Femtosecond-to-millisecond structural changes in a light-driven sodium pump.

Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.

Noncovalent interactions that tune the reactivities of the flavins in bifurcating electron transferring flavoprotein.

Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize noncovalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin (ETflavin) was calculated to stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers, whereas the Bf flavin (Bfflavin) was found to disfavor the ASQ state more than does free flavin and to be less susceptible to reduction. The stability of ETflavin ASQ was attributed in part to H-bond donation to the flavin O2 from a nearby His side chain, via comparison of models employing different tautomers of His. This H-bond between O2 and the ET site was uniquely strong in the ASQ state, whereas reduction of ETflavin to the anionic hydroquinone (AHQ) was associated with side chain reorientation, backbone displacement, and reorganization of its H-bond network including a Tyr from the other domain and subunit of the ETF. The Bf site was less responsive overall, but formation of the Bfflavin AHQ allowed a nearby Arg side chain to adopt an alternative rotamer that can H-bond to the Bfflavin O4. This would stabilize the anionic Bfflavin and rationalize effects of mutation at this position. Thus, our computations provide insights on states and conformations that have not been possible to characterize experimentally, offering explanations for observed residue conservation and raising possibilities that can now be tested.

Retracted: FoxP3 acts as a cotranscription factor with STAT3 in tumor-induced regulatory T cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the authors. This study provided an explanation for why loss of FoxP3 in inducible regulatory T cells results in reduced expression of interleukin (IL)-10 despite the absence of FoxP3 binding sites in the IL-10 promoter. STAT3 binding sites do exist in the promoter, and evidence for a direct molecular interaction between FoxP3 and STAT3 proteins was provided as an explanation of the effect of loss of FoxP3. As supporting evidence, we reported modeling of a structural interaction between these two transcription factors in Figure 4D. As the N-terminal region of FoxP3, which consists of the Exon-2 region, had not been solved at structural resolution, we mistakenly used what we deduced to be a FoxP3 related transcription factor, NFAT, in the modeling. The model depicted in Figure 4D therefore did not represent a putative interaction between FoxP3 and STAT3 as labeled, but rather a putative interaction between NFAT and STAT3. Given the incorrect labeling of Figure 4D, the lack of documentation in the paper describing exactly how the modeling was performed, the lack of evidence shown in the paper for the choice of NFAT as the modeling partner, and the limited supporting evidence for a cooperative interaction between FoxP3 and STAT3, the editors have concluded with the corresponding author that the appropriate course of action is to retract the paper. We apologize for any confusion and inconvenience caused to readers.

Two-photon conversion of a bacterial phytochrome.

In nature, sensory photoreceptors underlie diverse spatiotemporally precise and generally reversible biological responses to light. Photoreceptors also serve as genetically encoded agents in optogenetics to control by light organismal state and behavior. Phytochromes represent a superfamily of photoreceptors that transition between states absorbing red light (Pr) and far-red light (Pfr), thus expanding the spectral range of optogenetics to the near-infrared range. Although light of these colors exhibits superior penetration of soft tissue, the transmission through bone and skull is poor. To overcome this fundamental challenge, we explore the activation of a bacterial phytochrome by a femtosecond laser emitting in the 1 µm wavelength range. Quantum chemical calculations predict that bacterial phytochromes possess substantial two-photon absorption cross sections. In line with this notion, we demonstrate that the photoreversible Pr ↔ Pfr conversion is driven by two-photon absorption at wavelengths between 1170 and 1450 nm. The Pfr yield was highest for wavelengths between 1170 and 1280 nm and rapidly plummeted beyond 1300 nm. By combining two-photon activation with bacterial phytochromes, we lay the foundation for enhanced spatial resolution in optogenetics and unprecedented penetration through bone, skull, and soft tissue.

Doubling Förster Resonance Energy Transfer Efficiency in Proteins with Extrinsic Thioamide Probes: Implications for Thiomodified Nucleobases.

