Toward the Establishment of a Harmonized Physicochemical Profiling Platform for Therapeutic Oligonucleotides: A Case Study for Aptamers Where the Higher-Order Structure Influences Physical Properties.

Oligonucleotides are short nucleic acids that serve as one of the most promising classes of drug modality. However, attempts to establish a physicochemical evaluation platform of oligonucleotides for acquiring a comprehensive view of their properties have been limited. As the chemical stability and the efficacy as well as the solution properties at a high concentration should be related to their higher-order structure and intra-/intermolecular interactions, their detailed understanding enables effective formulation development. Here, the higher-order structure and the thermodynamic stability of the thrombin-binding aptamer (TBA) and four modified TBAs, which have similar sequences but were expected to have different higher-order structures, were evaluated using ultraviolet spectroscopy (UV), circular dichroism (CD), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). Then, the relationship between the higher-order structure and the solution properties including solubility, viscosity, and stability was investigated. The impact of the higher-order structure on the antithrombin activity was also confirmed. The higher-order structure and intra-/intermolecular interactions of the oligonucleotides were affected by types of buffers because of different potassium concentrations, which are crucial for the formation of the G-quadruplex structure. Consequently, solution properties, such as solubility and viscosity, chemical stability, and antithrombin activity, were also influenced. Each instrumental analysis had a complemental role in investigating the higher-order structure of TBA and modified TBAs. The utility of each physicochemical characterization method during the preclinical developmental stages is also discussed.

A Model Study to Assess Fibrillation and Product Stability to Support Peptide Drug Design.

The fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and can accelerate product development by guiding the selection of solution-stable candidates and formulations. The studies reported here investigated the effects of structural modifications on the fibrillation of a 29-residue peptide (PepA) and two sequence modified variants (PepB, PepC). The C-terminus of PepA was amidated, whereas both PepB and PepC retained the carboxylate, and Ser16 in PepA and PepB was substituted with a helix-stabilizing residue, α-aminoisobutyric acid (Aib), in PepC. In thermal denaturation studies by far-UV CD spectroscopy and fibrillation kinetic studies by fluorescence and turbidity measurements, PepA and PepB showed heat-induced conformational changes and were found to form fibrils, whereas PepC did not fibrillate and showed only minor changes in the CD signal. Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed a high degree of protection from HD exchange in mature PepA fibrils and its proteolytic fragments, indicating that most of the sequence had been incorporated into the fibril structure and occurred nearly simultaneously throughout the sequence. The effects of the net peptide charge and formulation pH on fibrillation kinetics were investigated. In real-time stability studies of two formulations of PepA at pH's 7.4 and 8.0, analytical methods detected significant changes in the stability of the formulations at different time points during the study, which were not observed during accelerated studies. Additionally, PepA samples were withdrawn from real-time stability and subjected to additional stress (40 °C, continuous shaking) to induce fibrillation; an approach that successfully amplified oligomers or prefibrillar species previously undetected in a thioflavin T assay. Taken together, these studies present an approach to differentiate and characterize fibrillation risk in structurally related peptides under accelerated and real-time conditions, providing a model for rapid, iterative structural design to optimize the stability of therapeutic peptides.

Multispectroscopic and Theoretical Investigation on the Binding Interaction of a Neurodegenerative Drug, Lobeline with Human Serum Albumin: Perturbation in Protein Conformation and Hydrophobic-Hydrophilic Surface.

