Conical Intersection Accessibility Dictates Brightness in Red Fluorescent Proteins.

Red fluorescent protein (RFP) variants are highly sought after for in vivo imaging since longer wavelengths improve depth and contrast in fluorescence imaging. However, the lower energy emission wavelength usually correlates with a lower fluorescent quantum yield compared to their green emitting counterparts. To guide the rational design of bright variants, we have theoretically assessed two variants (mScarlet and mRouge) which are reported to have very different brightness. Using an α-CASSCF QM/MM framework (chromophore and all protein residues within 6 Å of it in the QM region, for a total of more than 450 QM atoms), we identify key points on the ground and first excited state potential energy surfaces. The brighter variant mScarlet has a rigid scaffold, and the chromophore stays largely planar on the ground state. The dimmer variant mRouge shows more flexibility and can accommodate a pretwisted chromophore conformation which provides easier access to conical intersections. The main difference between the variants lies in the intersection seam regions, which appear largely inaccessible in mScarlet but partially accessible in mRouge. This observation is mainly related with changes in the cavity charge distribution, the hydrogen-bonding network involving the chromophore and a key ARG/THR mutation (which changes both charge and steric hindrance).

Exploring the Effects of Mutagenesis on FusionRed by Using Excited-State QM/MM Dynamics and Classical Force Field Simulations.

Fluorescent proteins (FPs) are a powerful tool for examining tissues, cells, and subcellular components in vivo and in vitro. FusionRed is a particular FP variant mutated from mKate2 that, in addition to lower cytotoxicity and aggregation rates, has shown potential for acting as a tunable photoswitch. This was posited to stem partially from the presence of a bulky side chain at position 158 and a further stabilizing residue at position 157. In this work, we apply computational techniques including classical molecular dynamics (MD) and combined quantum mechanics/molecular mechanics simulations (QM/MM) to explore the effect of mutagenesis at these locations in FusionRed on the chromophore structure, the excited-state surface, and relative positional stability of the chromophore in the protein pocket. We find specific connections between the statistical sampling of the underlying protein structure and the nonradiative decay mechanisms from excited-state dynamics. A single mutation (C158I) that restricts the motion of the chromophore through a favorable hydrophobic interaction corresponds to an increase in fluorescence quantum yield (FQY), while a second rescue mutation (C158I-A157N) partially restores the flexibility of the chromophore and photoswitchability with favorable water interactions on the surface of the protein that counteracts the original interaction. We suggest that applying this understanding of structural features that inhibit or favor rotation on the excited state can be applied for rational design of new, tunable and red photoswitches.

AutoParams: An Automated Web-Based Tool To Generate Force Field Parameters for Molecular Dynamics Simulations.

Many research questions benefit from molecular dynamics simulations to observe the motions and conformations of molecules over time, which rely on force fields that describe sets of common molecules by category. With the increase of importance for large data sets used in machine learning and growing computational efficiency, the ability to rapidly create large numbers of force field inputs is of high importance. Unusual molecules, such as nucleotide analogues, functionalized carbohydrates, and modified amino acids, are difficult to describe consistently using standard force fields, requiring the development of custom parameters for each unique molecule. While these parameters may be created by individual users, the process can become time-consuming or may introduce errors that may not be immediately apparent. We present an open-source automated parameter generation service, AutoParams, which requires minimal input from the user and creates useful Amber force field parameter sets for most molecules, particularly those that combine molecular types (e.g., a carbohydrate functionalized with a benzene). We include hierarchical atom-typing logic that makes it straightforward to expand with additional force fields and settings, and options for creating monomers in polymers, such as functionalized amino acids. It can be straightforwardly linked to any charge generation program and currently has interfaces to Psi4, PsiRESP, and TeraChem. It is open source and is available via GitHub. It includes error checking and testing protocols to ensure the parameters will be sufficient for subsequent molecular dynamics simulations and streamlines the creation of force field databases.

