Loop nucleobases-dependent folding of G-quadruplex in normal and cancer cell-mimicking KCl microenvironments.

In this study, the folding of G-quadruplex (G4) from the telomeric DNA sequences having loop nucleobases of different chemical natures, numbers, and arrangements in 10 mM and 100 mM KCl salt conditions mimicking the cancerous and normal KCl salt microenvironments have been investigated. The data suggest that the structure and stability of the G4 are highly dependent on the KCl salt concentration. In general, the conformational flexibility of the folded G4 is higher in KCl salt relevant to cancer than in the normal case for any loop arrangements with the same number of nucleobases. The stability of the G4 decreases with the increase in the number of loop nucleobases for both salt conditions. However, the decrease in the stability of G4 having adenine in the loop region is significantly higher than the case of thymine, particularly more prominent in the KCl salt relevant to the cancer. The topology of the folded G4 and its stability also depend delicately on the permutation of the nucleobases in the loop and the salt concentrations for a particular sequence. The findings indicate that the structure and stability of G4 are noticeably different in KCl salt relevant to physiological and cancer conditions.

Hydrophobic Interaction-Induced Topology-Independent Destabilization of G-Quadruplex.

Since the inception of the G-quadruplex (G4), enormous attention has been devoted to designing small molecules which can stabilize the G-quadruplex. In contrast, the knowledge about the molecules and mechanisms involved in the destabilization of G4 is sparse, although it is well recognized that destabilization of G4 is important in neurobiology and age-related genetic issues. In this study, it has been shown that amphiphilic molecules having a long hydrocarbon chain can destabilize G4, regardless of its topology, using various biophysical and molecular dynamics simulation methods. It has been observed that the hydrophobic interaction induced by the long hydrocarbon chain of amphiphilic molecules is the main contributor in triggering the destabilization of G4, although hydrogen bonding by the polar part of the molecules also cooperates in the destabilization process. The experiment and simulation studies suggest that a long hydrocarbon chain containing amphiphilic molecules gets aggregated, and their hydrocarbon chain as well as the polar group intrude in the quartet region from the 5' side and interact with guanine bases as well as nearby loops through hydrophobic and electrostatic interactions, which trigger the destabilization of G4.

Antimalarial Drugs Induce the Selective Folding of Human Telomeric G-Quadruplex in a Cancer-Mimicking Microenvironment.

Regulating the equilibrium between the duplex form of DNA and G-quadruplex (Gq) and stabilizing the folded Gq are the critical factors for any drug to be effective in cancer therapy due to the direct involvement of Gq in controlling the transcription process. Antimalarial drugs are in the trial stage for different types of cancer diseases; however, the plausible mechanism of action of these drug molecules is not well known. Hence, we investigate the plausible role of antimalarial drugs in the folding and stabilization of Gq-forming DNA sequences from the telomere and promoter gene regions by varying the salt (KCl) concentrations, mimicking the in vitro cancerous and normal cell microenvironments. The study reveals that antimalarial drugs fold and stabilize specifically to telomere Gq-forming sequences in the cancerous microenvironment than the DNA sequences located in the promoter region of the gene. Antimalarial drugs are not only able to fold Gq but also efficiently protect them from unfolding by their complementary strands, hence significantly biasing the equilibrium toward the Gq formation from the duplex. In contrast, in a normal cell microenvironment, K+ controls the folding of telomeres, and the role of antimalarial drugs is not prominent. This study suggests that the action of antimalarial drugs is sensitive to the cancer microenvironment as well as selective to the Gq-forming region.

Chemical Interaction Regulates the Folding and Topology of the G-Quadruplex Exclusively Induced by a Crowder.

The folding and stability of G-quadruplexes (Gq) are correlated with cancer and depend significantly on the chemical environment. Crowders are an important constituent of living cells. However, an understanding of the folding and topology of Gq induced exclusively by a crowder is lacking. Hence, folding and stabilization of the human telomere (htel) induced by polyethylene glycol and its derivative crowders have been studied using different biophysical techniques without the addition of salt. The data suggest that the crowder can alone induce the folding of the htel sequence into Gq and the topology of the folded structure depends on the composition of the crowder. Interestingly, a small chain size crowder favors the folding of the htel duplex to Gq, whereas a larger crowder prefers to stabilize the duplex form. Thermochemical data suggest that the nonlinear trend of the stability of folded Gq is modulated mainly by hydrogen bonding between the flexible part of the crowder and nucleobases, and the role of the excluded volume is not prominent. These findings might play an important role in improving our understanding of the folding and stabilization of htel in complex bimolecular environments.

