Biophysical journal最新文献

Gene transcription by an RNA polymerase (RNAP) enzyme requires that double-stranded DNA be locally and transiently opened, which results in an increase of DNA supercoiling downstream of the RNAP and a decrease of supercoiling upstream of it. When the DNA is initially torsionally relaxed and the RNAP experiences sufficiently large rotational drag, these variations lead to positively supercoiled plectonemes ahead of the RNAPs and negatively supercoiled ones behind it, a feature known as "twin supercoiled domain" (TSD). This work aims at deciphering into some more detail the torsional dynamics of circular DNA molecules being transcribed by RNAP enzymes. To this end, we performed Brownian dynamics simulations with a specially designed coarse-grained model. Depending on the superhelical density of the DNA molecule and the ratio of RNAP's twist injection rate and rotational relaxation speed, simulations reveal a rich panel of behaviors, which sometimes differ markedly from the crude TSD picture. In particular, for sufficiently slow rotational relaxation speed, positively supercoiled plectonemes never form ahead of an RNAP that transcribes a DNA molecule with physiological negative supercoiling. Rather, negatively supercoiled plectonemes form almost periodically at the upstream side of the RNAP and grow up to a certain length before detaching from the RNAP and destabilizing rapidly. The extent to which topological barriers hinder the dynamics of TSDs is also discussed.

Weak protein interactions are associated with a broad array of biological functions and are often implicated in molecular dysfunction accompanying human disease. In addition, these interactions are a critical determinant in the effective manufacturing, stability, and administration of biotherapeutic proteins. Despite their prominence, much remains unknown about how molecular attributes influence the hydrodynamic and thermodynamic contributions to the overall interaction mechanism. To systematically probe these contributions, we have evaluated self-interaction in a diverse set of proteins that demonstrate a broad range of behaviors from attractive to repulsive. Analysis of the composite trending in the data provides a convenient interconversion among interaction parameters measured from the concentration dependence of the molecular weight, diffusion coefficient, and sedimentation coefficient, as well as insight into the relationship between thermodynamic and hydrodynamic interactions. We find relatively good agreement between our data and a model for interacting hard spheres in the range of weak self-association. In addition, we propose an empirically derived, general scaling relationship applicable across a broad range of self-association and repulsive behaviors.

Many functions of ribonucleic acid (RNA) rely on its ability to assume specific sequence-structure motifs. Packaging signals found in certain RNA viruses are one such prominent example of functional RNA motifs. These signals are short hairpin loops that interact with coat proteins and drive viral self-assembly. As they are found in different positions along the much longer genomic RNA, the formation of their correct structure occurs as a part of a larger context. Any changes to this context can consequently lead to changes in the structure of the motifs themselves. In fact, previous studies have shown that structure and function of RNA motifs can be highly context sensitive to the flanking sequence surrounding them. However, in what ways different flanking sequences influence the structure of an RNA motif they surround has yet to be studied in detail. We focus on a hairpin-rich region of the RNA genome of bacteriophage MS2-a well-studied RNA virus with a wide potential for use in biotechnology-and systematically examine context-dependent structural stability of 14 previously identified hairpin motifs, which include putative and confirmed packaging signals. Combining secondary and tertiary RNA structure prediction of the hairpin motifs placed in different contexts, ranging from the native genomic sequence to random RNA sequences and unstructured poly-U sequences, we determine different measures of motif structural stability. In this way, we show that while some motif structures can be stable in any context, others require specific context provided by the genome. Our results demonstrate the importance of context in RNA structure formation and how changes in the flanking sequence of an RNA motif sometimes lead to drastic changes in its structure. Structural stability of a motif in different contexts could provide additional insights into its functionality as well as assist in determining whether it remains functional when intentionally placed in other contexts.

Recent experiments have demonstrated that the ejection velocity of different species of DNA viruses is temperature dependent, potentially influencing the cellular infection mechanisms of these viruses. However, due to the challenge in quantifying the multiscale characteristics of DNA virus systems, there is currently a lack of systematic theoretical research on the temperature-dependent evolution of ejection dynamics. This work presents a multiscale model to quantitatively explore the temperature-dependent mechanical properties during the virus ejection process, and unveil the underlying mechanisms. Two different assumptions of DNA structures, featuring two or single domains, are used for the early and later stages of ejection, respectively. Temperature is introduced as an influencing variable into the mesoscopic energy model by considering the temperature dependence of Debye length, DNA persistence length, molecular kinetic energy, and other parameters. The results indicate that temperature variations alter the energy landscape associated with DNA structure, leading to the changes in the energy minimum and corresponding DNA structure remaining in the capsid. These changes affect both the active ejection force and passive friction during the DNA ejection, ultimately leading to a significant increase in ejection velocity at higher temperatures. Furthermore, our model supports the previous hypothesis that temperature-induced changes in the size of viral portal pore could dramatically enhance DNA ejection velocity.