Lobeline (LOB), a naturally occurring alkaloid, has a broad spectrum of pharmacological activities and therapeutic potential, including applications in central nervous system disorders, drug misuse, multidrug resistance, smoking cessation, depression, and epilepsy. LOB represents a promising compound for developing treatments in various medical fields. However, despite extensive pharmacological profiling, the biophysical interaction between the LOB and proteins remains largely unexplored. In the current article, a range of complementary photophysical and cheminformatics methodologies were applied to study the interaction mechanism between LOB and the carrier protein HSA. Steady-state fluorescence and fluorescence lifetime experiments confirmed the static-quenching mechanisms in the HSA-LOB system. "K" (binding constant) of the HSA-LOB system was determined to be 105 M-1, with a single preferable binding site in HSA. The forces governing the HSA-LOB stable complex were analyzed by thermodynamic parameters and electrostatic contribution. The research also investigated how various metal ions affect complex binding. Site-specific binding studies depict Site I as probable binding in HSA by LOB. We conducted synchronous fluorescence, 3D fluorescence, and circular dichroism studies to explore the structural alteration occurring in the microenvironment of amino acids. To understand the robustness of the HSA-LOB complex, we used theoretical approaches, including molecular docking and MD simulations, and analyzed the principal component analysis and free energy landscape. These comprehensive studies of the structural features of biomolecules in ligand binding are of paramount importance for designing targeted drugs and delivery systems.

Angle-Resolved Linear Dichroism to Probe the Organization of Highly Ordered Collagen Biomaterials.

Controlling the assembly of high-order structures is central to soft-matter and biomaterial engineering. Angle-resolved linear dichroism can probe the ordering of chiral collagen molecules in the dense state. Collagen triple helices were aligned by solvent evaporation. Their ordering gives a strong linear dichroism (LD) that changes sign and intensity with varying sample orientations with respect to the beam linear polarization. Being complementary to circular dichroism, which probes the structure of chiral (bio)molecules, LD can shift from the molecular to the supramolecular scale and from the investigation of the conformation to interactions. Supported by multiphoton microscopy and X-ray scattering, we show that LD provides a straightforward route to probe collagen alignment, determine the packing density, and monitor denaturation. This approach could be adapted to any assembly of chiral (bio)macromolecules, with key advantages in detecting large-scale assemblies with high specificity to aligned and chiral molecules and improved sensitivity compared to conventional techniques.

Structural Investigations of Cargo Molecules Inside Icosahedrally Symmetric Encapsulin by VUVCD Spectroscopic Measurements.

Prokaryotes organize intracellular compartments with protein-based organelles called encapsulins. Encapsulins with icosahedral symmetry can encapsulate specific cargo proteins mediated by targeting peptides or encapsulation-mediating domains. Encapsulins have been used in eukaryotic cells for bioengineering, vaccine development, and nanoparticle alignment. Their versatility makes them attractive for research; however, detailed structural information on encapsulins is crucial for further applied research. However, cargo proteins are randomly oriented inside the icosahedral encapsulins. The random orientation of cargo proteins presents a challenge for structural analysis that relies on averaging processes such as x-ray crystallography and cryo-electron microscopy (cryo-EM) single-particle imaging. Therefore, we aimed to accurately estimate the secondary structure content and elucidate the structure of cargo proteins inside the particle by measuring the circular dichroism (CD) spectra using vacuum ultraviolet circular dichroism (VUVCD) spectroscopy. Thus, the structure of the cargo protein inside encapsulin was evaluated. This approach could potentially set a standard for evaluating cargo proteins inside particles in future applied research on encapsulins.

Chiroptically active copper clusters as platform for enantioselective detection of lysine.

Metal clusters have drawn considerable research attention over the years due to their fascinating optical properties. Owing to their appealing photophysical characteristics, these materials have drawn attention as potential candidates for various application in diverse fields, including disease detection, biosensing, chemical sensing, and the fabrication of light-harvesting materials. Presently, there is an increasing research focus on the use of clusters in biomedical research, both as biodetection platform and as bioimaging agents. Of special interest are chiral clusters, which can selectively interact with chiral biomolecules owing to their optical activity. Herein, we showcase the use of a pair of chiroptically active copper clusters for the enantioselective detection of lysine, an amino acid of vast biological relevance. Two techniques are concurrently employed for the detection of lysine at varying concentrations. Circular dichroism serves as a potent tool for detecting lysine at low concentrations, whereas luminescence is effectively employed as a detection method for high analyte concentrations. The combined electronic impact of clusters and lysine resulted in the emergence of an enhanced enantioselective Cotton effect at specific wavelength.