Rational Design and Expedient Synthesis of Heparan Sulfate Mimetics from Natural Aminoglycosides for Structure and Activity Relationship Studies.

Heparan sulfate (HS) contains variably repeating disaccharide units organized into high- and low-sulfated domains. This rich structural diversity enables HS to interact with many proteins and regulate key signaling pathways. Efforts to understand structure-function relationships and harness the therapeutic potential of HS are hindered by the inability to synthesize an extensive library of well-defined HS structures. We herein report a rational and expedient approach to access a library of 27 oligosaccharides from natural aminoglycosides as HS mimetics in 7-12 steps. This strategy significantly reduces the number of steps as compared to the traditional synthesis of HS oligosaccharides from monosaccharide building blocks. Combined with computational insight, we identify a new class of four trisaccharide compounds derived from the aminoglycoside tobramycin that mimic natural HS and have a strong binding to heparanase but a low affinity for off-target platelet factor-4 protein.

Rational Design of the ß-Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing.

Detection of anions in complex aqueous media is a fundamental challenge with practical utility that can be addressed by supramolecular chemistry. Biomolecular hosts such as proteins can be used and adapted as an alternative to synthetic hosts. Here, we report how the mutagenesis of the ß-bulge residues (D137 and W138) in mNeonGreen, a bright, monomeric fluorescent protein, unlocks and tunes the anion preference at physiological pH for sulfate, resulting in the turn-off sensor SulfOFF-1. This unprecedented sensing arises from an enhancement in the kinetics of binding, largely driven by position 138. In line with these data, molecular dynamics (MD) simulations capture how the coordinated entry and gating of sulfate into the ß-barrel is eliminated upon mutagenesis to facilitate binding and fluorescence quenching.

Computational investigations of selected enzymes from two iron and α-ketoglutarate-dependent families.

DNA alkylation is used as the key epigenetic mark in eukaryotes, however, most alkylation in DNA can result in deleterious effects. Therefore, this process needs to be tightly regulated. The enzymes of the AlkB and Ten-Eleven Translocation (TET) families are members of the Fe and alpha-ketoglutarate-dependent superfamily of enzymes that are tasked with dealkylating DNA and RNA in cells. Members of these families span all species and are an integral part of transcriptional regulation. While both families catalyze oxidative dealkylation of various bases, each has specific preference for alkylated base type as well as distinct catalytic mechanisms. This perspective aims to provide an overview of computational work carried out to investigate several members of these enzyme families including AlkB, ALKB Homolog 2, ALKB Homolog 3 and Ten-Eleven Translocate 2. Insights into structural details, mutagenesis studies, reaction path analysis, electronic structure features in the active site, and substrate preferences are presented and discussed.

Characterization of Nine Cancer-Associated Variants in Human DNA Polymerase κ.

Specialized DNA damage-bypass Y-family DNA polymerases contribute to cancer prevention by providing cellular tolerance to DNA damage that can lead to mutations and contribute to cancer progression by increasing genomic instability. Y-family polymerases can also bypass DNA adducts caused by chemotherapy agents. One of the four human Y-family DNA polymerases, DNA polymerase (pol) κ, has been shown to be specific for bypass of minor groove adducts and inhibited by major groove adducts. In addition, mutations in the gene encoding pol κ are associated with different types of cancers as well as with chemotherapy responses. We characterized nine variants of pol κ whose identity was inferred from cancer-associated single nucleotide polymorphisms for polymerization activity on undamaged and damaged DNA, their abilities to extend from mismatched or damaged base pairs at primer termini, and overall stability and dynamics. We find that these pol κ variants generally fall into three categories: similar activity to wild-type (WT) pol κ (L21F, I39T, P169T, F192C, and E292K), more active than WT pol κ (S423R), and less active than pol κ (R219I, R298H, and Y432S). Of these, only pol κ variants R298H and Y432S had markedly reduced thermal stability. Molecular dynamics (MD) simulations with undamaged DNA revealed that the active variant F192C and more active variant S423R with either correct or incorrect incoming nucleotide mimic WT pol κ with the correct incoming nucleotide, whereas the less active variants R219I, R298H, and Y432S with the correct incoming nucleotide mimic WT pol κ with the incorrect incoming nucleotide. Thus, the observations from MD simulations suggest a possible explanation for the observed experimental results that pol κ adopts specific active and inactive conformations that depend on both the protein variant and the identity of the DNA adduct.