Designing a potential protein-ligand pair is pivotal, not only to track the protein structure dynamics, but also to assist in an atomistic understanding of drug delivery. Herein, the potential of a small model thioamide probe being used to study albumin proteins is reported. By monitoring the Förster resonance energy transfer (FRET) dynamics with the help of fluorescence spectroscopic techniques, a twofold enhancement in the FRET efficiency of 2-thiopyridone (2TPY), relative to that of its amide analogue, is observed. Molecular dynamics simulations depict the relative position of the free energy minimum to be quite stable in the case of 2TPY through noncovalent interactions with sulfur, which help to enhance the FRET efficiency. Finally, its application is shown by pairing thiouracils with protein. It is found that the site-selective sulfur atom substitution approach and noncovalent interactions with sulfur can substantially enhance the FRET efficiency, which could be a potential avenue to explore in the design of FRET probes to study the structure and dynamics of biomolecules.

High-resolution structure of a partially folded insulin aggregation intermediate.

Insulin has long been served as a model for protein aggregation, both due to the importance of aggregation in the manufacture of insulin and because the structural biology of insulin has been extensively characterized. Despite intensive study, details about the initial triggers for aggregation have remained elusive at the molecular level. We show here that at acidic pH, the aggregation of insulin is likely initiated by a partially folded monomeric intermediate. High-resolution structures of the partially folded intermediate show that it is coarsely similar to the initial monomeric structure but differs in subtle details-the A chain helices on the receptor interface are more disordered and the B chain helix is displaced from the C-terminal A chain helix when compared to the stable monomer. The result of these movements is the creation of a hydrophobic cavity in the center of the protein that may serve as nucleation site for oligomer formation. Knowledge of this transition may aid in the engineering of insulin variants that retain the favorable pharamacokinetic properties of monomeric insulin but are more resistant to aggregation.

Nonproductive Binding Modes as a Prominent Feature of Aß40 Fiber Elongation: Insights from Molecular Dynamics Simulation.

The formation of amyloid fibers has been implicated in a number of neurodegenerative diseases. The growth of amyloid fibers is strongly thermodynamically favorable, but kinetic traps exist where the incoming monomer binds in an incompatible conformation that blocks further elongation. Unfortunately, this process is difficult to follow experimentally at the atomic level. It is also too complex to simulate in full detail and to date has been explored either through coarse-grained simulations, which may miss many important interactions, or full atomic simulations, in which the incoming peptide is constrained to be near the ideal fiber geometry. Here we use an alternate approach starting from a docked complex in which the monomer is from an experimental NMR structure of one of the major conformations in the unbound ensemble, a largely unstructured peptide with the central hydrophobic region in a 310 helix. A 1000 ns full atomic simulation in explicit solvent shows the formation of a metastable intermediate by sequential, concerted movements of both the fiber and the monomer. A Markov state model shows that the unfolded monomer is trapped at the end of the fiber in a set of interconverting antiparallel ß-hairpin conformations. The simulation here may serve as a model for the binding of other non-ß-sheet conformations to amyloid fibers.

Accelerated molecular dynamics simulation analysis of MSI-594 in a lipid bilayer.

Multidrug resistance against the existing antibiotics is one of the most challenging threats across the globe. Antimicrobial peptides (AMPs), in this regard, are considered to be one of the effective alternatives that can overcome bacterial resistance. MSI-594, a 24-residue linear alpha-helical cationic AMP, has been shown to function via the carpet mechanism to disrupt bacterial membrane systems. To better understand the role of lipid composition in the function of MSI-594, in the present study, eight different model membrane systems have been studied using accelerated molecular dynamics (aMD) simulations. The simulated results are helpful in discriminating the particular effects of cationic MSI-594 against zwitterionic POPC, anionic POPG and POPS, and neutral POPE lipid moieties. Additionally, the effects of various heterogeneous POPC/POPG (7 : 3), POPC/POPS (7 : 3), and POPG/POPE (1 : 3 and 3 : 1) bilayer systems on the dynamic interaction of MSI-594 have also been investigated. The effect on the lipid bilayer due to the interaction with the peptide is characterized by lipid acyl-chain order, membrane thickness, and acyl-chain dynamics. Our simulation results show that the lipid composition affects the membrane interaction of MSI-594, suggesting that membrane selectivity is crucial to its mechanism of action. The results reported in this study are helpful to obtain accurate atomistic-level information governing MSI-594 and its membrane disruptive antimicrobial mechanism of action, and to design next generation potent antimicrobial peptides.