Nucleobase Specific Understanding about the Interaction of Antimalarial Drug Chloroquine with Duplex DNA.

Antimalarial action of a drug is closely associated with the interaction with the parasite's DNA. Hence, in this study, the interaction of an important antimalarial drug, chloroquine (CLQ), has been investigated with six different sequences of DNA having pure adenine (A)-thymine (T) and pure cytosine (C)-guanine (G) as well as mixed nucleobases to achieve the nucleobase level of information in the binding of antimalarial drug with DNA along with binding induced stabilization/destabilization of DNA using different spectroscopic methods and molecular dynamics simulation technique. Further, the experiments have been also performed with 4-amino-7-chloroquinoline (7CLQ), an analogue of CLQ, to understand the role of the quinoline ring and side chain of CLQ in the binding with different sequences of DNA. The binding efficiency of CLQ with any sequence of DNA is higher than 7CLQ suggesting that the presence of charge on CLQ plays a prominent role in DNA binding. The data suggest that the binding of drug as well as induced stabilization of DNA depends significantly on the nature as well as the arrangement of the nucleobases. In general, the binding of CLQ with pure CG DNA is higher than with pure AT DNA; moreover, it prefers an alternate order of CG/AT than continual nucleobases in duplex DNA. CLQ predominately accommodates in the minor groove of AT DNA and prefers to form hydrogen bond mostly with the adenine nucleobase. In contrast to AT DNA, CLQ intrudes into the both major and minor grooves, but it is primarily accommodated into the major groove of CG DNA. CLQ forms a hydrogen bond mainly with guanine in the major groove and cytosine in the minor groove of CG DNA which enhances the binding of CLQ compared to AT DNA as well as induces higher stabilization in CG DNA. The molecular level information obtained about the functional group responsible for the interaction of CLQ as well as the role of chemical nature of nucleobases along with its ordering on the binding of CLQ with DNA may be useful in comprehensive understanding of its action mechanism.

Selective Action of Antimalarial Hydroxychloroquine on the Packing of Phospholipids and Interfacial Water Associated with Lysosomal Model Membranes: A Vibrational Sum Frequency Generation Study.

Understanding the structural change of lysosomal membranes induced by hydroxychloroquine (HCQ) drug is essential as it has been considered as one of the probable mechanisms of its antimalarial action. In this context, vibrational sum frequency generation (VSFG) spectra of the O-H region of water and C-H of the hydrocarbon chain of negatively charged and zwitterionic phospholipids associated with the lysosomal membrane in the absence and presence of different concentrations of HCQ have been measured at the air/water interface. The interfacial water at the negatively charged and zwitterionic lipids gets restructured in the presence of HCQ; however, the mechanism of restructuring is different due to the charge of the head groups of lipids. Interestingly, the presence of HCQ leads to a disorder in the negatively charged lipids, irrespective of their chemical nature, mainly by creating the gauche defect in the hydrocarbon chain of the lipid. In contrast, the ordering of the zwitterionic lipid does not show any appreciable change with the addition of HCQ. The finding on the selectivity of HCQ in affecting the ordering of the lipid depending on its head group charge and restructuring of interfacial water may be useful in understanding the molecular level mechanism of the antimalarial action of HCQ.

pH Regulates Ligand Binding to an Enzyme Active Site by Modulating Intermediate Populations.