ALKBH7 Variant Related to Prostate Cancer Exhibits Altered Substrate Binding.

The search for prostate cancer biomarkers has received increased attention and several DNA repair related enzymes have been linked to this dysfunction. Here we report a targeted search for single nucleotide polymorphisms (SNPs) and functional impact characterization of human ALKBH family dioxygenases related to prostate cancer. Our results uncovered a SNP of ALKBH7, rs7540, which is associated with prostate cancer disease in a statistically significantly manner in two separate cohorts, and maintained in African American men. Comparisons of molecular dynamics (MD) simulations on the wild-type and variant protein structures indicate that the resulting alteration in the enzyme induces a significant structural change that reduces ALKBH7's ability to bind its cosubstrate. Experimental spectroscopy studies with purified proteins validate our MD predictions and corroborate the conclusion that this cancer-associated mutation affects productive cosubstrate binding in ALKBH7.

Reduced structural flexibility for an exonuclease deficient DNA polymerase III mutant.

DNA synthesis, carried out by DNA polymerases, requires balancing speed and accuracy for faithful replication of the genome. High fidelity DNA polymerases contain a 3'-5' exonuclease domain that can remove misincorporated nucleotides on the 3' end of the primer strand, a process called proofreading. The E. coli replicative polymerase, DNA polymerase III, has spatially separated (∼55 Å apart) polymerase and exonuclease subunits. Here, we report on the dynamics of E. coli DNA polymerase III proofreading in the presence of its processivity factor, the ß2-sliding clamp, at varying base pair termini using single-molecule FRET. We find that the binding kinetics do not depend on the base identity at the termini, indicating a tolerance for DNA mismatches. Further, our single-molecule data and MD simulations show two previously unobserved features: (1) DNA Polymerase III is a highly dynamic protein that adopts multiple conformational states while bound to DNA with matched or mismatched ends, and (2) an exonuclease-deficient DNA polymerase III has reduced conformational flexibility. Overall, our single-molecule experiments provide high time-resolution insight into a mechanism that ensures high fidelity DNA replication to maintain genome integrity.

Computational Simulations of DNA Polymerases: Detailed Insights on Structure/Function/Mechanism from Native Proteins to Cancer Variants.

Genetic information is vital in the cell cycle of DNA-based organisms. DNA polymerases (DNA Pols) are crucial players in transactions dealing with these processes. Therefore, the detailed understanding of the structure, function, and mechanism of these proteins has been the focus of significant effort. Computational simulations have been applied to investigate various facets of DNA polymerase structure and function. These simulations have provided significant insights over the years. This perspective presents the results of various computational studies that have been employed to research different aspects of DNA polymerases including detailed reaction mechanism investigation, mutagenicity of different metal cations, possible factors for fidelity synthesis, and discovery/functional characterization of cancer-related mutations on DNA polymerases.

LICHEM: A QM/MM program for simulations with multipolar and polarizable force fields.

We introduce an initial implementation of the LICHEM software package. LICHEM can interface with Gaussian, PSI4, NWChem, TINKER, and TINKER-HP to enable QM/MM calculations using multipolar/polarizable force fields. LICHEM extracts forces and energies from unmodified QM and MM software packages to perform geometry optimizations, single-point energy calculations, or Monte Carlo simulations. When the QM and MM regions are connected by covalent bonds, the pseudo-bond approach is employed to smoothly transition between the QM region and the polarizable force field. A series of water clusters and small peptides have been employed to test our initial implementation. The results obtained from these test systems show the capabilities of the new software and highlight the importance of including explicit polarization. © 2016 Wiley Periodicals, Inc.

