Exploring the Specific Role of Iron Center in the Catalytic Activity of Human Serum Transferrin: CTAB-Induced Conformational Changes and Sequestration by Mixed Micelles.

Conformational changes play a seminal role in modulating the activity of proteins. This concept becomes all the more relevant in the context of metalloproteins, owing to the formation of specific conformation(s) induced by internal perturbations (like a change in pH, ligand binding, or receptor binding), which may carry out the binding and release of the metal ion/ions from the metal binding center of the protein. Herein, we investigated the conformational changes of an iron-binding protein, monoferric human serum transferrin (Fe-hTF), using several spectroscopic approaches. We could reversibly tune the cetyltrimethylammonium bromide (CTAB)-induced conformation of the protein, exploiting the concept of mixed micelles formed by three sequestrating agents: (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) hydrate (CHAPS) and two bile salts, namely, sodium cholate (NaC) and sodium deoxycholate (NaDC). The formation of mixed micelles between CTAB and these reagents (CHAPS/NaC/NaDC) results in the sequestration of CTAB molecules from the protein environment and aids the protein in reattaining its native-like structure. However, the guanidinium hydrochloride-induced denatured Fe-hTF did not acquire its native-like structure using these sequestrating agents, which substantiates the exclusive role of mixed micelles in the present study. Apart from this, we found that the conformation of transferrin (adopted in the presence of CTAB) displays pronounced esterase-like activity toward the para-nitrophenyl acetate (PNPA) substrate as compared to native transferrin. We also outlined the impact of the iron center and amino acids surrounding the iron center on the effective catalytic activity in the CTAB medium. We estimated ∼3 times higher specific catalytic efficiency for the iron-depleted Apo-hTF compared to the fully iron-saturated Fe2-hTF in the presence of CTAB.

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.

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.

Contrasting Effect of Salts on the Binding of Antimalarial Drug Hydroxychloroquine with Different Sequences of Duplex DNA.

Hydroxychloroquine (HCQ) is an important antimalarial drug which functions plausibly by targeting the DNA of parasites. Salts play a crucial role in the functionality of various biological processes. Hence, the effect of salts (NaCl and MgCl2) on the binding of HCQ with AT- and CG-DNAs as well as the binding-induced stability of both sequences of DNAs have been investigated using the spectroscopic and molecular dynamics (MD) simulation methods. It has been found that the effect of salts on the binding of HCQ is highly sensitive to the nature of ions as well as DNA sequences. The effect of ions is opposite for the binding of AT- and CG-DNAs as the presence of Mg2+ ions enhances the binding of HCQ with AT-DNA, whereas the binding of HCQ with CG-DNA gets decreased on the addition of both ions. Similarly, the presence of Mg2+ enhances the stabilization of HCQ-bound AT-DNA, whereas the effect is opposite for the CG-DNA in the presence of both the ions. The MD simulation study suggests that the hydration states of both ions are different and they interact differently in the minor and major grooves of both the sequences of DNA which may be one of the reasons for the different binding of HCQ with these two sequences of DNA in the presence of salts. The information about the effect of salts on the binding of HCQ with DNAs in a sequence-specific manner may be useful in understanding the mechanism of the action and toxicity effect of HCQ against malaria.