Mode of Action of a Designed Antimicrobial Peptide: High Potency against Cryptococcus neoformans.

There is a significant need for developing compounds that kill Cryptococcus neoformans, the fungal pathogen that causes meningoencephalitis in immunocompromised individuals. Here, we report the mode of action of a designed antifungal peptide, VG16KRKP (VARGWKRKCPLFGKGG) against C. neoformans. It is shown that VG16KRKP kills fungal cells mainly through membrane compromise leading to efflux of ions and cell metabolites. Intracellular localization, inhibition of in vitro transcription, and DNA binding suggest a secondary mode of action for the peptide, hinting at possible intracellular targets. Atomistic structure of the peptide determined by NMR experiments on live C. neoformans cells reveals an amphipathic arrangement stabilized by hydrophobic interactions among A2, W5, and F12, a conventional folding pattern also known to play a major role in peptide-mediated Gram-negative bacterial killing, revealing the importance of this motif. These structural details in the context of live cell provide valuable insights into the design of potent peptides for effective treatment of human and plant fungal infections.

Structural Elucidation of the Cell-Penetrating Penetratin Peptide in Model Membranes at the Atomic Level: Probing Hydrophobic Interactions in the Blood-Brain Barrier.

Cell-penetrating peptides (CPPs) have shown promise in nonpermeable therapeutic drug delivery, because of their ability to transport a variety of cargo molecules across the cell membranes and their noncytotoxicity. Drosophila antennapedia homeodomain-derived CPP penetratin (RQIKIWFQNRRMKWKK), being rich in positively charged residues, has been increasingly used as a potential drug carrier for various purposes. Penetratin can breach the tight endothelial network known as the blood-brain barrier (BBB), permitting treatment of several neurodegenerative maladies, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. However, a detailed structural understanding of penetratin and its mechanism of action is lacking. This study defines structural features of the penetratin-derived peptide, DK17 (DRQIKIWFQNRRMKWKK), in several model membranes and describes a membrane-induced conformational transition of the DK17 peptide in these environments. A series of biophysical experiments, including high-resolution nuclear magnetic resonance spectroscopy, provides the three-dimensional structure of DK17 in different membranes mimicking the BBB or total brain lipid extract. Molecular dynamics simulations support the experimental results showing preferential binding of DK17 to particular lipids at atomic resolution. The peptide conserves the structure of the subdomain spanning residues Ile6-Arg11, despite considerable conformational variation in different membrane models. In vivo data suggest that the wild type, not a mutated sequence, enters the central nervous system. Together, these data highlight important structural and functional attributes of DK17 that could be utilized in drug delivery for neurodegenerative disorders.

Deciphering the role of the AT-rich interaction domain and the HMG-box domain of ARID-HMG proteins of Arabidopsis thaliana.

ARID-HMG DNA-binding proteins represent a novel group of HMG-box containing protein family where the AT-rich interaction domain (ARID) is fused with the HMG-box domain in a single polypeptide chain. ARID-HMG proteins are highly plant specific with homologs found both in flowering plants as well as in moss such as Physcomitrella. The expression of these proteins is ubiquitous in plant tissues and primarily localises in the cell nucleus. HMGB proteins are involved in several nuclear processes, but the role of ARID-HMG proteins in plants remains poorly explored. Here, we performed DNA-protein interaction studies with Arabidopsis ARID-HMG protein HMGB11 (At1g55650) to understand the functionality of this protein and its individual domains. DNA binding assays revealed that AtHMGB11 can bind double-stranded DNA with a weaker affinity (Kd = 475 ± 17.9 nM) compared to Arabidopsis HMGB1 protein (Kd = 39.8 ± 2.68 nM). AtHMGB11 also prefers AT-rich DNA as a substrate and shows structural bias for supercoiled DNA. Molecular docking of the DNA-AtHMGB11 complex indicated that the protein interacts with the DNA major groove, mainly through its ARID domain and the junction region connecting the ARID and the HMG-box domain. Also, predicted by the docking model, mutation of Lys(85) from the ARID domain and Arg(199) & Lys(202) from the junction region affects the DNA binding affinity of AtHMGB11. In addition, AtHMGB11 and its truncated form containing the HMG-box domain can not only promote DNA mini-circle formation but are also capable of inducing negative supercoils into relaxed plasmid DNA suggesting the involvement of this protein in several nuclear events. Overall, the study signifies that both the ARID and the HMG-box domain contribute to the optimal functioning of ARID-HMG protein in vivo.