Understanding the mechanism of ligands binding to their protein targets and the influence of various factors governing the binding thermodynamics is essential for rational drug design. The solution pH is one of the critical factors that can influence ligand binding to a protein cavity, especially in enzymes whose function is sensitive to the pH. Using computer simulations, we studied the pH effect on the binding of a guanidinium ion (Gdm+) to the active site of hen egg-white lysozyme (HEWL). HEWL serves as a model system for enzymes with two acidic residues in the active site and ligands with Gdm+ moieties, which can bind to the active sites of such enzymes and are present in several approved drugs treating various disorders. The computed free energy surface (FES) shows that Gdm+ binds to the HEWL active site using two dominant binding pathways populating multiple intermediates. We show that the residues close to the active site that can anchor the ligand could play a critical role in ligand binding. Using a Markov state model, we quantified the lifetimes and kinetic pathways connecting the different states in the FES. The protonation and deprotonation of the acidic residues in the active site in response to the pH change strongly influence the Gdm+ binding. There is a sharp jump in the ligand-binding rate constant when the pH approaches the largest pKa of the acidic residue present in the active site. The simulations reveal that, at most, three Gdm+ can bind at the active site, with the Gdm+ bound in the cavity of the active site acting as a scaffold for the other two Gdm+ ions binding. These results can aid in providing greater insights into designing novel molecules containing Gdm+ moieties that can have high binding affinities to inhibit the function of enzymes with acidic residues in their active site.

Spectroscopic and Molecular Dynamics Aspect of Antimalarial Drug Hydroxychloroquine Binding with Human Telomeric G-Quadruplex.

Hydroxychloroquine (HCQ) is an important drug that is in the trial stage for different types of cancer diseases; however, insight about the mechanism of its action is almost unknown. G-quadruplex (Gq) has been considered one of the potential targets for the cure of cancer; hence, it is essential to understand the possibility of the binding of HCQ with Gq to get a better understanding of its action. In this study, the molecular insight into the possibility of the binding of HCQ with different topological forms of Gq of the human telomere (htel) has been investigated using spectroscopic, thermochemical, and molecular dynamics simulation techniques. The spectroscopic and thermochemical studies clearly suggest that HCQ has a topological preference in the binding with htel in the form of a hybrid structure rather than the antiparallel form and the binding of HCQ stabilizes preferably to the hybrid form. The molecular dynamics simulation study suggests that the interaction of HCQ in the groove and loop regions of the hybrid structure is more stable compared to the antiparallel form, which is the probable reason for the topological preference of HCQ. This study depicts that HCQ has a topological preference in the binding and stabilization of the Gq of htel, which makes it potentially an important drug for targeting the telomere region associated with cancer disease.

Protonation State of Dopamine Neurotransmitter at the Aqueous Interface: Vibrational Sum Frequency Generation Spectroscopy Study.

Dopamine is an important amine-based chemical neurotransmitter whose protonated state plays a crucial role in the recognition process. Understanding the structure and protonated state of dopamine at the aqueous interface is desired as the diffusion as well as binding of dopamine with the receptors take place frequently in the aqueous interface region. Vibrational sum frequency generation (VSFG) study of the OH stretch of water at the air/water interface in the presence of dopamine is performed and compared with its analog, phenylethylamine, and catechol. The VSFG data suggest that, unlike the bulk case, the population of the deprotonated amine group of dopamine is higher at the aqueous interface. This study suggests that the structure of dopamine at the aqueous interface is different from the bulk which may be useful in understanding the recognition process of dopamine in the interfacial region.

DNA-Induced Reorganization of Water at Model Membrane Interfaces Investigated by Heterodyne-Detected Vibrational Sum Frequency Generation Spectroscopy.

Lipid-DNA complexes are important nonviral vectors to be used in gene therapy, which is one of the promising strategies for the cure of many diseases. Although interfacial water is expected to play a significant role in lipid-DNA complexation, a molecular-level understanding about the role of interfacial water in the DNA-lipid complexation is still sparse. In this study, the structure and orientation of water at cationic and zwitterionic lipid monolayer/water interfaces in the presence of DNA are studied by the use of interface-selective heterodyne-detected vibrational sum frequency generation (HD-VSFG) spectroscopy. It is found that the adsorption of DNA at a cationic lipid interface drastically decreases the orientation of interfacial water reflecting the neutralization of the positively charged interface, whereas the adsorption of DNA at a zwitterionic lipid interface makes interfacial water become more "H-up", indicating that the originally zwitterionic interface becomes negatively charged due to the DNA adsorption. Furthermore, interfacial water having relatively strong hydrogen bonds is observed at both the cationic and zwitterionic lipid interfaces in the presence of DNA.

The enhanced dissociation and associated surface structure of the anesthetic propofol at the water interface: vibrational sum frequency generation study.

