Biophysical chemistry最新文献

{"title":"The Drosophila RNA binding protein Hrp48 binds a specific RNA sequence of the msl-2 mRNA 3’ UTR to regulate translation","authors":"","doi":"10.1016/j.bpc.2024.107346","DOIUrl":"10.1016/j.bpc.2024.107346","url":null,"abstract":"<div><div>Repression of <em>msl-2</em> mRNA translation is essential for viability of <em>Drosophila melanogaster</em> females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3’ untranslated region (UTR) of the <em>msl-2</em> transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with <em>msl-2</em> are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of <em>msl-2</em> 3’ UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to <em>msl-2</em>.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142590115","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Understanding Cu+2 binding with DNA: A molecular dynamics study comparing Cu2+ and Mg2+ binding to the Dickerson DNA","authors":"","doi":"10.1016/j.bpc.2024.107347","DOIUrl":"10.1016/j.bpc.2024.107347","url":null,"abstract":"<div><div>Cu²⁺ ions led DNA damage by reactive oxygen species (ROS) is widely known biological phenomena. The ionic radii of Cu²⁺ and Mg²⁺ being similar, the binding of Cu²⁺ ions to DNA is expected to be similar to that of the Mg²⁺ ions. However, little is known how Cu²⁺ ions bind in different parts (phosphate, major and minor grooves) of a double-strand (ds) DNA, especially at atomic level. In the present study, we employ molecular dynamic (MD) simulations to investigate the binding of Cu²⁺ ions with the Dickerson DNA, a B-type dodecamer double stranded (ds) DNA. The binding characteristics of Cu²⁺ and Mg²⁺ ions with this dsDNA are compared to get an insight into the differences and similarities in binding behavior of both ions. Unlike Mg²⁺ ions, the first hydration shell of Cu²⁺ is found to be labile, thus it shows both direct and indirect binding with the dsDNA, i.e., binding through displacement of water from the hydration shell or through the hydration shell. Though the binding propensity of Cu²⁺ ions with dsDNA is observed relatively stronger, the binding order to phosphates, major groove, and minor groove is found qualitatively similar (phosphates &gt; major groove &gt; minor groove) for both ions. The study gives a deep understanding of Cu²⁺ binding to DNA, which could be helpful in rationalizing the Cu²⁺ led ROS-mediated DNA damage.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142567741","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biophysical significance of fluorescence spectroscopy in deciphering nucleic acid dynamics: From fundamental to recent advancements","authors":"","doi":"10.1016/j.bpc.2024.107345","DOIUrl":"10.1016/j.bpc.2024.107345","url":null,"abstract":"<div><div>Fluorescence spectroscopy has revolutionized the study of nucleic acids, providing invaluable insights into the dynamic processes that underpin gene expression, replication, and repair. This review explores the application of fluorescence probes in monitoring the conformational changes, interactions, and regulatory mechanisms of DNA and RNA. We discuss the utility of intercalating and non-intercalating fluorescent probes in real-time tracking of nucleic acid dynamics, highlighting their role in elucidating the molecular mechanisms of DNA replication, transcriptional regulation, and DNA repair. By offering a detailed analysis of recent advancements, this review underscores the significance of fluorescence-based techniques in enhancing our understanding of nucleic acid behavior and their implications for genomic stability and therapeutic interventions.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142534624","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"How fingers affect folding of a thumb: Inter-subdomain cooperation in the folding of SARS-CoV-2 RdRp protein","authors":"","doi":"10.1016/j.bpc.2024.107342","DOIUrl":"10.1016/j.bpc.2024.107342","url":null,"abstract":"<div><div>The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is a critical enzyme essential for the virus's replication and transcription, making it a key therapeutic target. The RdRp protein exhibits a characteristic cupped right-hand shaped structure with two vital subdomains: the fingers and the thumb. Despite being distinct, biophysical experiments suggest that these subdomains cooperate to facilitate RNA accommodation, ensuring RdRp functionality. To investigate the structure-based mechanisms underlying the fingers-thumb interaction in both apo and RNA-bound RdRp, we constructed a coarse-grained