Evidence for Inhibition of Lysozyme Amyloid Fibrillization by Peptide Fragments from Human Lysozyme: A Combined Spectroscopy, Microscopy, and Docking Study.

Degenerative diseases, such as Alzheimer's and prion diseases, as well as type II diabetes, have a pathogenesis associated with protein misfolding, which routes with amyloid formation. Recent strategies for designing small-molecule and polypeptide antiamyloid inhibitors are mainly based on mature fibril structures containing cross ß-sheet structures. In the present study, we have tackled the hypothesis that the rational design of antiamyloid agents that can target native proteins might offer advantageous prospect to design effective therapeutics. Lysozyme amyloid fibrillization was treated with three different peptide fragments derived from lysozyme protein sequence R(107)-R(115). Using low-resolution spectroscopic, high-resolution NMR, and STD NMR-restrained docking methods such as HADDOCK, we have found that these peptide fragments have the capability to affect lysozyme fibril formation. The present study implicates the prospect that these peptides can also be tested against other amyloid-prone proteins to develop novel therapeutic agents.

Biophysical insights into the membrane interaction of the core amyloid-forming Aß40 fragment K16-K28 and its role in the pathogenesis of Alzheimer's disease.

The aggregation of amyloid-ß (Aß) on neuronal membranes is implicated in both neuronal toxicity and the progression of Alzheimer's disease. Unfortunately, the heterogeneous environment that results from peptide aggregation in the presence of lipids makes the details of these pathways difficult to interrogate. In this study, we report an investigation of the membrane interaction of an Aß fragment (K16LVFFAEDVGSNK28, KK13), which maintains the amyloidogenic nature of the full-length peptide and is implicated in membrane-mediated folding, through a combination of NMR spectroscopy and molecular dynamics simulations. Despite KK13's ability to form amyloids in solution, the monomer remains unstructured in the presence of lipid bilayers, unlike its full-length parent peptide. Additionally, NMR and molecular dynamics simulation results support that the presence of GM1 ganglioside, a lipid which strongly promotes binding between Aß and lipid bilayers, promotes KK13 binding to but not folding on the membrane. Finally, we show that the peptide partitions between the membrane and aqueous solution based on the hydrophobicity of the N-terminal residues, regardless of lipid composition. These results support previous discoveries suggesting the importance of GM1 ganglioside in exacerbating membrane-driven aggregation while identifying the potential importance of C-terminal residues in membrane binding and folding, which has previously been unclear.

Plant alkaloid chelerythrine induced aggregation of human telomere sequence--a unique mode of association between a small molecule and a quadruplex.

Small molecules that interact with G-quadruplex structures formed by the human telomeric region and stabilize them have the potential to evolve as anticancer therapeutic agents. Herein we report the interaction of a putative anticancer agent from a plant source, chelerythrine, with the human telomeric DNA sequence. It has telomerase inhibitory potential as demonstrated from telomerase repeat amplification assay in cancer cell line extract. We have attributed this to the quadruplex binding potential of the molecule and characterized the molecular details of the interaction by means of optical spectroscopy such as absorbance and circular dichroism and calorimetric techniques such as isothermal titration calorimetry and differential scanning calorimetry. The results show that chelerythrine binds with micromolar dissociation constant and 2:1 binding stoichiometry to the human telomeric DNA sequence. Chelerythrine association stabilizes the G-quadruplex. Nuclear magnetic resonance spectroscopy ((1)H and (31)P) shows that chelerythrine binds to both G-quartet and phosphate backbone of the quadruplex leading to quadruplex aggregation. Molecular dynamics simulation studies support the above inferences and provide further insight into the mechanism of ligand binding. The specificity toward quartet binding for chelerythrine is higher compared to that of groove binding. MM-PBSA calculation mines out the energy penalty for quartet binding to be -4.7 kcal/mol, whereas that of the groove binding is -1.7 kcal/mol. We propose that the first chelerythrine molecule binds to the quartet followed by a second molecule which binds to the groove. This second molecule might bring about aggregation of the quadruplex structure which is evident from the results of nuclear magnetic resonance.

