, Innate/drug effects , Introns , Nucleic Acid Conformation , Protein Kinase Inhibitors/immunology , RNA Ligase (ATP)/genetics , RNA Ligase (ATP)/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Circular/genetics , RNA, Circular/immunology , Thymidylate Synthase/genetics , Thymidylate Synthase/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , eIF-2 Kinase/metabolismABSTRACT
Helicases are molecular motors with many activities. They use the energy from ATP hydrolysis to unwind double-stranded nucleic acids while translocating on the single-stranded DNA. In addition to unwinding, many helicases are able to remove proteins from nucleic acids. Bacteriophage T4 Dda is able to displace a variety of DNA binding proteins and streptavidin bound to biotinylated oligonucleotides. We have identified a subdomain of Dda that when deleted, results in a protein variant that has nearly wild type activity for unwinding double-stranded DNA but exhibits greatly reduced streptavidin displacement activity. Interestingly, this domain has little effect on displacement of either gp32 or BamHI bound to DNA but does affect displacement of trp repressor from DNA. With this variant, we have identified residues which enhance displacement of some proteins from DNA.
Subject(s)
Bacteriophage T4 , DNA Helicases , Viral Proteins , Bacterial Proteins , Bacteriophage T4/enzymology , DNA/chemistry , DNA Helicases/chemistry , DNA, Single-Stranded/genetics , Repressor Proteins , Streptavidin/metabolism , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
We describe an approach for integrating distance restraints from Double Electron-Electron Resonance (DEER) spectroscopy into Rosetta with the purpose of modeling alternative protein conformations from an initial experimental structure. Fundamental to this approach is a multilateration algorithm that harnesses sets of interconnected spin label pairs to identify optimal rotamer ensembles at each residue that fit the DEER decay in the time domain. Benchmarked relative to data analysis packages, the algorithm yields comparable distance distributions with the advantage that fitting the DEER decay and rotamer ensemble optimization are coupled. We demonstrate this approach by modeling the protonation-dependent transition of the multidrug transporter PfMATE to an inward facing conformation with a deviation to the experimental structure of less than 2Å Cα RMSD. By decreasing spin label rotamer entropy, this approach engenders more accurate Rosetta models that are also more closely clustered, thus setting the stage for more robust modeling of protein conformational changes.
Subject(s)
Algorithms , Models, Molecular , Protein Conformation , Bacteriophage T4/enzymology , Computational Biology , Electron Spin Resonance Spectroscopy/statistics & numerical data , Methionine Adenosyltransferase/chemistry , Molecular Dynamics Simulation/statistics & numerical data , Multidrug Resistance-Associated Proteins/chemistry , Muramidase/chemistry , Pyrococcus furiosus/enzymology , Software , Spin LabelsABSTRACT
Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site. Gln 118 forms a hydrogen-bonded junction in a helical unit that we term the central coupler, because it connects the catalytic centers to DNA and the sliding clamp. A suppressor mutation indicates that hydrogen bonding in the junction is important, and molecular dynamics simulations reveal that it maintains rigidity in the central coupler. The glutamine-mediated junction is preserved in diverse AAA+ ATPases, suggesting that a connected network of hydrogen bonds that links ATP molecules is an essential aspect of allosteric communication in these proteins.
Subject(s)
ATPases Associated with Diverse Cellular Activities/metabolism , Adenosine Triphosphate/metabolism , Bacteriophage T4/enzymology , DNA-Directed DNA Polymerase/metabolism , ATPases Associated with Diverse Cellular Activities/chemistry , ATPases Associated with Diverse Cellular Activities/genetics , Allosteric Regulation , Bacteriophage T4/genetics , Bacteriophage T4/growth & development , Catalysis , DNA Replication , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , Glutamine/metabolism , Hydrogen Bonding , Molecular Dynamics Simulation , Mutation , Protein Conformation , Structure-Activity Relationship , Virus ReplicationABSTRACT
All-inorganic lead halide perovskites have become promising alternatives to traditional semiconductor electrochemiluminescence (ECL) emitters because of their appealing optoelectronic attributes, but major challenges remain in improving their stability and enhancing charge injection/transfer capacities. Herein, a self-sustaining suprastructure was constructed by successively loading aminated carbon dots (NCDs) and CsPbBr3 perovskite quantum dots (PeQDs) in situ into hierarchical zeolite imidazole framework-8 (HZIF-8). The elaborated architecture guarantees not only improved stability via the peripheral HZIF-8 protective barrier but also accelerated charge transport and efficient self-enhanced ECL between PeQDs and the surrounding NCDs in a confined structure. As a result, the ternary nanocomposite is endowed with greatly improved stability and ECL efficiency. Based on this ternary nanocomposite as an electrode substrate, a novel ECL sensing strategy is further proposed for the first time to evaluate T4 polynucleotide kinase activity and screen its inhibitors. This work opens an avenue for the advancement of perovskite-based ECL emitters as well as the development of corresponding applications in the ECL domain.