structure-based model based on recent cryo-electron microscopy data. The simulations reveal frequent open-to-closed conformational transitions in apo RdRp, akin to a breathing-like motion. These conformational changes are regulated by the fingers-thumb association and the folding dynamics of the thumb subdomain. The thumb adopts a stable fold only when tethered by the fingers-thumb interface; when these subdomains are disconnected, the thumb transitions into an open state. A significant number of open-to-closed transition events were analyzed to generate a transition contact probability map, which highlights a few specific residues at the thumb-fingers interface, distant from the RNA accommodation sites, as essential for inducing the thumb's folding process. Given that thumb subdomain folding is critical for RNA binding and viral replication, the study proposes that these interfacial residues may function as remote regulatory switches and could be targeted for the development of allosteric drugs against SARS-CoV-2 and similar RNA viruses.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142534111","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vitro and in silico effect of meldrum's acid-derived compounds on Staphylococcus aureus strains as NorA efflux pump inhibitors","authors":"","doi":"10.1016/j.bpc.2024.107344","DOIUrl":"10.1016/j.bpc.2024.107344","url":null,"abstract":"<div><div>The misuse of antibiotics has led to an alarming increase in bacterial strains resistant to these drugs. Efflux pumps, which expel antibiotics from bacterial cells, have emerged as one of the key mechanisms of bacterial resistance. In the quest to combat and mitigate bacterial resistance, researchers have turned their attention to efflux pump inhibitors as a potential solution. Meldrum's acid, a synthetic molecule widely utilized in the synthesis of bioactive compounds, has garnered significant interest in this regard. Hence, this study aims to investigate the antibacterial activity and evaluate the efficacy of three derivatives of meldrum's acid in inhibiting efflux mechanisms, employing both in silico and in vitro approaches. The antibacterial activity of the derivatives was assessed through rigorous broth microdilution testing. While the derivatives themselves did not exhibit direct antibacterial activity, they demonstrated remarkable potential in potentiating the effects of antibiotics. Additionally, fluorescence emission assays using ethidium bromide (EtBr) revealed fluorescence levels comparable to the positive control, indicating a possible blockade of efflux pumps. Molecular docking studies conducted in silico further supported these findings by revealing binding interactions similar to norfloxacin and CCCP, known efflux pump inhibitors. These results underscore the potential of meldrum's acid derivatives as effective inhibitors of efflux pumps. By inhibiting these mechanisms, the derivatives hold promise in enhancing the effectiveness of antibiotics and combatting bacterial resistance. This study contributes valuable insights into the development of novel strategies to address the pressing issue of bacterial resistance and paves the way for further research and exploration in this field.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142494593","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Solubilisation & purification of membrane proteins using benzylamine-modified SMA polymers","authors":"","doi":"10.1016/j.bpc.2024.107343","DOIUrl":"10.1016/j.bpc.2024.107343","url":null,"abstract":"<div><div>Extraction of proteins from the membrane using styrene maleic acid <em>co</em>-polymers (SMA), forming SMA lipid particles (SMALPs), has allowed for the first time the purification of membrane proteins with their lipid bilayer environment. To date, SMA2000 has been the most effective polymer used for this purpose, with a 2:1 ratio of styrene:maleic acid, and styrene and maleic acid moieties spread statistically throughout the chain. However, SMA2000 is a highly polydisperse polymer that contains an array of different polymer lengths and sequences. RAFT polymerisation offers much better control over the polymer length; however, homogeneous distribution of styrene and maleic acid throughout the polymer is difficult to achieve. Instead, here RAFT polymerisation was used to produce a 1:1 styrene:maleic anhydride polymer, which was then modified with benzylamine. This mimics the 2:1 hydrophobic:hydrophilic nature of SMA2000, while controlling the length and obtaining a homogeneous distribution of the hydrophobic moieties (styrene and <em>N</em>-benzylmaleimide). SMA-benzylamine (SMA-BA) polymers of three different lengths (2, 4, and 7 kDa) were all able to solubilise purified lipids, cellular membranes, and a range of specific proteins. However, the larger 7 kDa polymer solubilised membranes more slowly and less efficiently than the shorter polymers. This also affected the yield of purified protein obtained by affinity purification with this polymer. The smallest 2 kDa polymer solubilised membranes the fastest but appeared to offer less stability to the extracted proteins. The SMA-BA polymers were more sensitive to Mg²⁺ ions than SMA2000. SMA-BA 4 kDa was otherwise comparable to SMA2000 and even gave a higher degree of purity.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142494594","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"On the production of singlet oxygen by the isoalloxazine ring in free and protein-bound flavin cofactors","authors":"","doi":"10.1016/j.bpc.2024.107333","DOIUrl":"10.1016/j.bpc.2024.107333","url":null,"abstract":"<div><div>Flavin cofactors, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), as a part of flavoenzymes play a critical role in the catalysis of multiple reactions predominantly of a redox nature. Question arises why nature developed two very similar cofactors with an identical functional part – isoalloxazine ring. We believe that an answer is related to the fact that the isoalloxazine ring belongs to endogenous photosensitizers able to produce reactive and potentially harmful singlet oxygen, ¹O₂, with high efficiency, Φ_Δ,FMN ∼ 0.6. In fact, in contrast with one main conformation of FMN in water, the presence of the adenosine mononucleotide in FAD induces a dynamic equilibrium of two main conformations – closed (∼80 %) and open (∼20 %). The presence of predominant closed conformation of FAD in water has a significant impact on the Φ_Δ,FAD value, which is nearly 10-fold lower, Φ_Δ,FAD ∼ 0.07, than that of FMN. On the other hand, based on our analysis of a non-homologous dataset of FAD containing 105 proteins, ∼75 % enzyme-bound FAD exists predominantly in open conformations but the Φ_Δ values are significantly decreased, Φ_Δ &lt; 0.03. We addressed these contradictory observations by analysis of: (i) dependence of Φ_Δ,FAD value on opening the FAD conformation by urea and (ii) amino acid propensities for isoalloxazine binding site. We demonstrated that urea-induced destabilization, in 7 M vs 0 M urea, of the closed FAD conformation leads to a ∼ 3-fold increase of Φ_Δ, proving the causative relation between Φ_Δ value and the flavin cofactor conformation. Detailed examination of the flavoproteins dataset clearly indicated positive propensities of three amino acids: glycine, cysteine, and tryptophan for isoalloxazine ring binding site. We hypothesize that both the closed conformation of free FAD and the arrangement of the isoalloxazine binding site is important for prevention of potentially harmful ¹O₂ production in cells.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142436535","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Stability and conformation of DNA-hairpin in cylindrical confinement","authors":"","doi":"10.1016/j.bpc.2024.107331","DOIUrl":"10.1016/j.bpc.2024.107331","url":null,"abstract":"<div><div>We conducted atomistic Molecular Dynamics (MD) simulations of DNA-Hairpin molecules encapsulated within Single-Walled Carbon Nanotubes (SWCNTs) at a temperature of 300 K. Our investigation revealed that the structural integrity of the DNA-Hairpin can be maintained within SWCNTs, provided that the diameter of the SWCNT exceeds a critical threshold value. Conversely, when the SWCNT diameter falls below this critical threshold, the DNA-Hairpin undergoes denaturation, even at a temperature of 300 K. The DNA-Hairpin model we employed consisted of a 12-base pair stem and a 3-base loop, and we studied various SWCNTs with different diameters. Our analyses identified a critical SWCNT diameter of 3.39 nm at 300 K. Examination of key structural features, such as hydrogen bonds (H-bonds), van der Waals (vdW) interactions, and other inter-base interactions, demonstrated a significant reduction in the number of H-bonds, vdW energy, and electrostatic energies among the DNA hairpin's constituent bases when confined within narrower SWCNTs (with diameters of 2.84 nm and 3.25 nm). However, it was observed that the increased interaction energy between the DNA-Hairpin and the inner surface of narrower SWCNTs promoted the denaturation of the DNA-Hairpin. In-depth analysis of electrostatic mapping and hydration status