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Ribonucleic acid (RNA) is exceedingly sensitive to degradation compared to DNA. The current protocol for storage of purified RNA requires freezing conditions below −20 °C. Recent advancements in biological chemistry have identified amino acid-based ionic liquids as suitable preservation media for RNA, even in the presence of degrading enzymes. However, the mechanistic insight into the interaction between ILs and RNA is unclear. To the best of our knowledge, no attempts are made so far to provide a molecular view. This work aims to establish a detailed understanding of how ILs enable structural stability to RNA sourced from Torula yeast. Herein, we manifest the hypothesis of multimodal binding of IL and its minimal perturbation to the macromolecular structure, with several spectroscopic techniques such as time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) aided with molecular dynamics at microsecond time scales. Relevant structural and thermodynamic details from biophysical experiments confirm that even long-term RNA preservation with ILs is a possible alternative devoid of any structural deformation. These results establish a unifying mechanism of how ILs are maintaining conformational integrity and thermal stability. The atomistic insights are transferable for their potential applications in drug delivery and biomaterials by considering the advantages of having maximum structural retention and minimum toxicity., Electrostatic interactions drive the multimodal binding between ChAAILs and RNA. Biocompatible ChAAILs can be used as a green preservative medium for long-term storage of RNA at ambient conditions.

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.

Microtubule affinity regulating kinase (MARK4) is considered as a potential drug target for diabetes, cancer, and neurodegenerative diseases. Since the role of MARK4 in the phosphorylation of tau protein and subsequently Alzheimer's disease has been established, therefore, we have investigated the binding affinity and MARK4 inhibitory potential of cholic acid (CHA) using both computational and spectroscopic methods. Molecular docking suggested a strong binding of CHA to the functionally important residues of MARK4. We further performed 500 ns molecular dynamics simulation which suggested the MARK4-CHA system was quite stable throughout the simulation trajectory. CHA potential binds to the MARK4 with a binding constant (K) of 107 M−1 at 288 K. Further, MARK4 activity was inhibited by CHA with an IC50 = 5.5 μM. Further insights into the thermodynamic parameters suggested that MARK4-CHA complex formation is driven by both electrostatic and van der Waals interactions. Overall study provides a rationale to use CHA in the drug development via MARK4 inhibition, towards possible therapeutic implications in neurodegenerative diseases.

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.

Insulin, a simple polypeptide hormone with huge biological importance, has long been known to self-assemble in vitro and form amyloid-like fibrillar aggregates. Utilizing high-resolution NMR, Raman spectroscopy, and computational analysis, we demonstrate that the fluctuation of the carboxyl terminal (C-ter) residues of the insulin B-chain plays a key role in the growth phase of insulin aggregation. By comparing the insulin sourced from bovine, human, and the modified glargine (GI), we observed reduced aggregation propensity in the GI variant, resulting from two additional Arg residues at its C-ter. NMR analysis showed atomic contacts and residue-specific interactions, particularly the salt bridge and H-bond formed among the C-ter residues Arg31B, Lys29B, and Glu4A. These inter-residue interactions were reflected in strong nuclear Overhauser effects among Arg31BδH-Glu4AδH and Lys29BδHs-Glu4AδH in GI, as well as the associated downfield chemical shift of several A-chain amino terminal (N-ter) residues. The two additional Arg residues of GI, Arg31B and Arg32B, enhanced the stability of the GI native structure by strengthening the Arg31B, Lys29B, and Glu4A salt bridge, thus reducing extensive thermal distortion and fluctuation of the terminal residues. The high stability of the salt bridge retards tertiary collapse, a crucial biochemical event for oligomerization and subsequent fibril formation. Circular dichroism and Raman spectroscopic measurement also suggest slow structural distortion in the early phase of the aggregation of GI because of the restricted mobility of the C-ter residues as explained by NMR. In addition, the structural and dynamic parameters derived from molecular dynamics simulations of insulin variants highlight the role of residue-specific contacts in aggregation and amyloid-like fibril formation.

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 β-hairpin conformations. The simulation here may serve as a model for the binding of other non-β-sheet conformations to amyloid fibers.

Pyruvate dehydrogenase kinase-3 (PDK3) plays important role in the glucose metabolism and is associated with cancer progression, and thus being considered as an attractive target for cancer therapy. In this study, we employed spectroscopic techniques to study the structural and conformational changes in the PDK3 at varying pH conditions ranging from pH 2.0 to 12.0. UV/Vis, fluorescence and circular dichroism spectroscopic measurements revealed that PDK3 maintains its native-like structure (both secondary and tertiary) in the alkaline conditions (pH 7.0–12.0). However, a significant loss in the structure was observed under acidic conditions (pH 2.0–6.0). The propensity of aggregate formation at pH 4.0 was estimated by thioflavin T fluorescence measurements. To further complement structural data, kinase activity assay was performed, and maximum activity of PDK3 was observed at pH 7.0–8.0 range; whereas, its activity was lost under acidic pH. To further see conformational changes at atomistic level we have performed all-atom molecular dynamics at different pH conditions for 150 ns. A well defined correlation was observed between experimental and computational studies. This work highlights the significance of structural dependence of pH for wide implications in protein-protein interaction, biological function and drug design procedures.