Circular dichroism spectrum of (R)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol in albumin: Alterations caused by complexation-Experimental and in silico investigation.

This study describes the interaction of human serum albumin (HSA) with the binol derivative (R)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol (R-BrB), which has its optical activity based on the prohibitive energetic barrier for conversion into the enantiomer (S)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol (S-BrB). The objective was to assess the ability of HSA to differentiate axial enantiomers based on their binding efficiency and their impact on the CD spectra. We discovered that both enantiomers were effective ligands, and the CD signal disappeared when equimolar amounts of R-BrB and S-BrB were simultaneously added, indicating no preference for either enantiomer. The complexation resulted in a significant signal increase at 250 nm and a bathochromic effect at 370 nm. Molecular docking simulations were performed, and the lower energy pose of R-BrB was selected for DFT calculations. The theoretical CD spectra of free and complexed R-BrB were obtained and showed alterations corroborating the experimental results. By comparing the difference spectrum (HSA:R-BrB minus HSA) with the spectrum of free RBrB in water or ethyl alcohol, we concluded that the CD signal intensification was due to the increased solubilization of R-BrB upon binding to HSA.

Testing the Simplified Molecular Dynamics Approach to Improve the Reproduction of ECD Spectra and Monitor Aggregation.

A simplified molecular-dynamics-based electronic circular dichroism (ECD) approach was tested on three condensed derivatives with limited conformational flexibility and an isochroman-2H-chromene hybrid, the ECD spectra of which could not be precisely reproduced by the conventional ECD calculation protocol. Application of explicit solvent molecules at the molecular mechanics (MD) level in the dynamics simulations and subsequent TDDFT-ECD calculation for the unoptimized MD structures was able to improve the agreements between experimental and computed spectra. Since enhancements were achieved even for molecules with limited conformational flexibility, deformations caused by the solvent molecules and multitudes of conformers produced with unoptimized geometries seem to be key factors for better agreement. The MD approach could confirm that aggregation of the phenanthrene natural product luzulin A had a significant contribution to a specific wavelength range of the experimental ECD. The MD approach has proved that dimer formation occurred in solution and this was responsible for the anomalous ECD spectrum. The scope and limitations of the method have also been discussed.

Zippered G-quadruplex/hemin DNAzyme: exceptional catalyst for universal bioanalytical applications.

G-quadruplex (G4)/hemin DNAzyme is promising horseradish peroxidase (HRP)-mimic candidate in the biological field. However, its relatively unsatisfactory catalytic capacity limits the potential applications. Inspired by nature protease, we conducted a proximity-enhanced cofactor assembly strategy (PECA) to form an exceptional HRP mimic, namely zippered G4/hemin DNAzyme (Z-G4/H). The hybridization of short oligonucleotides induced proximity assembly of the DNA-grafted hemin (DGH) with the complementary G4 sequences (cG4s), mimicking the tight configuration of protease cofactor and apoenzyme. The detailed investigations of catalytic efficiency and mechanism verified the higher activity, more rapid catalytic rate and high environmental tolerance of the Z-G4/H than the classical G4/hemin DNAzymes (C-G4/H). Furthermore, a proximity recognition transducer has been developed based on the PECA for sensitive detection of gene rearrangement and imaging human epidermal growth factor receptor 2 protein (HER2) dimerization on cell surfaces. Our studies demonstrate the high efficiency of Z-G4/H and its universal application potential in clinical diagnostics and biomolecule interaction research. It also may offer significant opportunities and inspiration for the engineering of the protease-free mimic enzyme.

Riboflavin Stabilizes Abasic, Oxidized G-Quadruplex Structures.