further revealed that the DNA-Hairpin experienced inadequate hydration and non-uniform distribution of counter ions within SWCNTs having diameters below the critical value of 3.39 nm. Our inference is that the inappropriate hydration of counter ions, along with their non-uniform spatial distribution around the DNA hairpin, contributes to the denaturation of the molecule within SWCNTs of smaller diameters. For DNA-Hairpin molecules that remained undenatured within SWCNTs, we investigated their mechanical properties, particularly the elastic properties. Our findings demonstrated an increase in the persistence length of the DNA-Hairpin with increasing SWCNT diameter. Additionally, the stretch modulus and torsional stiffness of the DNA-Hairpin were observed to increase as a function of SWCNT diameter, indicating that confinement within SWCNTs enhances the mechanical flexibility of the DNA-Hairpin.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142457279","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of PARP1 binding to c-KIT1 G-quadruplex DNA: Insights into domain-specific interactions","authors":"","doi":"10.1016/j.bpc.2024.107330","DOIUrl":"10.1016/j.bpc.2024.107330","url":null,"abstract":"<div><div>Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme involved in catalyzing Poly-(ADP-ribosyl)ation. PARP1 binds to different forms of DNA and DNA breaks and thus plays important roles in several cellular processes, including DNA damage repair, cell cycle regulation, chromatin remodeling, and maintaining genomic stability. In this study, we conducted biochemical and biophysical characterization of PARP1 binding to G-quadruplex DNA (G4-DNA). Our investigation identified ZnF1, ZnF3, and WGR as the critical domains to mediate PARP1 binding to G4-c-KIT1. Also, our results show that these domains together show cooperativity for G4-c-KIT1 recognition. Further, we establish that the presence of an oxidized (5-carboxylcytosine) base in the loop region of G4-c-KIT1 (G4-5caC-cKIT1) does not affect its recognition by PARP1. Both G4-c-KIT1 and G4-5caC-cKIT1 are potent stimulators of PARP1's catalytic activity. Our study advances the understanding of PARP1's versatile DNA binding capabilities for G4-c-KIT1 DNA irrespective of the oxidation/ modification in the DNA base. These insights into PARP1's domain-specific contributions to G4-c-KIT1 DNA recognition and catalysis expand our knowledge of its multifaceted roles in DNA repair and genome maintenance.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142341033","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy","authors":"","doi":"10.1016/j.bpc.2024.107329","DOIUrl":"10.1016/j.bpc.2024.107329","url":null,"abstract":"<div><div><em>Natronomonas pharaonis</em> halorhodopsin (<em>Np</em>HR) is a light-driven Cl⁻ inward pump that is widely used as an optogenetic tool. Although <em>Np</em>HR is previously extensively studied, its Cl⁻ uptake process is not well understood from the protein structure perspective, mainly because in crystalline lattice, it has been difficult to analyze the structural changes associated with the Cl⁻ uptake process. In this study, we used solid-state NMR to analyze <em>Np</em>HR both in the Cl⁻-bound and -free states under near-physiological transmembrane condition. Chemical shift perturbation analysis suggested that while the structural change caused by the Cl⁻ depletion is widespread over the <em>Np</em>HR molecule, residues in the extracellular (EC) part of helix D exhibited significant conformational changes that may be related to the Cl⁻ uptake process. By combining photochemical analysis and dynamic nuclear polarization (DNP)-enhanced solid-state NMR measurement on <em>Np</em>HR point mutants for the suggested residues, we confirmed their importance in the Cl⁻ uptake process. In particular, we found the mutation at Ala165 position, located at the trimer interface, to an amino acid with bulky sidechain (A165V) significantly perturbs the late photocycle and disrupts its trimeric assembly in the Cl⁻-free state as well as during the ion-pumping cycle under the photo-irradiated condition. This strongly suggested an outward movement of helix D at EC part, disrupting the trimer integrity. Together with the spectroscopic data and known <em>Np</em>HR crystal structures, we proposed a model that this helix movement is required for creating the Cl⁻ entrance path on the extracellular surface of the protein and is crucial to the Cl⁻ uptake process.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142380057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}