Synthesis of the pentasaccharide with a 2-aminoethyl linker attached to the reducing end corresponding to the cell wall O-antigen of Escherichia coli O86 strain is reported. The synthetic strategy involves sequential glycosylation of suitably protected monosaccharide intermediates under similar glycosylation reaction conditions. Thioglycosides have been used as glycosyl donor throughout the synthetic strategy. Conformational analysis of the synthesized pentasaccharide has been carried out using 2D ROESY NMR spectral analysis and all atom explicit molecular dynamics (MD) simulation technique. Graphical abstract Facile synthesis of the pentasaccharide with a 2-aminoethyl linker attached to the reducing end corresponding to the cell wall O-antigen of Escherichia coli O86 strain is reported. Conformational analysis of the synthesized pentasaccharide has been carried out using 2D ROESY NMR spectral analysis and all atom explicit molecular dynamics (MD) simulation technique.

Amyloid formation has been implicated in a number of neurodegenerative diseases. The elongation of amyloid fibers is thermodynamically strongly 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 thus so far 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 monomer. A Markov state model shows the unfolded monomer is trapped at the end of the fiber in a set of interconverting anti-parallel β-hairpin conformations. The simulation here may serve as a model for the binding of other non-β-sheet conformations to amyloid fibers.

Antifreeze proteins (AFPs) are the key biomolecules that enable species to survive under subzero temperature conditions. The physiologically relevant activities of AFPs are based on the adsorption to ice crystals, followed by the inhibition of subsequent crystal layer growth of ice, routed with depression in freezing point in a noncolligative manner. The functional attributes governing the mechanism by which AFPs inhibit freezing of body fluids in bacteria, fungi, plants, and fishes are mainly attributed to their adsorption onto the surface of ice within the physiological system. Importantly, AFPs are also known for their application in cryopreservation of biological samples that might be related to membrane interaction. To date, there is a paucity of information detailing the interaction of AFPs with membrane structures. Here, we focus on elucidating the biophysical properties of the interactions between AFPs and micelle models that mimic the membrane system. Micelle model systems of zwitterionic DPC and negatively charged SDS were utilized in this study, against which a significant interaction is experienced by two AFP molecules, namely, Peptide 1m and wfAFP (the popular AFP sourced from winter flounder). Using low- and high-resolution biophysical characterization techniques, such as circular dichroism (CD) and NMR spectroscopy, a strong evidence for the interactions of these AFPs with the membrane models is revealed in detail and is corroborated by in-depth residue-specific information derived from molecular dynamics simulation. Altogether, these results not only strengthen the fact that AFPs interact actively with membrane systems, but also demonstrate that membrane-associated AFPs are dynamic and capable of adopting a number of conformations rendering fluidity to the system.

Antifreeze proteins (AFPs) enable the polar living species to survive subzero temperature conditions through effective lowering of the freezing point of body fluids. At the molecular level, AFPs directly interact with the growing seeds of ice crystals to inhibit their formation. To understand the structural and dynamic aspects of this interaction at the atomistic level, molecular dynamics (MD) simulations were carried out on several type I AFPs at multiple temperatures, including the physiologically relevant temperature of 273 K, a lower temperature of 227 K, and the conventional 300 K. A comparison of the principal component analysis (PCA) and mean squared deviation plots for Winter flounder AFP, HPLC6 (mutant of winter flounder AFP), Sculpin, and peptide 1m AFPs reveals that simulations at 273 and 227 K result in the formation of more conserved metastable states than at 300 K. Other parameters such as root-mean-square deviation (rmsd), solvent accessibility surface area (SASA), H-bonding and residual density function (RDF) also suggest the same. MD simulations with ice crystal, where AFPs are complexed to ice plane with TIP4P/ice water model, help in finding relevance of dynamic behavior, and physiological temperature becomes more pronounced. Additionally, a control study on a nonantifreeze protein (LL37) is included, which aids in exploring significant information. On the basis of this approach, it was found that AFPs at 273 and 227 K display relevant dynamic properties that appear at 300 K for nonantifreeze proteins. The present study hence emphasizes the importance of performing computational simulations for antifreeze proteins at the physiologically relevant temperature (273 K), and even at lower temperatures (like 227 K), rather than at room temperatures (300 K).