The G-quadruplex is a noncanonical fold of DNA commonly found at telomeres and within gene promoter regions of the genome. These guanine-rich sequences are highly susceptible to damages such as base oxidation and depurination, leading to abasic sites. In the present work, we address whether a vacancy, such as an abasic site, in a G-quadruplex serves as a specific ligand recognition site. When the G-tetrad is all guanines, the vacant (abasic) site is recognized and bound by free guanine nucleobase. However, we aim to understand whether the preference for a specific ligand recognition changes with the presence of a guanine oxidation product 8-oxo-7,8-dihydroguanine (OG) adjacent to the vacancy in the tetrad. Using molecular dynamics simulation, circular dichroism, and nuclear magnetic resonance, we examined the ability for riboflavin to stabilize abasic site-containing G-quadruplex structures. Through structural and free energy binding analysis, we observe riboflavin's ability to stabilize an abasic site-containing G-quadruplex only in the presence of an adjacent OG-modified base. Further, when compared to simulation with the vacancy filled by free guanine, we observe that the free guanine nucleobase is pushed outside of the tetrad by OG to interact with other parts of the structure, including loop residues. These results support the preference of riboflavin over free guanine to fill an OG-adjacent G-quadruplex abasic vacancy.

Spectroscopic and Computational Investigation of the Epoxyqueuosine Reductase QueG Reveals Intriguing Similarities with the Reductive Dehalogenase PceA.

Queuosine (Q) is a highly modified nucleoside of transfer RNA that is formed from guanosine triphosphate over the course of eight steps. The final step in this process, involving the conversion of epoxyqueuosine (oQ) to Q, is catalyzed by the enzyme QueG. A recent X-ray crystallographic study revealed that QueG possesses the same cofactors as reductive dehalogenases, including a base-off Co(II)cobalamin (Co(II)Cbl) species and two [4Fe-4S] clusters. While the initial step in the catalytic cycle of QueG likely involves the formation of a reduced Co(I)Cbl species, the mechanisms employed by this enzyme to accomplish the thermodynamically challenging reduction of base-off Co(II)Cbl to Co(I)Cbl and to convert oQ to Q remain unknown. In this study, we have used electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopies in conjunction with whole-protein quantum mechanics/molecular mechanics (QM/MM) computations to further characterize wild-type QueG and select variants. Our data indicate that the Co(II)Cbl cofactor remains five-coordinate upon substrate binding to QueG. Notably, during a QM/MM optimization of a putative QueG reaction intermediate featuring an alkyl-Co(III) species, the distance between the Co ion and coordinating C atom of oQ increased to >3.3 Å and the C-O bond of the epoxide reformed to regenerate the oQ-bound Co(I)Cbl reactant state of QueG. Thus, our computations indicate that the QueG mechanism likely involves single-electron transfer from the transient Co(I)Cbl species to oQ rather than direct Co-C bond formation, similar to the mechanism that has recently been proposed for the tetrachloroethylene reductive dehalogenase PceA.

A Disulfide-Stabilized Aß that Forms Dimers but Does Not Form Fibrils.