Many proteins with intrinsically disordered regions undergo liquid-liquid phase separation under specific conditions in vitro and in vivo. These complex biopolymers form a metastable phase with distinct mechanical properties defining the timescale of their biological functions. However, determining these properties is nontrivial, even in vitro, and often requires multiple techniques. Here we report the measurement of both viscosity and surface tension of biomolecular condensates via correlative fluorescence microscopy and atomic force microscopy (AFM) in a single experiment (fluorescence recovery after probe-induced dewetting, FRAP-ID). Upon surface tension evaluation via regular AFM-force spectroscopy, controlled AFM indentations induce dry spots in fluorescent condensates on a glass coverslip. The subsequent rewetting exhibits a contact line velocity that is used to quantify the condensed-phase viscosity. Therefore, in contrast with fluorescence recovery after photobleaching (FRAP), where molecular diffusion is observed, in FRAP-ID fluorescence recovery is obtained through fluid rewetting and the subsequent morphological relaxation. We show that the latter can be used to cross-validate viscosity values determined during the rewetting regime. Making use of fluid mechanics, FRAP-ID is a valuable tool to evaluate the mechanical properties that govern the dynamics of biomolecular condensates and determine how these properties impact the temporal aspects of condensate functionality.

Signaling through the Ras-MAPK pathway can exhibit switch-like activation, which has been attributed to the underlying positive feedback and bimodality in the activation of RasGDP to RasGTP by SOS. SOS contains both catalytic and allosteric Ras binding sites, and a common assumption is that allosteric activation selectively by RasGTP provides the mechanism of positive feedback. However, recent single-molecule studies have revealed that SOS catalytic rates are independent of the nucleotide state of Ras in the allosteric binding site, raising doubt about this as a positive feedback mechanism. Here, we perform detailed kinetic analyses of receptor-mediated recruitment of full-length SOS to the membrane while simultaneously monitoring its catalytic activation of Ras. These results, along with kinetic modeling, expose the autoinhibition release step in SOS, rather than either recruitment or allosteric activation, as the underlying mechanism giving rise to positive feedback in Ras activation.

T. maritima and B. subtilis are bacteria that inhabit significantly different thermal environments, ∼80 vs. ∼40°C, yet employ similar lysine riboswitches to aid in the transcriptional regulation of the genes involved in the synthesis and transport of amino acids. Despite notable differences in G-C basepair frequency and primary sequence, the aptamer moieties of each riboswitch have striking similarities in tertiary structure, with several conserved motifs and long-range interactions. To explore genetic adaptation in extreme thermal environments, we compare the kinetic and thermodynamic behaviors in T. maritima and B. subtilis lysine riboswitches via single-molecule fluorescence resonance energy transfer analysis. Kinetic studies reveal that riboswitch folding rates increase with lysine concentration while the unfolding rates are independent of lysine. This indicates that both riboswitches bind lysine through an induced-fit ("bind-then-fold") mechanism, with lysine binding necessarily preceding conformational changes. Temperature-dependent van't Hoff studies reveal qualitative similarities in the thermodynamic landscapes for both riboswitches in which progression from the open, lysine-unbound state to both transition states (‡) and closed, lysine-bound conformations is enthalpically favored yet entropically penalized, with comparisons of enthalpic and entropic contributions extrapolated to a common [K⁺] = 100 mM in quantitative agreement. Finally, temperature-dependent Eyring analysis reveals the TMA and BSU riboswitches to have remarkably similar folding/unfolding rate constants when extrapolated to their respective (40 and 80°C) environmental temperatures. Such behavior suggests a shared strategy for ligand binding and aptamer conformational change in the two riboswitches, based on thermodynamic adaptations in number of G-C basepairs and/or modifications in tertiary structure that stabilize the ligand-unbound conformation to achieve biocompetence under both hyperthermophilic and mesothermophilic conditions.

Phycocyanobilin (PCB)-binding proteins, including cyanobacteriochromes and phytochromes, function as photoreceptors and exhibit a wide range of absorption maximum wavelengths. To elucidate the color-tuning mechanisms among these proteins, we investigated seven crystal structures of six PCB-binding proteins: Anacy_2551g3, AnPixJg2, phosphorylation-responsive photosensitive histidine kinase, RcaE, Sb.phyB(PG)-PCB, and Slr1393g3. Employing a quantum chemical/molecular mechanical approach combined with a polarizable continuum model, our analysis revealed that differences in absorption wavelengths among PCB-binding proteins primarily arise from variations in the shape of the PCB molecule itself, accounting for a ∼150 nm difference. Remarkably, calculated excitation energies sufficiently reproduced the absorption wavelengths of these proteins spanning ∼200 nm, including 728 nm for Anacy_2551g3. However, assuming the hypothesized lactim conformation resulted in a significant deviation from the experimentally measured absorption wavelength for Anacy_2551g3. The significantly red-shifted absorption wavelength of Anacy_2551g3 can unambiguously be explained by the significant overlap of molecular orbitals between the two pyrrole rings at both edges of the PCB chromophore without the need to hypothesize lactim formation.