European Biophysics Journal最新文献

The thiamine (vitamin B1) biosynthesis pathway is essential for most prokaryotes and some eukaryotes, including yeasts and plants. The 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase (HMPP kinase), encoded by the thiD gene, catalyzes two phosphorylation reactions involving intermediates in this pathway, ultimately producing thiamine pyrophosphate, the active form of thiamine. Here, we present the crystal structure of HMPP kinase from Thermus thermophilus HB8 (TtHMPPK), resolved at a resolution of 2.05 Å. The asymmetric unit of the TtHMPPK structure includes one protomer, though it functions as a homodimer in its active form, like the HMPP kinase from Salmonella typhimurium. The TtHMPPK protomer is an α/β protein featuring nine β-sheets flanked by eight structurally conserved α-helices, which are characteristic of the ribokinase family. The structure reveals a Rossmann β–α–β motif, commonly found in nucleotide-binding proteins. Structural analysis of TtHMPPK, compared to the Salmonella typhimurium HMPP kinase, indicates that Ala16, Thr40, Gln42, Ala78, and Val105 are active site residues involved in catalysis. The structural studies suggest that TtHMPPK belongs to the ribokinase superfamily and exhibits structural similarities with an enzyme containing a Rossmann-like structural motif (RLM). This Rossmann fold enables HMPP kinase to catalyze the phosphorylation of HMPP, a critical step in thiamine production.

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Comparing experimental and calculated hydrodynamic properties of (bio)-macromolecules, such as the translational diffusion coefficient (D_{t(20,w)}^{0}) and the intrinsic viscosity [η], is a useful strategy for the validation of predicted and/or solved atomic-level structures. Bead modeling is a prominent methodology, with several computational tools available. The program GRPY (Generalized Rotne–Prager–Yamakawa) allows the hydrodynamic calculations to be performed at the one-atom one-bead scale, allowing overlaps, but it is computer intensive with CPU requirements depending on the number of beads N as ~ N³. The program ZENO, based on the electrostatics–hydrodynamics analogy and using a Monte Carlo numerical path integration, can compute (D_{t(20,w)}^{0}) and [η] directly on bead models, and it is almost independent of the target size. Since bead models are a very efficient way to include the hydration effect when dealing with bio-macromolecules, we present here an in-depth comparison between GRPY and ZENO, both as implemented in the US-SOMO suite. Relatively low but systematic differences (0.2–2%, increasing with model size) appear when using bead models of proteins at the residue- or atomic-level scales. When comparing the results provided on a restricted set of bead models by two other computationally intensive methods having other drawbacks, the very accurate but not handling overlaps HYDROMULTIPOLE, and the boundary elements BEST requiring extrapolation, GRPY was found to fare better than ZENO. While efforts are in progress to directly improve the ZENO performance, a heuristic correction based on the results for a series of protein bead models is proposed, allowing for a better consistency with GRPY.

Recombinant adeno-associated virus (rAAV) has been widely used as an effective delivery tool in gene therapy. One of the challenges facing the production of high-quality rAAV is optimization of the production and purification methodologies. Cesium chloride density gradient ultracentrifugation (CsCl-DGUC) has been traditionally utilized for rAAV purification; however, few studies have focused on CsCl-DGUC for rAAV purification despite this technique having a great potential for clinical-grade or large-scale rAAV production. In this study, we aimed to explore the unaddressed challenges associated with rAAV purification using CsCl-DGUC. We clarified that AAV capsids assembled by the different stoichiometries of three viral proteins (VP1, VP2, and VP3) showed heterogeneous population in the CsCl density gradient and encapsidated DNA increased the buoyant density differences of these capsids, resulting in the wider distribution. We implemented CsCl-DGUC using a vertical rotor which improved throughput and enhanced the separation of desired AAV particles from impurities. Furthermore, we examined the effect of CsCl exposure during purification, and the presence of residual CsCl in the purified rAAV. This study provides valuable insights into the application of CsCl-DGUC in the manufacturing of rAAV while ensuring adequate efficacy and safety for gene therapy.

In the study of protein self-assembly, knowledge of the extent of electrical and hydrophobic interactions is important. In previous work our group deduced an expression for the hydrophobic energy between the monomers of an assembly. This energy decays exponentially with a characteristic distance r_H. The object of this work is to obtain a more precise physical interpretation of r_H. In very simple systems, according to our model, r_H turns out to be the distance between the hydrophobic dipole moment vectors H. As systems become more complex and the action of the electrostatic dipole moment vectors D appear, discrepancies begin to be seen between the values obtained for r_H and the distances between vectors. It is observed that the simple application of Coulomb’s law is not sufficient to explain these discrepancies. We introduce the (D–H) factor into the electrostatic interaction, since proteins interact within an ionic medium. This formulation implies the appearance of an exponential decay factor r_D, which is the thickness of the ionic atmosphere surrounding protein molecules. The distance adopted by two interacting monomers in a protein assembly is affected by both types of interaction and therefore is a function of both r_H and r_D. In a number of cases, the electrostatic interaction between D vectors is repulsive, generating a potential barrier that monomers are able to cross thanks to an overwhelmingly attractive hydrophobic potential well. In other cases both interactions are attractive and the distance between monomers appears as a compromise of both r_H and r_D.

The simulation of analytical ultracentrifugation data in the sedimentation velocity (SV) mode is extremely useful for experimental planning and hypothesis testing. However, undertaking such simulations can be daunting, especially if one is unpracticed in SV analytic software and the underlying hydrodynamic precepts of the method. Recently, to address this need, the software SViMULATE was introduced. This software featured a simple user interface and facile, on-the-fly conversions of familiar macromolecular properties (e.g., molar mass, shape) to the quantities needed for a successful SV simulation (the sedimentation coefficient, s, and the translational diffusion coefficient, D_T). The software offered an easy route to simulate an unlimited number of species, and two experimental modes, normal and difference SV, were enabled. In the current work, features added to SViMULATE since its initial release are detailed. These include new experimental modes: two interacting systems, nonideal sedimentation, flotation, and band (or “analytical zone”) SV. Further, the modeling of polydisperse species as a series of related individual species has been enabled, and more sophisticated radial and time discretizations enhance the numerical stability of the simulation engine. These features significantly expand the scope and utility of the software, and the advances described herein are immediately available in version 1.4.0 of SViMULATE.

After the elastic regime is surpassed, cortical bone exhibits significant microcracking in its post-elastic mechanical behavior. This work develops a thermodynamically consistent, nonlinear constitutive model based on statistical mechanics, designed to predict the stress–strain relationship and the progression of inter-osteon microcracking. To assess the model’s sufficiency, precise tensile and bending tests were performed in comparison to empirical curves that illustrated theoretical predictions of constitutive relationships. Moreover, entropy increases were quantitatively assessed using model parameters refined through experimental data. A large-size sample was utilized, comprising 51 dog-bone-shaped cortical bone specimens from the 4th ribs of various subjects for uniaxial tensile tests, and 15 complete fourth ribs for bending tests. Displacement and strain fields were meticulously recorded using digital image correlation and video analysis. The model demonstrated robustness, accurately fitting the data from all experimental specimens and revealing correlations between constitutive parameters and anthropometric variables. Entropy calculations provide insights into the behavior of the bone under varying strains: microcracking is minimal at low strains with stress nearly proportional to strain, escalating significantly beyond a critical threshold, thus challenging the linear relationship between stress and strain.