Subject(s)
Biosensing Techniques , Electrochemical Techniques , Luminescent Measurements , Metal-Organic Frameworks/chemistry , Polynucleotide 5'-Hydroxyl-Kinase/analysis , Quantum Dots/chemistry , Bacteriophage T4/enzymology , Bromides/chemistry , Carbon , Cesium/chemistry , Enzyme Inhibitors/pharmacology , Lead/chemistry , Metal-Organic Frameworks/chemical synthesis , Particle Size , Polynucleotide 5'-Hydroxyl-Kinase/antagonists & inhibitors , Polynucleotide 5'-Hydroxyl-Kinase/metabolism , Surface PropertiesABSTRACT
Sampling multiple binding modes of a ligand in a single molecular dynamics simulation is difficult. A given ligand may have many internal degrees of freedom, along with many different ways it might orient itself in a binding site or across several binding sites, all of which might be separated by large energy barriers. We have developed a novel Monte Carlo move called molecular darting (MolDarting) to reversibly sample between predefined binding modes of a ligand. Here, we couple this with nonequilibrium candidate Monte Carlo (NCMC) to improve acceptance of moves. We apply this technique to a simple dipeptide system, a ligand binding to T4 lysozyme L99A, and ligand binding to HIV integrase to test this new method. We observe significant increases in acceptance compared to uniformly sampling the internal and rotational/translational degrees of freedom in these systems.
Subject(s)
Bacteriophage T4/enzymology , HIV Integrase/metabolism , HIV/enzymology , Muramidase/metabolism , Binding Sites , Dipeptides/metabolism , HIV Integrase/chemistry , HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/pharmacology , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Monte Carlo Method , Muramidase/chemistry , Protein Binding , ThermodynamicsABSTRACT
Lysozyme is widely used as a model protein in studies of structure-function relationships. Recently, lysozyme has gained attention for use in accelerating the degradation of secondary sludge, which mainly consists of bacteria. However, a high-throughput screening system for lysozyme engineering has not been reported. Here, we present a lysozyme screening system using a genetically encoded biosensor. We first cloned bacteriophage T4 lysozyme (T4L) into a plasmid under control of the araBAD promoter. The plasmid was expressed in Escherichia coli with no toxic effects on growth. Next, we observed that increased soluble T4L expression decreased the fluorescence produced by the genetic enzyme screening system. To investigate T4L evolution based on this finding, we generated a T4L random mutation library, which was screened using the genetic enzyme screening system. Finally, we identified two T4L variants showing 1.4-fold enhanced lytic activity compared to native T4L. To our knowledge, this is the first report describing the use of a genetically encoded biosensor to investigate bacteriophage T4L evolution. Our approach can be used to investigate the evolution of other lysozymes, which will expand the applications of lysozyme.
Subject(s)
Bacteriophage T4 , Biosensing Techniques , Directed Molecular Evolution , Escherichia coli , Muramidase , Viral Proteins , Bacteriophage T4/enzymology , Bacteriophage T4/growth & development , Escherichia coli/enzymology , Escherichia coli/genetics , Muramidase/genetics , Muramidase/metabolism , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
This protocol describes procedures for cloning blunt-ended DNA fragments into linearized plasmid vectors. To obtain the maximum number of "correct" ligation products when cloning blunt-ended target fragments, the two components of DNA in the ligation reaction must be present at an appropriate ratio. If the molar ratio of plasmid vector to target DNA is too high, then the ligation reaction may generate an undesirable number of circular empty plasmids, both monomeric and polymeric; if too low, the ligation reaction may generate an excess of linear and circular homopolymers and heteropolymers of varying sizes, orientations, and compositions. For this reason, the orientation of the foreign DNA and the number of inserts in each recombinant clone must always be validated by restriction endonuclease mapping or some other means.