Aß dimers are a basic building block of many larger Aß oligomers and are among the most neurotoxic and pathologically relevant species in Alzheimer's disease. Homogeneous Aß dimers are difficult to prepare, characterize, and study because Aß forms heterogeneous mixtures of oligomers that vary in size and can rapidly aggregate into more stable fibrils. This paper introduces AßC18C33 as a disulfide-stabilized analogue of Aß42 that forms stable homogeneous dimers in lipid environments but does not aggregate to form insoluble fibrils. The AßC18C33 peptide is readily expressed in Escherichia coli and purified by reverse-phase HPLC to give ca. 8 mg of pure peptide per liter of bacterial culture. SDS-PAGE establishes that AßC18C33 forms homogeneous dimers in the membrane-like environment of SDS and that conformational stabilization of the peptide with a disulfide bond prevents the formation of heterogeneous mixtures of oligomers. Mass spectrometric (MS) studies in the presence of dodecyl maltoside (DDM) further confirm the formation of stable noncovalent dimers. Circular dichroism (CD) spectroscopy establishes that AßC18C33 adopts a ß-sheet conformation in detergent solutions and supports a model in which the intramolecular disulfide bond induces ß-hairpin folding and dimer formation in lipid environments. Thioflavin T (ThT) fluorescence assays and transmission electron microscopy (TEM) studies indicate that AßC18C33 does not undergo fibril formation in aqueous buffer solutions and demonstrate that the intramolecular disulfide bond prevents fibril formation. The recently published NMR structure of an Aß42 tetramer (PDB: 6RHY) provides a working model for the AßC18C33 dimer, in which two ß-hairpins assemble through hydrogen bonding to form a four-stranded antiparallel ß-sheet. It is anticipated that AßC18C33 will serve as a stable, nonfibrilizing, and noncovalent Aß dimer model for amyloid and Alzheimer's disease research.

Influence of the heme distal pocket on nitrite binding orientation and reactivity in Sperm Whale myoglobin.

Nitrite binding to recombinant wild-type Sperm Whale myoglobin (SWMb) was studied using a combination of spectroscopic methods including room-temperature magnetic circular dichroism. These revealed that the reactive species is free nitrous acid and the product of the reaction contains a nitrite ion bound to the ferric heme iron in the nitrito- (O-bound) orientation. This exists in a thermal equilibrium with a low-spin ground state and a high-spin excited state and is spectroscopically distinct from the purely low-spin nitro- (N-bound) species observed in the H64V SWMb variant. Substitution of the proximal heme ligand, histidine-93, with lysine yields a novel form of myoglobin (H93K) with enhanced reactivity towards nitrite. The nitrito-mode of binding to the ferric heme iron is retained in the H93K variant again as a thermal equilibrium of spin-states. This proximal substitution influences the heme distal pocket causing the pKa of the alkaline transition to be lowered relative to wild-type SWMb. This change in the environment of the distal pocket coupled with nitrito-binding is the most likely explanation for the 8-fold increase in the rate of nitrite reduction by H93K relative to WT SWMb.

PDBMD2CD: providing predicted protein circular dichroism spectra from multiple molecular dynamics-generated protein structures.

PDBMD2CD is a new web server capable of predicting circular dichroism (CD) spectra for multiple protein structures derived from molecular dynamics (MD) simulations, enabling predictions from thousands of protein atomic coordinate files (e.g. MD trajectories) and generating spectra for each of these structures provided by the user. Using MD enables exploration of systems that cannot be monitored by direct experimentation. Validation of MD-derived data from these types of trajectories can be difficult via conventional structure-determining techniques such as crystallography or nuclear magnetic resonance spectroscopy. CD is an experimental technique that can provide protein structure information from such conditions. The website utilizes a much faster (minimum ∼1000×) and more accurate approach for calculating CD spectra than its predecessor, PDB2CD (1). As well as improving on the speed and accuracy of current methods, new analysis tools are provided to cluster predictions or compare them against experimental CD spectra. By identifying a subset of the closest predicted CD spectra derived from PDBMD2CD to an experimental spectrum, the associated cluster of structures could be representative of those found under the conditions in which the MD studies were undertaken, thereby offering an analytical insight into the results. PDBMD2CD is freely available at: https://pdbmd2cd.cryst.bbk.ac.uk.

Tools and methods for circular dichroism spectroscopy of proteins: a tutorial review.