Propofol, the most administered drug for general anesthesia, affects the acid-base equilibrium at the interfacial region of arterial blood. Hence, the structure of propofol at the water interface under different pH conditions has been measured using the surface-selective vibrational sum frequency generation (VSFG) technique to understand the hydration as well as the dissociation of propofol at the water interface. Propofol remains in its neutral form at pH ≤ 5.8 in which the OH group of propofol forms a hydrogen bond with interfacial water molecules, where a few interfacial water molecules also interact with the π electron density of propofol. By contrast, propofol prefers to be in the deprotonated state at pH ≥ 7, due to which the surface of water becomes negatively charged and hence the interfacial water becomes oriented and the intensity of the OH stretch of water is enhanced. The pKa of propofol at the water interface is ∼three units lower than in the bulk medium indicating that the dissociation of propofol is notably enhanced at the water interface. These VSFG studies suggest that, unlike the bulk, propofol prefers to be in the charged state at the water interface under physiological conditions, which may be important in understanding its diffusion and acid-base equilibrium in the interfacial arterial blood region.

The combined action of cations and anions of ionic liquids modulates the formation and stability of G-quadruplex DNA.

G-Quadruplex (Gq) formation and stabilization by any molecule is an essential requirement for its application in therapy, especially in oncology. Metal cations have shown higher propensity of the formation of the Gq structure and its stabilization. In this study, the role of both cations and anions of ionic liquids (ILs) on the Gq formation of human telomere (hTeloG) and its stability was investigated using spectroscopic and molecular dynamics simulation techniques. Irrespective of the nature of anions of ILs, tetramethylguanidinium (TMG) cations associated with different anions can form an antiparallel Gq structure in hTeloG. However, the propensity of the formation of an antiparallel Gq structure and its stability depend on the chain length of anions of ILs. Gq is significantly less stable in ILs having longer hydrocarbon chain anions compared to the short chain anions suggesting that the hydrophobicity of the anion plays a critical role in the stability and formation of the Gq structure by ILs. The data indicate that longer hydrocarbon chain anions of ILs preferably interact in the loop region of Gq through hydrophobic interaction which enhances the overall binding of the cation of ILs with Gq causing a decrease in the stacking energy between the G-quartets as well as Hoogsteen hydrogen bonds between the guanine bases leading to the destabilization of the antiparallel Gq structure.

Signature of the surface hydrated proton and associated restructuring of water at model membrane interfaces: a vibrational sum frequency generation study.

Hydrated proton at membrane interfaces plays an important role in the bioenergetic process of almost all organisms. Herein, the signature of the hydrated proton at membrane interfaces has been investigated by measuring the vibrational sum frequency generated (VSFG) spectra of negatively charged and zwitterionic lipids in the presence of different concentrations of acids. The addition of acids decreases the intensity of the OH stretch of the VSFG signal of water present at the negatively charged and zwitterionic lipids along with the enhanced intensity of the broad VSFG signal in the range of 2500-2800 cm-1. The enhanced intensity of the broad continuum observed in the range of 2500-2800 cm-1 has been assigned to the signature of the hydrated proton at the lipid interfaces. The decrease in the VSFG signal of the OH stretch of water along with the appearance of the broad signal suggests that the hydrated proton exists in the vicinity of the lipid interfaces and restructures the interaction between the interfacial water molecules.

Groove Switching of Hydroxychloroquine Modulates the Efficacy of Binding and Induced Stability to DNA.

Hydroxychloroquine (HCQ) is an important drug for the treatment of rheumatoid arthritis and malaria. HCQ targets specifically to nucleic acids for its action. However, the mechanism of HCQ binding and the effect of its binding on the stability of DNA are elusive. In this study, the binding mechanism of HCQ and the effect of binding on stability of different sequences of DNA have been investigated using spectroscopic and molecular dynamics (MD) simulation techniques. HCQ binds with all of the sequences of DNA and stabilizes them. However, binding efficacy of HCQ with DNA depends on its sequences as the binding constant is highest for pure guanine-cytosine (G-C) rich DNA and decreases with the increase of adenine-thymine (A-T) bases. HCQ prefers to interact with AT DNA through the minor groove whereas the major groove along with intercalation are the favorable binding mode in the case of GC DNA. The binding of HCQ in the major groove of GC DNA enhances the stacking between the bases compared to the case of AT DNA which leads to higher stability for GC DNA. It appears that the groove switching of HCQ is correlated with binding affinity as well as stability of different sequences of DNA.