An idea to explore: Cultivating the art of proposal writing among graduate students.

Proposal writing is an essential requirement for making progress in academics. Learning this skill necessitates support from a mentor to cultivate effective habits. It entails effective strategies from graduate students, such as literature reading and using online tools. Additionally, they must develop an understanding of resource accountability, system thinking, and considering deadlines as a driving force. Good practices for effective proposal writing also involve planning to summarize the work done in the field. Moreover, it requires ideal mentor support by providing timely assistance, helping students overcome impostor syndrome, sharing successful proposals, and creating a cooperative environment.

Lysine acetylation of Hsp16.3: Effect on its structure, chaperone function and influence towards the growth of Mycobacterium tuberculosis.

Hsp16.3 plays a vital role in the slow growth of Mycobacterium tuberculosis via its chaperone function. Many secretory proteins, including Hsp16.3 undergo acetylation in vivo. Seven lysine (K) residues (K64, K78, K85, K114, K119, K132 and K136) in Hsp16.3 are acetylated inside pathogen. However, how lysine acetylation affects its structure, chaperone function and pathogen's growth is still elusive. We examined these aspects by executing in vitro chemical acetylation (acetic anhydride modification) and by utilizing a lysine acetylation mimic mutant (Hsp16.3-K64Q/K78Q/K85Q/K114Q/K119Q/K132Q/K136Q). Far- and near-UV CD measurements revealed that the chemically acetylated proteins(s) and acetylation mimic mutant has altered secondary and tertiary structure than unacetylated/wild-type protein. The chemical modification and acetylation mimic mutation also disrupted the oligomeric assembly, increased surface hydrophobicity and reduced stability of Hsp16.3, as revealed by GF-HPLC, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid binding and urea denaturation experiments, respectively. These structural changes collectively led to an enhancement in chaperone function (aggregation and thermal inactivation prevention ability) of Hsp16.3. Moreover, when the H37Rv strain expressed the acetylation mimic mutant protein, its growth was slower in comparison to the strain expressing the wild-type/unacetylated Hsp16.3. Altogether, these findings indicated that lysine acetylation improves the chaperone function of Hsp16.3 which may influence pathogen's growth in host environment.

Benefits of hybrid QM/MM over traditional classical mechanics in pharmaceutical systems.

Hybrid quantum mechanics/molecular mechanics (QM/MM) is one of the most reliable approaches for accurately modeling and studying the complex pharmaceutical discovery system. Classical mechanics has significantly accelerated the drug discovery process in the past decade. However, the current challenge is the large pool of false positives, which require extensive validation. Hybrid QM/MM is an effective solution for accurately studying ligand binding, structural mechanisms, free energy evaluation, and spectroscopic characterization. This article highlights the methodological details relevant to cost-effective hybrid QM/MM methods. This approach, combined with traditional pharmacoinformatics methods, could be a reliable strategy to balance the cost and accuracy of the calculations.

Network analysis of chromophore binding site in LOV domain.

Photoreceptor proteins are versatile toolbox for developing biosensors for optogenetic applications. These molecular tools get activated upon illumination of blue light, which in turn offers a non-invasive method for gaining high spatiotemporal resolution and precise control of cellular signal transduction. The Light-Oxygen-Voltage (LOV) domain family of proteins is a well-recognized system for constructing optogenetic devices. Translation of these proteins into efficient cellular sensors is possible by tuning their photochemistry lifetime. However, the bottleneck is the need for more understanding of the relationship between the protein environment and photocycle kinetics. Significantly, the effect of the local environment also modulates the electronic structure of chromophore, which perturbs the electrostatic and hydrophobic interaction within the binding site. This work highlights the critical factors hidden in the protein networks, linking with their experimental photocycle kinetics. It presents an opportunity to quantitatively examine the alternation in chromophore's equilibrium geometry and identify details which have substantial implications in designing synthetic LOV constructs with desirable photocycle efficiency.