Subject(s)
Cloning, Molecular/methods , DNA/genetics , Genetic Vectors/genetics , Plasmids/genetics , Bacteriophage T4/enzymology , Buffers , DNA Ligases/metabolism , DNA, Recombinant/genetics , DNA, Recombinant/isolation & purification , DNA, Recombinant/metabolism , Escherichia coli/genetics , Viral Proteins/metabolismABSTRACT
The bacteriophage T4 genome contains two genes that code for proteins with lysozyme activity-e and 5. Gene e encodes the well-known T4 lysozyme (commonly called T4L) that functions to break the peptidoglycan layer late in the infection cycle, which is required for liberating newly assembled phage progeny. Gene product 5 (gp5) is the tail-associated lysozyme, a component of the phage particle. It forms a spike at the tip of the tail tube and functions to pierce the outer membrane of the Escherichia coli host cell after the phage has attached to the cell surface. Gp5 contains a T4L-like lysozyme domain that locally digests the peptidoglycan layer upon infection. The T4 Spackle protein (encoded by gene 61.3) has been thought to play a role in the inhibition of gp5 lysozyme activity and, as a consequence, in making cells infected by bacteriophage T4 resistant to later infection by T4 and closely related phages. Here we show that (1) gp61.3 is secreted into the periplasm where its N-terminal periplasm-targeting peptide is cleaved off; (2) gp61.3 forms a 1:1 complex with the lysozyme domain of gp5 (gp5Lys); (3) gp61.3 selectively inhibits the activity of gp5, but not that of T4L; (4) overexpression of gp5 causes cell lysis. We also report a crystal structure of the gp61.3-gp5Lys complex that demonstrates that unlike other known lysozyme inhibitors, gp61.3 does not interact with the active site cleft. Instead, it forms a "wall" that blocks access of an extended polysaccharide substrate to the cleft and, possibly, locks the enzyme in an "open-jaw"-like conformation making catalysis impossible.
Subject(s)
Bacteriophage T4/enzymology , Muramidase/antagonists & inhibitors , Viral Proteins/metabolism , Bacteriophage T4/genetics , Crystallography, X-Ray , Escherichia coli/virology , Genome, Viral/genetics , Protein Conformation , Viral Proteins/geneticsABSTRACT
The detailed understanding of the binding of small molecules to proteins is the key for the development of novel drugs or to increase the acceptance of substrates by enzymes. Nowadays, computer-aided design of protein-ligand binding is an important tool to accomplish this task. Current approaches typically rely on high-throughput docking essays or computationally expensive atomistic molecular dynamics simulations. Here, we present an approach to use the recently re-parametrized coarse-grained Martini model to perform unbiased millisecond sampling of protein-ligand interactions of small drug-like molecules. Remarkably, we achieve high accuracy without the need of any a priori knowledge of binding pockets or pathways. Our approach is applied to a range of systems from the well-characterized T4 lysozyme over members of the GPCR family and nuclear receptors to a variety of enzymes. The presented results open the way to high-throughput screening of ligand libraries or protein mutations using the coarse-grained Martini model.
Subject(s)
Molecular Dynamics Simulation , Protein Binding , Proteins/chemistry , Bacteriophage T4/enzymology , Biophysics , Computational Biology , High-Throughput Screening Assays , Ligands , Molecular Docking Simulation , Muramidase/chemistry , Protein Conformation , ThermodynamicsABSTRACT
Molecular dynamics (MD) simulations of explicit representations of fluorescent dyes attached via a linker to a protein allow, e.g., probing commonly used approximations for dye localization and/or orientation or modeling Förster resonance energy transfer. However, setting up and performing such MD simulations with the AMBER suite of biomolecular simulation programs has remained challenging due to the unavailability of an easy-to-use set of parameters within AMBER. Here, we adapted the AMBER-DYES parameter set derived by Graen et al. [J. Chem. Theory Comput. 10, 5505 (2014)] into "AMBER-DYES in AMBER" to generate a force field applicable within AMBER for commonly used fluorescent dyes and linkers attached to a protein. In particular, the computationally efficient graphics processing unit (GPU) implementation of the AMBER MD engine can now be exploited to overcome sampling issues of dye movements. The implementation is compatible with state-of-the-art force fields such as GAFF, GAFF2, ff99SB, ff14SB, lipid17, and GLYCAM_06j, which allows simulating post-translationally modified proteins and/or protein-ligand complexes and/or proteins in membrane environments. It is applicable with frequently used water models such as TIP3P, TIP4P, TIP4P-Ew, and OPC. For ease of use, a LEaP-based workflow was created, which allows attaching (multiple) dye/linker combinations to a protein prior to further system preparation steps. Following the parameter development described by Graen et al. [J. Chem. Theory Comput. 10, 5505 (2014)] and the adaptation steps described here, AMBER-DYES in AMBER can be extended by additional linkers and fluorescent molecules.