Photophysical Properties of Noncanonical Amino Acid 7-Fluorotryptophan Sharply Different from Those of Canonical Derivative Tryptophan: Spectroscopic and Quantum Chemical Calculations.

Due to the limited number of naturally existing canonical amino acids, several noncanonical amino acids have been designed to understand the diverse complex biological functions. Fluorinated amino acids are one of the important noncanonical amino acids that have been used to understand the different complex processes of proteins. In this study, the photophysical properties of the noncanonical amino acid 7-fluorotryptophan (7F-Trp) in different solvents have been investigated using extensive spectroscopic as well as quantum chemical calculation methods and compared with those of tryptophan (Trp). The spectroscopic and quantum chemical calculation data suggest that unlike Trp, 7F-Trp can be used to detect the excited-state proton transfer from solvents depending on its acidity, which makes 7F-Trp a potential candidate for sensing the excited-state proton transfer from the solvent molecules.

Restructuring of Membrane Water and Phospholipids in Direct Interaction of Neurotransmitters with Model Membranes Associated with Synaptic Signaling: Interface-Selective Vibrational Sum Frequency Generation Study.

Comprehensive molecular-level understanding of the role of interfacial water and phospholipids associated with synaptic membranes during their direct interaction with neurotransmitters is essential because of their involvement in synaptic signaling. Herein, the interfacial regions of the synaptic membranes mimicking anionic and zwitterionic phospholipids are probed in the presence of dopamine and serotonin neurotransmitters using surface-specific vibrational sum frequency generation technique. Neurotransmitters intrude into the headgroup region of both zwitterionic and anionic lipids by restructuring the interfacial water associated with the phospholipids, although the restructuring mechanism is different for both lipids. Neurotransmitters also decrease the overall ordering of both the phospholipids probably by creating gauche defects. Neurotransmitters restructure the surface water, conformation, and the ordering of the hydrocarbon chains of the zwitterionic and anionic phospholipids associated with synaptic membranes, which could be potentially an important step for synaptic signaling.

Sequence specific hydrogen bond of DNA with denaturants affects its stability: Spectroscopic and simulation studies.

BACKGROUND: Several different small molecules have been used to target the DNA helix in order to treat the diseases caused by its mutation. Guanidinium(Gdm+) and urea based drugs have been used for the diseases related to central nervous system, also as the anti-inflammatory and chemotherapeutic agent. However, the role of Gdm+ and urea in the stabilization/destabilization of DNA is not well understood. METHODS: Spectroscopic techniques along with molecular dynamics (MD) simulation have been performed on different sequences of DNA in the presence of guanidinium chloride (GdmCl) and urea to decode the binding of denaturants with DNA and the role of hydrogen bond with the different regions of DNA in its stability/destability. RESULTS AND CONCLUSION: Our study reveals that, Gdm+ of GdmCl and urea both intrudes into the groove region of DNA along with the interaction with its phosphate backbone. However, interaction of Gdm+ and urea with the nucleobases in the groove region is different. Gdm+ forms the intra-strand hydrogen bond with the central region of the both sequences of DNA whereas inter-strand hydrogen bond along with water assisted hydrogen bond takes place in the case of urea. The intra-strand hydrogen bond formation capability of Gdm+ with the nucleobases in the minor groove of DNA decreases its groove width which probably causes the stabilization of B-DNA in GdmCl. In contrast, the propensity of the formation of inter-strand hydrogen bond of urea with the nucleobases in the groove region of DNA without affecting the groove width destabilizes B-DNA as compared to GdmCl. This study depicts that the opposite effect of GdmCl and urea on the stability is a general property of B-DNA. However, the extent of stabilization/destabilization of DNA in Gdm+ and urea depend on its sequence probably due to the difference in the intra/inter-strand hydrogen bonding with different bases present in both the sequences of DNA. GENERAL SIGNIFICANCE: The information obtained from this study will be useful for the designing of Gdm+ based drug molecule which can target the DNA more specifically and selectively.

Interaction of α-Synuclein with Phospholipids and the Associated Restructuring of Interfacial Lipid Water: An Interface-Selective Vibrational Spectroscopic Study.