Circular dichroism (CD) spectroscopy is a widely-used method in biochemistry, structural biology and pharmaceutical chemistry. More than 24 000 papers published in the past decade have included CD characterisations of proteins; many of those studies have also included other complementary chemical, biophysical, and computational chemistry methods. This tutorial review describes the background to the technique of CD spectroscopy and good practice methods for high quality data collection. It specifically focuses on both established and new methods and tools available for experimental design and interpretation, data processing, visualisation, analysis, validation, archiving, and accession, including tools developed to enhance the complementarity of this method with other structural and chemical biology studies.

Interaction between miR4749 and Human Serum Albumin as Revealed by Fluorescence, FRET, Atomic Force Spectroscopy and Computational Modelling.

The interaction of Human Serum Albumin (HSA) with the microRNA, miR4749, was investigated by Atomic Force Spectrscopy (AFS), static and time-resolved fluorescence spectroscopy and by computational methods. The formation of a HSA/miR4749 complex with an affinity of about 104 M-1 has been assessed through a Stern-Volmer analysis of steady-state fluorescence quenching of the lone Trp residue (Trp214) emission of HSA. Förster Resonance Energy Transfer (FRET) measurements of fluorescence lifetime of the HSA/miR4749 complex were carried out in the absence and in the presence of an acceptor chromophore linked to miR4749. This allowed us to determine a distance of 4.3 ± 0.5 nm between the lone Trp of HSA and the dye bound to miR4749 5p-end. Such a distance was exploited for a screening of the possible binding sites between HSA and miR4749, as predicted by computational docking. Such an approach, further refined by binding free energy calculations, led us to the identification of a consistent model for the structure of the HSA/miR4749 complex in which a positively charged HSA pocket accommodates the negatively charged miRNA molecule. These results designate native HSA as a suitable miRNA carrier under physiological conditions for delivering to appropriate targets.

Probing the E/K Peptide Coiled-Coil Assembly by Double Electron-Electron Resonance and Circular Dichroism.

Double electron-electron resonance (DEER, also known as PELDOR) and circular dichroism (CD) spectroscopies were explored for the purpose of studying the specificity of the conformation of peptides induced by their assembly into a self-recognizing system. The E and K peptides are known to form a coiled-coil heterodimer. Two paramagnetic TOAC α-amino acid residues were incorporated into each of the peptides (denoted as K** and E**), and a three-dimensional structural investigation in the presence or absence of their unlabeled counterparts E and K was performed. The TOAC spin-labels, replacing two Ala residues in each compound, are covalently and quasi-rigidly connected to the peptide backbone. They are known not to disturb the native structure, so that any conformational change can easily be monitored and assigned. DEER spectroscopy enables the measurement of the intramolecular electron spin-spin distance distribution between the two TOAC labels, within a length range of 1.5-8 nm. This method allows the individual conformational changes for the K**, K**/E, E**, and E**/K molecules to be investigated in glassy frozen solutions. Our data reveal that the conformations of the E** and K** peptides are strongly influenced by the presence of their counterparts. The results are discussed with those from CD spectroscopy and with reference to the already reported nuclear magnetic resonance data. We conclude that the combined DEER/TOAC approach allows us to obtain accurate and reliable information about the conformation of the peptides before and after their assembly into coiled-coil heterodimers. Applications of this induced fit method to other two-component, but more complex, systems, like a receptor and antagonists, a receptor and a hormone, and an enzyme and a ligand, are discussed.

Cα-Methyl-l-valine: A Preferential Choice over α-Aminoisobutyric Acid for Designing Right-Handed α-Helical Scaffolds.