Dimer Interface Destabilization of Photodissociative Dronpa Driven by Asymmetric Monomer Dynamics.

Photoswitchable Dronpa (psDronpa) is a unique member of the fluorescent protein family that can undergo reversible photoinduced switching between fluorescent and dark states and has recently been engineered into a dimer (pdDronpaV) that can dissociate and reassociate as part of its photoswitchable pathway. However, the specific details of the protein structure-function relationship of the dimer interface along with how the dimer proteins interact with each other upon chromophore isomerization are not yet clear. Classical molecular dynamics simulations were performed on psDronpa as monomers and dimers as well as the pdDronpaV dimer and with cis/trans chromophore structures. Analysis of the cis and trans isomers of the chromophore illustrated key differences between their interactions with residues in the protein in both the monomer and dimer forms of psDronpa. Examination of the psDronpa dimer showed nonidentical chromophore interactions between the domains, indicating domain directional favoring. Examination of the trans form of pdDronpaV illuminated the importance of hydrogen bonding between the monomeric domains in maintaining their association, as well as illustrating the motion of dissociation of the domains. This discovery offers important information for possible future mutations of pdDronpaV that might be made to accelerate dissociation.

Simulation-guided engineering of split GFPs with efficient ß-strand photodissociation.

Green fluorescent proteins (GFPs) are ubiquitous for protein tagging and live-cell imaging. Split-GFPs are widely used to study protein-protein interactions by fusing proteins of interest to split GFP fragments that create a fluorophore upon typically irreversible complementation. Thus, controlled dissociation of the fragments is desirable. Although we have found that split strands can be photodissociated, the quantum efficiency of light-induced photodissociation of split GFPs is low. Traditional protein engineering approaches to increase efficiency, including extensive mutagenesis and screening, have proved difficult to implement. To reduce the search space, key states in the dissociation process are modeled by combining classical and enhanced sampling molecular dynamics with QM/MM calculations, enabling the rational design and engineering of split GFPs with up to 20-fold faster photodissociation rates using non-intuitive amino acid changes. This demonstrates the feasibility of modeling complex molecular processes using state-of-the-art computational methods, and the potential of integrating computational methods to increase the success rate in protein engineering projects.

Proton Transfer from a Photoacid to a Water Wire: First Principles Simulations and Fast Fluorescence Spectroscopy.

Proton transfer reactions are ubiquitous in chemistry, especially in aqueous solutions. We investigate photoinduced proton transfer between the photoacid 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) and water using fast fluorescence spectroscopy and ab initio molecular dynamics simulations. Photoexcitation causes rapid proton release from the HPTS hydroxyl. Previous experiments on HPTS/water described the progress from photoexcitation to proton diffusion using kinetic equations with two time constants. The shortest time constant has been interpreted as protonated and photoexcited HPTS evolving into an "associated" state, where the proton is "shared" between the HPTS hydroxyl and an originally hydrogen bonded water. The longer time constant has been interpreted as indicating evolution to a "solvent separated" state where the shared proton undergoes long distance diffusion. In this work, we refine the previous experimental results using very pure HPTS. We then use excited state ab initio molecular dynamics to elucidate the detailed molecular mechanism of aqueous excited state proton transfer in HPTS. We find that the initial excitation results in rapid rearrangement of water, forming a strong hydrogen bonded network (a "water wire") around HPTS. HPTS then deprotonates in ≤3 ps, resulting in a proton that migrates back and forth along the wire before localizing on a single water molecule. We find a near linear relationship between the emission wavelength and proton-HPTS distance over the simulated time scale, suggesting that the emission wavelength can be used as a ruler for the proton distance. Our simulations reveal that the "associated" state corresponds to a water wire with a mobile proton and that the diffusion of the proton away from this water wire (to a generalized "solvent-separated" state) corresponds to the longest experimental time constant.