Interaction of α-Synuclein (αS) with biological lipids is crucial for the onset of its fibrillation at the cell membrane/water interface. Probed herein is the interaction of αS with membrane-mimicking lipid monolayer/water interfaces. The results depict that αS interacts negligibly with zwitterionic lipids, but strongly affects the pristine air/water and charged lipid/water interfaces by perturbing the structure and orientation of the interfacial water. The net negative αS (-9 in bulk water; pH 7.4) reorients the water as hydrogen-up (H-up) at the air/water interface, and electrostatically interacts with positively charged lipids, making the interface nearly net neutral. αS also interacts with negatively charged lipids: the net H-up orientation of the interfacial water decreases at the anionic lipid/water interface, revealing a domain-specific interaction of net negative αS with the negatively charged lipids at the membrane surface.

Hydrogen bonding of ionic liquids in the groove region of DNA controls the extent of its stabilization: synthesis, spectroscopic and simulation studies.

Ionic liquids (ILs) have been extensively used for stabilization and long-term DNA storage. However, molecular level understanding of the role of the hydrogen bond of DNA with ILs in its stabilization is still inadequate. Two ILs, namely, 1,1,3,3-tetramethylguanidinium acetate (TMG) and 2,2-diethyl-1,1,3,3-tetramethylguanidinium acetate (DETMG), have been synthesized, of which TMG has a hydrogen bonding N-H group whereas DETMG does not contain any hydrogen bonding site. It has been found that both TMG and DETMG cations interact in the groove region of DNA; however, their mode of interaction is distinctly different, which causes the stabilization of DNA in the presence of TMG, whereas the effect is opposite in the case of DETMG. It is apparent from the data that only the accommodation of ILs in the groove region is not enough for the stabilization of DNA. MD simulation and spectroscopic studies combinedly indicate that the hydrogen bonding capability of the TMG cation enhances the hydrogen bonding between the Watson-Crick base pairs of DNA, resulting in its stabilization. In contrast, the bigger size as well as the absence of the hydrogen bonding site of the DETMG cation perturbs the minor groove width and base pair step parameters of DNA during its intrusion into the minor groove, which decreases the hydrogen bond between the Watson-Crick base pairs of DNA, leading to destabilization.

Alteration of the groove width of DNA induced by the multimodal hydrogen bonding of denaturants with DNA bases in its grooves affects their stability.

BACKGROUND: Denaturants, namely, urea and guanidinium chloride (GdmCl) affect the stability as well as structure of DNA. Critical assessment of the role of hydrogen bonding of these denaturants with the different regions of DNA is essential in terms of its stability and structural aspect. However, the understanding of the mechanistic aspects of structural change of DNA induced by the denaturants is not yet well understood. METHODS: In this study, various spectroscopic along with molecular dynamics (MD) simulation techniques were employed to understand the role of hydrogen bonding of these denaturants with DNA bases in their stability and structural change. RESULTS AND CONCLUSION: It has been found that both, GdmCl and urea intrude into groove region of DNA by striping surrounding water. The hydrogen bonding pattern of Gdm+ and urea with DNA bases in its groove region is multimodal and distinctly different from each other. The interaction of GdmCl with DNA is stabilized by electrostatic interaction whereas electrostatic and Lennard-Jones interactions both contribute for urea. Gdm+ forms direct hydrogen bond with the bases in the minor groove of DNA whereas direct and water assisted hydrogen bond takes place with urea. The hydrogen bond formed between Gdm+ with bases in the groove region of DNA is stronger than urea due to strong electrostatic interaction along with less self-aggregation of Gdm+ than urea. The distinct hydrogen bonding capability of Gdm+ and urea with DNA bases in its groove region affects its width differently. The interaction of Gdm+ decreases the width of the minor and major groove which probably increases the strength of hydrogen bond between the Watson-Crick base pairs of DNA leading to its stability. In contrast, the interaction of urea does not affect much to the width of the grooves except the marginal increase in the minor groove width which probably decreases the strength of hydrogen bond between Watson Crick base pairs leading to the destabilization of DNA. GENERAL SIGNIFICANCE: Our study clearly depicts the role of hydrogen bonding between DNA bases and denaturants in their stability and structural change which can be used further for designing of the guanidinium based drug molecules.