Molecular Insight into the Structural Dynamics of Holliday Junctions Modulated by Integration Host Factor.

The integration host factor (IHF) in Escherichia coli is a nucleoid-associated protein with multifaceted roles that encompass DNA packaging, viral DNA integration, and recombination. IHF binds to double-stranded DNA featuring a 13-base pair (bp) consensus sequence with high affinity, causing a substantial bend of approximately 160° upon binding. Although wild-type IHF (WtIHF) is principally involved in DNA bending to facilitate foreign DNA integration into the host genome, its engineered counterpart, single-chain IHF (ScIHF), was specifically designed for genetic engineering and biotechnological applications. Our study delves into the interactions of both IHF variants with Holliday junctions (HJs), pivotal intermediates in DNA repair, and homologous recombination. HJs are dynamic structures capable of adopting open or stacked conformations, with the open conformation facilitating processes such as branch migration and strand exchange. Using microscale thermophoresis, we quantitatively assessed the binding of IHF to four-way DNA junctions that harbor specific binding sequences H' and H1. Our findings demonstrate that both IHF variants exhibit a strong affinity for HJs, signifying a structure-based recognition mechanism. Circular dichroism (CD) experiments unveiled the impact of the protein on the junction's conformation. Furthermore, single-molecule Förster resonance energy transfer (smFRET) confirmed the influence of IHF on the junction's dynamicity. Intriguingly, our results revealed that WtIHF and ScIHF binding shifts the population toward the open conformation of the junction and stabilizes it in that state. In summary, our findings underscore the robust affinity of the IHF for HJs and its capacity to stabilize the open conformation of these junctions.

G-quadruplex modulation by E. coli SSB: A comprehensive study on binding affinities and modes using single-molecule FRET.

G-quadruplexes (GQs) are essential guanine-rich secondary structures found in DNA and RNA, playing crucial roles in genomic maintenance and stability. Recent studies have unveiled GQs in the intergenic regions of the E. coli genome, suggesting their biological significance and potential as anti-microbial targets. Here, we investigated the interaction between homo-tetrameric E. coli SSB and GQ-forming single-stranded DNA (ssDNA) sequence with varying lengths. Combining Microscale Thermophoresis (MST) and conventional spectroscopic techniques, we explored E. coli SSB binding to ssDNA and the structural changes of these secondary DNA structures upon protein binding. Subsequently, we have utilized smFRET to probe the conformational changes of GQ-ssDNA structures upon SSB binding. Our results provide detailed insights into SSB's access to various GQ-ssDNA sequencies and the wrapping of this homo-tetrameric protein around GQ-ssDNA in multiple distinct binding modalities. This study sheds light on the intricate details of E. coli SSB's interaction with ssDNA and the resulting widespread conformational changes within these oligonucleotide structures after protein binding. It offers a thorough insight into SSB's accesses to various GQ-ssDNA architectures. The finding demonstrates the multifaceted binding methods through which this homo-tetrameric protein envelops GQ-ssDNA and could prove valuable in deciphering biological processes that involve DNA G-quadruplexes.

Insights into the Dynamics and Helicase Activity of RecD2 of Deinococcus radiodurans during DNA Repair: A Single-Molecule Perspective.

While the double helix is the most stable conformation of DNA inside cells, its transient unwinding and subsequent partial separation of the two complementary strands yields an intermediate single-stranded DNA (ssDNA). The ssDNA is involved in all major DNA transactions such as replication, transcription, recombination, and repair. The process of DNA unwinding and translocation is shouldered by helicases that transduce the chemical energy derived from nucleotide triphosphate (NTP) hydrolysis to mechanical energy and utilize it to destabilize hydrogen bonds between complementary base pairs. Consequently, a comprehensive understanding of the molecular mechanisms of these enzymes is essential. In the last few decades, a combination of single-molecule techniques (force-based manipulation and visualization) have been employed to study helicase action. These approaches have allowed researchers to study the single helicase-DNA complex in real-time and the free energy landscape of their interaction together with the detection of conformational intermediates and molecular heterogeneity during the course of helicase action. Furthermore, the unique ability of these techniques to resolve helicase motion at nanometer and millisecond spatial and temporal resolutions, respectively, provided surprising insights into their mechanism of action. This perspective outlines the contribution of single-molecule methods in deciphering molecular details of helicase activities. It also exemplifies how each technique was or can be used to study the helicase action of RecD2 in recombination DNA repair.