Proton Transfer Dynamics in the Aprotic Proton Accepting Solvent 1-Methylimidazole.

The dynamics of proton transfer to the aprotic solvent 1-methylimidazole (MeIm, proton acceptor) from the photoacid 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) was investigated using fast fluorescence measurements. The closely related molecule, 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt (MPTS), which is not a photoacid, was also studied for comparison. Following optical excitation, the wavelength-dependent population dynamics of HPTS in MeIm resulting from the deprotonation process were collected over the entire fluorescence emission window. Analysis of the time-dependent fluorescence spectra revealed four distinct fluorescence bands that appear and decay on different time scales. We label these four states as protonated (P), associated I (AI), associated II (AII), and deprotonated (D). We find that the simple kinetic scheme of P → AI → AII → D is not consistent with the data. Instead, the kinetic scheme that describes the data has P decaying into AI, which mainly goes on to deprotonation (D), but AI can also feed into AII. AII can return to AI or decay to the ground state, but does not deprotonate within experimental error. Quantum chemistry and excited state QM/MM Born-Oppenheimer molecular dynamics simulations indicate that AI and AII are two H-bonding conformations of MeIm to the HPTS hydroxyl, axial, and equatorial, respectively.

Unfolding Pathways of Hen Egg-White Lysozyme in Ethanol.

The aggregation of amyloid fibrils can lead to various diseases including Alzheimer's, Parkinson's disease, and transmissible spongiform encephalopathy. Amyloid fibrils can develop from a variety of proteins in the body as they misfold into a primarily ß-sheet structure and aggregate. Human lysozyme has been shown to have far reaching effects in the human health-a homologous enzyme, hen egg-white lysozyme, has been shown to denature to a primarily ß-sheet structure at low pH and high alcohol content solution. We have studied these systems in atomic-level detail with a combination of constant pH and microsecond long molecular dynamics simulation in explicit solvent, which cumulatively total over 10 µs of simulation time. These studies have allowed us to determine two potential unfolding pathways depending on the protonation state of a key glutamic acid residue as well as the effect of solution dynamics and pH on the unfolding process.

Bulky Lesion Bypass Requires Dpo4 Binding in Distinct Conformations.

Translesion DNA synthesis is an essential process that helps resume DNA replication at forks stalled near bulky adducts on the DNA. Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) that can be metabolically activated to benzo[a]pyrene diol epoxide (BPDE), which then can react with DNA to form carcinogenic DNA adducts. Here, we have used single-molecule florescence resonance energy transfer (smFRET) experiments, classical molecular dynamics simulations, and nucleotide incorporation assays to investigate the mechanism by which the model Y-family polymerase, Dpo4, bypasses a (+)-cis-B[a]P-N 2-dG adduct in DNA. Our data show that when (+)-cis-B[a]P-N 2-dG is the templating base, the B[a]P moiety is in a non-solvent exposed conformation stacked within the DNA helix, where it effectively blocks nucleotide incorporation across the adduct by Dpo4. However, when the media contains a small amount of dimethyl sulfoxide (DMSO), the adduct is able to move to a solvent-exposed conformation, which enables error-prone DNA replication past the adduct. When the primer terminates across from the adduct position, the addition of DMSO leads to the formation of an insertion complex capable of accurate nucleotide incorporation.

Investigating carbohydrate based ligands for galectin-3 with docking and molecular dynamics studies.

Galectin-3 (Gal-3) is a carbohydrate binding protein that is overexpressed in several types of cancers, including pancreatic cancer, which makes it a good target for both imaging and therapeutic drug design. A ligand library specialized for 18F positron emission tomography (PET) has been investigated with molecular dynamics (MD) and free energy methods to determine the relative binding energies of various potential ligands. Our results suggest that traditional docking methods can give good results when complemented by molecular dynamics and free energy methods for these types of ligands. Available experimental binding affinities for a small number of the tested compounds show very good agreement with the calculated energies and provide the rational approach for design of Gal-3 ligands with even higher affinity.