Biology subjects

Krupa, P.

Publications and source records attributed to Krupa, P..

4 recordsLinked to original sources

bioRxiv2024

Serotonin Promotes Vesicular Association and Fusion by Modifying Lipid Bilayers

The primary event in chemical neurotransmission involves the fusion of a membrane-limited vesicle at the plasma membrane and the subsequent release of its chemical neurotransmitter cargo. The cargo itself is not known to have any effect on the fusion event. However, amphiphilic monoamine neurotransmitters (e.g. serotonin and dopamine) are known to strongly interact with lipid bilayers and to affect their mechanical properties, which can in principle impact membrane-mediated processes. Here we probe whether serotonin can enhance the association and fusion of artificial lipid vesicles in vitro. We employ Fluorescence Correlation Spectroscopy and Total Internal Reflection Fluorescence microscopy to measure the attachment and fusion of vesicles whose lipid compositions mimic the major lipid components of synaptic vesicles. We find that association between vesicles and supported lipid bilayers are strongly enhanced in a serotonin dose-dependent manner, and this drives an increase in the rate of spontaneous fusion. Molecular dynamics simulations and fluorescence spectroscopy data show that serotonin insertion increases the water content of the hydrophobic part of the bilayer. This suggests that the enhanced membrane association is likely driven by an energetically favourable drying transition. Other monoamines such as dopamine and norepinephrine, but not other related species such as tryptophan, show similar effects on membrane association. Our results reveal a lipid bilayer-mediated mechanism by which monoamines can themselves modulate vesicle fusion, potentially adding to the control toolbox for the tightly regulated process of neurotransmission in vivo. TOC graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/576155v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@2717c5org.highwire.dtl.DTLVardef@899c21org.highwire.dtl.DTLVardef@697232org.highwire.dtl.DTLVardef@c850a0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Protocols for Multi-Scale Molecular Dynamics Simulations in Amber and Gromacs: a Case Study of Intrinsically Disordered Amyloid Beta

Intrinsically disordered proteins (IDPs) present challenges to conventional experimental techniques due to their large-scale conformational fluctuations and the transient occurrence of structural elements. This work illustrates computational methods for studying IDPs at various levels of resolution. The included simulation protocol offers a step-by-step guide on how to conduct molecular dynamics (MD) simulations and analyze the results using the Amber and Gromacs packages, employing both all-atom and coarse-grained approaches. This protocol can be easily adapted to study other biomacromolecules, including folded and disordered proteins and peptides. Furthermore, it is discussed in this work how to perform standard molecular modeling operations, such as amino-acid substitutions (mutagenesis) and insertions of residues missing in a protein structure, as well as how to incorporate post-translational modifications into the simulations, such as disulfide bonds, which are often crucial for proteins to attain their physiologically functional structure. In conventional MD studies, disulfide bonds are typically fixed at the preparation step and remain unchanged throughout the simulations, unable to break or reform. Here, in contrast, a dynamic approach is presented. It involves adequate distance restraints applied to the sulfur atoms of selected cysteine residues, allowing disulfide bonds to break and reform during the simulation. The effectiveness of these methodologies is demonstrated by examining a model IDP, the monomeric form of 1-42 amyloid-{beta} (A{beta}42), both with and without disulfide bonds, at different levels of resolution. This study not only contributes to our understanding of the role of disulfide bonds but also provides detailed simulation protocols that can serve as a foundation for future investigations. SUMMARYGiven the challenges of experimental studies on intrinsically disordered proteins, this manuscript demonstrates step-by-step protocols for conducting all-atom and coarse-grained molecular dynamics simulations using two widespread packages, Amber and Gromacs. The monomeric form of 1-42 amyloid-{beta} (A{beta}42) is used as an example, from which insights into the structure, dynamics and physicochemical properties of this protein can be obtained.

molecular biology↗

bioRxiv2023

Computational Design of a Highly-Specific HVEM-Based Inhibitor of LIGHT Protein

MotivationHVEM-LIGHT binding regulates the immune system response in various ways: it co-stimulates T cell proliferation; promotes B cell differentiation and secretion of immunoglobulins; and enhances dendritic cell maturation. Strong and prolonged stimulation of T cells to proliferate causes high levels of IFN-{gamma}, which leads to chronic inflammation and is the reason for various autoimmune diseases. Therefore, blocking HVEM-LIGHT interaction may be a way to cure these diseases and prevent adverse reaction in organ and tissue transplantation. ResultsIn this work, we designed 62 peptides based on the CRDs of the HVEM structure, differentiating in the number and combination of disulfide bonds present. Based on extensive all-atom MD simulations in state-of-the-art force fields, followed by MM-GBSA binding energy estimation, we selected the most promising CRD2 variants interacting with LIGHT. Several point mutations of these variants provided us with the most strongly binding moiety: the CRD2 with a single disulfide bond (C58-C73) and K54E substitution. This result was supprased only by the truncated variants of CRD2(39-73) with the same disulfide bond present. The binding mechanism was investigated by the use of steered MD simulations, which showed the increased binding affinity of the abovementioned variants, while experimental circular dichroism was used to determine their structural properties. Availability and ImplementationThree PDB models of the LIGHT inhibitors: PM0084527, PM0084528, and PM0084592. Contactpkrupa@ifpan.edu.pl Supplementary informationOnline supplementary data is available at: .

bioinformatics↗

bioRxiv2020

A computational model to unravel the function of amyloid-β peptide in contact with a phospholipid membrane

The normal synapse activity involves the release of copper and other divalent cations in the synaptic region. These ions have a strong impact on the membrane properties, especially when the membrane has charged groups, like it is the case of synapse. In this work we use an atomistic computational model of dimyristoyl-phosphatidylcholine (DMPC) membrane bilayer. We perturb this model with a simple model of divalent cation (Mg2+), and with a single amyloid-{beta} (A{beta}) peptide of 42 residues, both with and without a single Cu2+ ion bound to the N-terminus. In agreement with experimental results reported in the literature, the model confirms that divalent cations locally destabilize the DMPC membrane bilayer, and, for the first time, that the monomeric form of A{beta} helps in avoiding the interactions between divalent cations and DMPC, preventing significant effects on the DMPC bilayer properties. These results are discussed in the frame of a protective role of diluted A{beta} peptide floating in the synaptic region. Author summaryWe modelled the behavior of a Mg2+ divalent cation, with the size of Zn2+ and Cu2+, in contact with a phosphatidyl lipid bilayer. We also modelled the monomeric amyloid-{beta} peptide 1-42, both free and Cu-loaded, the latter mimicking the final step of the binding between the peptide and the divalent cation. On the basis of the simulation results, we propose that the peptide hinders the strong interactions between the divalent cation and the membrane.

biophysics↗