Revealing the DNA Unwinding Activity and Mechanism of Fork Reversal by RecG While Exposed to Variants of Stalled Replication-fork at Single-Molecular Resolution.

RecG, belonging to the category of Superfamily-2 plays a vital role in rescuing different kinds of stalled fork. The elemental mechanism of the helicase activity of RecG with several non-homologous stalled fork structures resembling intermediates formed during the process of DNA repair has been investigated in the present study to capture the dynamic stages of genetic rearrangement. The functional characterization has been exemplified through quantifying the response of the substrate in terms of their molecular heterogeneity and dynamical response by employing single-molecule fluorescence methods. An elevated processivity of RecG is observed for the stalled fork where progression of lagging daughter strand is ahead as compared to that of the leading strand. Through precise alteration of its function in terms of unwinding, depending upon the substrate DNA, RecG catalyzes the formation of Holliday junction from a stalled fork DNA. RecG is found to adopt an asymmetric mode of locomotion to unwind the lagging daughter strand for facilitating formation of Holliday junction that acts as a suitable intermediate for recombinational repair pathway. Our results emphasize the mechanism adopted by RecG during its 'sliding back' mode along the lagging daughter strand to be 'active translocation and passive unwinding'. This also provide clues as to how this helicase decides and controls the mode of translocation along the DNA to unwind.

Kineto-Mechanistic Investigation of Effect of Macromolecular Crowding on the Breathing of DNA Bubble.

Macromolecular crowding along with hydrogen bonding or stacking interactions and hydration reportedly has enormous repercussions on elementary biochemical processes, such as the folding of proteins or nucleic acids involving the stability of DNA base pairing. By using the mismatch-induced DNA bubble as a mesoscopic model, the complex interplay of macromolecular crowding on the dynamical fluctuations at the bubble region within the thermodynamic limit has been monitored using single-molecule fluorescence resonance energy transfer (sm-FRET). These single-molecule experimental results have been further corroborated using physical models such as "scaled particle theory" (SPT) and "Gaussian cloud model" (GCM), to predict the biological activity of DNA. The two-state fluctuation of the DNA bubble has been visualized as a function of the nature, size, and concentration of the crowder. The influence of crowders on the DNA conformation has been investigated with the help of the m-factor, the eccentricity, and the kinetic and thermodynamic parameters without any prior assumption. The clear effect of crowding on the dynamics of such a simple biomolecular system emphasizes the power of single-molecule methods and the dependency of the radius of gyration of the co-solute as well as the preferential interaction with the crowder on the distinct conformational states adopted by the bubble. This study provides an idea and hypothesizes the preferential propensity of the DNA bubble to adopt a conformation with the single-stranded domains being far apart, independent of the crowder size, that may be beneficial for efficient recognition by proteins for an uninterrupted procession of the biological process of the central dogma.

Conformational dynamics of myoglobin in the presence of vitamin B12: A spectroscopic and in silico investigation.

Myoglobin is an essential transport protein of heart and muscle tissues that acts as a local oxygen reservoir and a marker in different diseased conditions. On the other hand, Vitamin B12 is a vital nutrient that helps synthesize red blood cells, DNA, and proteins. To understand the ability of vitamin B12 to bind to the excess of myoglobin produced in the body under certain conditions (muscle injuries, severe trauma, etc.), it is essential to dig into the interaction between them. Therefore, the present study reports the binding interaction of vitamin B12 and myoglobin employing different spectroscopic and computational methods. The myoglobin's intrinsic fluorescence is quenched by vitamin B12 via static nature as observed from steady-state as well as time-resolved fluorescence measurements. The microenvironment of myoglobin's tryptophan residue gets affected, but there is no change observed in its α-helical content by vitamin B12 as seen from synchronous fluorescence and circular dichroism measurements. The probable binding of vitamin B12 on myoglobin was elucidated through molecular docking, and the interaction stability was studied by molecular dynamics simulation. The determination of vitamin B12's affinity to myoglobin and its effect on the conformational transitions of myoglobin might afford valuable insight for clinical pharmacology.

Spectroscopic and computational insight into the conformational dynamics of hemoglobin in the presence of vitamin B12.