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 carpet mechanism to disrupt the bacterial membrane systems. To better understand the role of lipid composition on the function of MSI-594, in the present study, eight different model membrane systems have been studied using accelerated molecular dynamics (aMD) simulation. 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 interaction with the peptide is characterized by lipid acyl-chain order, membrane thickness, as well as 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 resullts reported in this study are helpful to obtain accurate atomistic-level information governing MSI-594 and its membrane disruptive antimicrobial mechanism of action, as well as to design next generation potent antimicrobial peptides.

Synthesis of a pentasaccharide repeating unit of the cell wall O-antigenic polysaccharide of enteroaggregative Escherichia coli O175 has been achieved using one-pot, three components [1+2+2] block glycosylations. The intermediate steps were good yielding and satisfactory stereoselectivity was observed in the glycosylations. The synthesized pentasaccharide was subjected to conformational analysis in aqueous environment using 2D ROESY NMR spectral analysis in conjugation with molecular dynamics (MD) simulation technique.

A hexasaccharide repeating unit of the O-antigen of the cell wall of Shigella flexneri type 1d has been synthesized using a stereoselective [3+3] block glycosylation approach. Recently developed glycosylation conditions were used in the synthesis. A thioglycoside was used as an orthogonal glycosyl donor during the synthesis. The synthesized hexasaccharide was subjected to detailed NMR spectroscopic and molecular modeling studies to determine its conformational behavior in water. The NOE-based two-dimensional NOSEY experiment and all-atom explicit molecular dynamics (MD) simulation studies suggest that the oligosaccharide is not very flexible, that it remains rigid with respect to the glycosidic linkages between the sugar moieties.

A straightforward synthesis of the tetrasaccharide repeating unit of the O-antigen of the Escherichia coli O69 strain as its 2-aminoethyl glycoside has been accomplished by carrying out two iterative glycosylations in one pot. The stereochemical outcome of the glycosylations was very good. The conformational analysis of the synthesized tetrasaccharide was carried out using NOE based two-dimensional ROESY spectroscopy in conjugation with all atom explicit molecular dynamics simulation studies. The solvent-perturbation induced conformations of the tetrasaccharide 1 were found quite stable in solution.

Synthesis of the pentasaccharide repeating unit of the O-antigen of Escherichia coli O13 strain has been achieved using a straightforward linear synthetic strategy. Similar reaction conditions have been used for all glycosylations as well as protective group manipulations. All intermediate steps are high yielding and the glycosylation steps are stereoselective. The synthesized pentasaccharide was subjected to conformational analysis using 2D ROESY NMR spectral analysis and molecular dynamics (MD) simulation to get detailed information on conformation of the molecule in aqueous solution.

The stabilization of overhang G-rich repetitive DNA units at the 3′-end of telomeres, which are well known to form functionally important G-quadruplex structures, is a current goal in designing novel anticancer drugs. In the present study, we have undertaken an in silico approach by molecular docking using a small molecule library to find potential G-quadruplex stabilizing agents. Two molecules, A, [N′1-imino(2-pyridyl)methyl-3,4,5-trimethoxybenzene-1-carbohydrazide] and B, [(3-[4-({[3-({4-[(2cyanoethyl)(methyl)amino]benzylidene}amino)propyl]imino}methyl)(methyl) anilino]propanenitrile)], that had good docking scores have been investigated for interaction with G-quadruplexes in a Molecular Dynamics simulation study. Fluorescence spectroscopy of G-quadruplexes bound to the screened molecules A and B was used to experimentally validate the theoretical results. The binding of ligands A and B to G-quadruplexes resulted in blue shifts of 10–18 nm, respectively, in the fluorescence emission spectra of the G-quadruplexes, demonstrating that both molecules bind to the G-face of the quadruplex. The same experiment was performed for the complexation of these small molecules with a G-rich DNA duplex, . Interestingly, no blue shift was observed in the fluorescence emission spectra of the DNA duplex in the presence of these small molecules. Thus, these findings indicated that these ligands very selectively bind to G-quadruplexes instead of the duplex DNA. In addition, a one-dimensional water ligand observed via a gradient spectroscopy Nuclear Magnetic Resonance (NMR) experiment showed that both molecules bound to the 23-mer G-quadruplex DNA. The molecular properties of the ligand–quadruplex complex have been analyzed with the help of the Adaptive Poisson-Boltzmann Solver, revealing that electrostatics govern the binding of the small molecules to G-quadruplexes. Both molecules were investigated in detail using solvation free energy calculations and Absorption, Distribution, Metabolism, Elimination and Toxicity (ADMET) predictions, which provide insight into lead optimization for designing G-quadruplex stabilizing agents; therefore, these molecules have potential as new therapeutic agents.

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.