Subject(s)
Fluorescent Dyes/chemistry , Muramidase/chemistry , Viral Proteins/chemistry , Bacteriophage T4/enzymology , Carbocyanines/chemistry , Databases, Chemical , Datasets as Topic , Fluoresceins/chemistry , Molecular Dynamics Simulation , Sulfonic Acids/chemistryABSTRACT
Protein-ligand recognition is dynamic and complex. A key approach in deciphering the mechanism underlying the recognition process is to capture the kinetic process of the ligand in its act of binding to its designated protein cavity. Toward this end, ultralong all-atom molecular dynamics simulation has recently emerged as a popular method of choice because of its ability to record these events at high spatial and temporal resolution. However, success via this route comes at an exorbitant computational cost. Herein, we demonstrate that coarse-grained models of the protein, when systematically optimized to maintain its tertiary fold, can capture the complete process of spontaneous protein-ligand binding from bulk media to the cavity at crystallographic precision and within wall clock time that is orders of magnitude shorter than that of all-atom simulations. The exhaustive sampling of ligand exploration in protein and solvent, harnessed by coarse-grained simulation, leads to elucidation of new ligand recognition pathways and discovery of non-native binding poses.
Subject(s)
Benzamidines/metabolism , Benzene/metabolism , Camphor/metabolism , Cytochrome P-450 Enzyme System/metabolism , Muramidase/metabolism , Trypsin/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacteriophage T4/enzymology , Benzamidines/chemistry , Benzene/chemistry , Camphor/chemistry , Catalytic Domain , Cytochrome P-450 Enzyme System/chemistry , Ligands , Molecular Dynamics Simulation , Muramidase/chemistry , Protein Binding , Pseudomonas putida/enzymology , Trypsin/chemistry , Viral Proteins/chemistry , Viral Proteins/metabolismABSTRACT
Chemical exchange saturation transfer (CEST) NMR experiments have emerged as a powerful tool for characterizing dynamics in proteins. We show here that the CEST approach can be extended to systems with symmetrical exchange, where the NMR signals of all exchanging species are severely broadened. To achieve this, multiquantum CEST (MQ-CEST) is introduced, where the CEST pulse is applied to a longitudinal multispin order density element and the CEST profiles are encoded onto nonbroadened nuclei. The MQ-CEST approach is demonstrated on the restricted rotation of guanidinium groups in arginine residues within proteins. These groups and their dynamics are essential for many enzymes and for noncovalent interactions through the formation of hydrogen bonds, salt-bridges, and π-stacking interactions, and their rate of rotation is highly indicative of the extent of interactions formed. The MQ-CEST method is successfully applied to guanidinium groups in the 19 kDa L99A mutant of T4 lysozyme.
Subject(s)
Arginine/chemistry , Guanidines/chemistry , Muramidase/chemistry , Viral Proteins/chemistry , Bacteriophage T4/enzymology , Molecular Structure , Muramidase/genetics , Mutation , Nuclear Magnetic Resonance, Biomolecular , Viral Proteins/geneticsABSTRACT
A nanoplatform based on metal-organic frameworks (MOFs) and lambda exonuclease (λ exo) for the fluorimetric determination of T4 polynucleotide kinase (T4 PNK) activity and inhibition is described. Fe-MIL-88 was selected as the nanomaterial because of its significant preferential binding ability to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) and its quenching property. The synthesized Fe-MIL-88 was characterized by transmission electron microscope, scanning electron microscope, and X-ray photoelectron spectroscopy. In the presence of T4 PNK, FAM-labeled dsDNA (FAM-dsDNA) is phosphorylated on its 5'-terminal. λ exo then recognizes and cleaves the phosphorylated strand yielding FAM-labeled ssDNA (FAM-ssDNA). The fluorescence of the produced FAM-ssDNA is quenched due to Fe-MIL-88's absorbing on FAM-ssDNA. On the contrary, in the absence of T4 PNK, the phosphorylation and cleavage processes cannot take place. Therefore, the fluorescence of FAM-dsDNA still remains. The fluorescence intensity is detected at the maximum emission wavelength of 524 nm using the maximum excitation wavelength of 488 nm. The assay of T4 PNK based on the fluorescence quenching of FAM-ssDNA achieves a linear relationship in the range 0.01-5.0 U mL-1 with a detection limit of 0.0089 U mL-1 in buffer. The assay exhibits excellent performance for T4 PNK activity determination in a complex biological matrix. The results also reveal the ability of the assay for T4 PNK inhibitor screening. Graphical abstract Schematic presentation of a nanoplatform based on Fe-MIL-88 and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity. FAM-ssDNA, FAM-labeled single-stranded DNA; cDNA, complementary DNA; λ exo, lambda exonuclease;T4 PNK, T4 polynucleotide kinase.