In synthetic peptides containing Gly and coded α-amino acids, one of the most common practices to enhance their helical extent is to incorporate a large number of l-Ala residues along with noncoded, strongly foldameric α-aminoisobutyric acid (Aib) units. Earlier studies have established that Aib-based peptides, with propensity for both the 310- and α-helices, have a tendency to form ordered three-dimensional structure that is much stronger than that exhibited by their l-Ala rich counterparts. However, the achiral nature of Aib induces an inherent, equal preference for the right- and left-handed helical conformations as found in Aib homopeptide stretches. This property poses challenges in the analysis of a model peptide helical conformation based on chirospectroscopic techniques like electronic circular dichroism (ECD), a very important tool for assigning secondary structures. To overcome such ambiguity, we have synthesized and investigated a thermally stable 14-mer peptide in which each of the Aib residues of our previously designed and reported analogue ABGY (where B stands for Aib) is replaced by Cα-methyl-l-valine (L-AMV). Analysis of the results described here from complementary ECD and 1H nuclear magnetic resonance spectroscopic techniques in a variety of environments firmly establishes that the L-AMV-containing peptide exhibits a significantly stronger preference compared to that of its Aib parent in terms of conferring α-helical character. Furthermore, being a chiral α-amino acid, L-AMV shows an intrinsic, extremely strong bias for a quite specific (right-handed) screw sense. These findings emphasize the relevance of L-AMV as a more appropriate unit for the design of right-handed α-helical peptide models that may be utilized as conformationally constrained scaffolds.

Long Tracts of Guanines Drive Aggregation of RNA G-Quadruplexes in the Presence of Spermine.

G-Quadruplexes (GQs) are compact, stable structures in DNA and RNA comprised of two or more tiers of quartets whose G-rich motif of tracts of two or more G's occurs commonly within genomes and transcriptomes. While thermodynamically stable in vitro, these structures remain difficult to study in vivo. One approach to understanding GQ in vivo behavior is to test whether conditions and molecules found in cells facilitate their folding. Polyamines are biogenic polycations that interact with RNA. Among common polyamines, spermine contains the highest charge and is found in eukaryotes, making it a good candidate for association with high-charge density nucleic acid structures like GQs. Using a variety of techniques, including ultraviolet-detected thermal denaturation, circular dichroism, size exclusion chromatography, and confocal microscopy, on an array of quadruplex sequence variants, we find that eukaryotic biological concentrations of spermine induce microaggregation of three-tiered G-rich sequences, but not of purely two-tiered structures, although higher spermine concentrations induce aggregation of even these. The formation of microaggregates can also be induced by addition of as little as a single G to a two-tiered structure; moreover, they form at biological temperatures, are sensitive to salt, and can form in the presence of at least some flanking sequence. Notably, GQ aggregation is not observed under prokaryotic-like conditions of no spermine and higher NaCl concentrations. The sequence, polyamine, and salt specificity of microaggregation reported herein have implications for the formation and stability of G-rich nucleic acid aggregates in vivo and for functional roles for understudied GQ sequences with only two quadruplex tiers.

Molecular Insights into Conformational Heterogeneity and Enhanced Structural Integrity of Helicobacter pylori DNA Binding Protein Hup at Low pH.

The summarized amalgam of internal relaxation modulations and external forces like pH, temperature, and solvent conditions determine the protein structure, stability, and function. In a free-energy landscape, although conformers are arranged in vertical hierarchy, there exist several adjacent parallel sets with conformers occupying equivalent energy cleft. Such conformational states are pre-requisites for the functioning of proteins that have oscillating environmental conditions. As these conformational changes have utterly small re-arrangements, nuclear magnetic resonance (NMR) spectroscopy is unique in elucidating the structure-dynamics-stability-function relationships for such conformations. Helicobacter pylori survives and causes gastric cancer at extremely low pH also. However, least is known as to how the genome of the pathogen is protected from reactive oxygen species (ROS) scavenging in the gut at low pH under acidic stress. In the current study, biophysical characteristics of H. pylori DNA binding protein (Hup) have been elucidated at pH 2 using a combination of circular dichroism, fluorescence, NMR spectroscopy, and molecular dynamics simulations. Interestingly, the protein was found to have conserved structural features, differential backbone dynamics, enhanced stability, and DNA binding ability at low pH as well. In summary, the study suggests the partaking of Hup protein even at low pH in DNA protection for maintaining the genome integrity.