Protein-ligand interactions play a significant role in all living organisms, thereby affecting the design and application of drugs and other biomaterials. The current study reports the binding of vitamin B12 to hemoglobin, employing optical spectroscopy and computational methods. It is observed that vitamin B12 quenched the intrinsic fluorescence of hemoglobin. The nature of quenching appears to be static according to the steady-state and time-resolved fluorescence measurements. The conformational changes of hemoglobin caused by vitamin B12 interactions were studied by synchronous fluorescence spectroscopy and protein secondary structure analyses. The synchronous fluorescence spectra indicate the tryptophan residue microenvironment change while no secondary structural change is observed from circular dichroism spectra and molecular dynamics (MD) simulation study. The computational molecular docking elucidated the probable binding of vitamin B12 at the active site of hemoglobin, whereas the stability of the hemoglobin-vitamin B12 complex was studied by MD simulation. The study might be helpful for the treatment of pernicious anemia, hereditary transcobalamin deficiency, and performance enhancement of elite athletes.

A spectroscopic and computational intervention of interaction of lysozyme with 6-mercaptopurine.

In the present work, biophysical insight into the binding interactions of the protein, hen egg white (HEW) lysozyme (Lyz) with an anticancer drug, 6-mercaptopurine (6-MP)' was investigated by using a combination of spectroscopic and computational tools. 6-MP, a synthetic analog of natural purines, is a well-known anticancer drug and antiviral agent that inhibits the synthesis of RNA, DNA, and proteins. Lysozyme is a single-chain protein that can combine with endogenous and exogenous substances to exert its antiviral, antibacterial, and antitumor effects. The intrinsic fluorescence of lysozyme was quenched with the increased addition of 6-MP. The quenching mechanism was found to be static in nature as shown by the fluorescence lifetime and excitation spectrum measurements. The conformational changes of Lyz in the presence of 6-MP were monitored both at the ensemble and single-molecule level by using synchronous fluorescence spectroscopy, circular dichroism (CD), and fluorescence correlation spectroscopy (FCS). Molecular docking results predicted the probable binding sites for 6-MP on Lyz. The experimental findings are in good agreement with the results obtained by the molecular dynamics (MD) simulation study. Graphical abstract.

Biophysical study on complex formation between ß-Lactoglobulin and vitamin B12.

Biophysical insight into the binding interaction between the major whey protein, ß-Lactoglobulin (ßLG) and vitamin B12, was studied using different spectroscopic tools such as steady-state & time-resolved fluorescence spectroscopy, Circular Dichroism (CD) and Fluorescence Correlation Spectroscopy (FCS). The intrinsic fluorescence of ßLG was quenched by vitamin B12. From the time-resolved fluorescence experiment, the nature of quenching was found to be static suggesting ground-state complex formation between ßLG and vitamin B12, which was also supported by the excitation spectra. Synchronous fluorescence spectra revealed that the tryptophan residue microenvironment of ßLG was affected by the vitamin B12. The CD spectra suggested that the secondary structure of the ßLG remains unaffected by vitamin B12. From the FCS experiment, the tertiary structure of ßLG was observed to be stable in the presence of vitamin B12 at the single-molecule level. The outcome of this study might have potential applications in the food and pharmaceutical industry.

Spectroscopic insight into the interaction of dopamine with spherical gold nanoparticles.

Dopamine (DA) is a monoamine neurotransmitter of phenethylamine and catecholamine families, which is present in the central nervous system (CNS) and its periphery. Since DA is associated with several functions in the brain and body (motivational salience, reward, motor control, paracrine messenger, etc.), any imbalance in the DA level can trigger several neurodegenerative and other diseases. On the other hand, the spherical gold nanoparticles (AuNPs) can be used for drug delivery in several parts of the body. In addition, AuNPs also have the potentiality to penetrate through the blood-brain barrier and interact with the central nervous system without causing any toxicity. In view of many applications, it is important to look into the interaction between DA and AuNPs for a potential drug delivery model in DA related diseases. Here, we have used the steady-state and time-resolved fluorescence spectroscopic tools to investigate the binding interaction of DA with AuNPs. The nature of the quenching mechanism was confirmed through both steady-state and time-resolved fluorescence measurements. The binding constants along with the number of binding sites were estimated from the steady-state fluorescence measurements. The distance between DA and AuNPs was calculated using Förster's theory to verify the possibility of fluorescence resonance energy transfer (FRET) from DA to AuNPs.