Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP

Swain, Brendan M; Guo, Dawei; Singh, Himansha; Rawlins, Philip; McAlister, M.; Veen, Hendrik W. van

doi:10.1038/s41598-020-76943-0

Cited by 8 publications

(6 citation statements)

References 37 publications

(50 reference statements)

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“…The partition coefficient obtained at neutral pH is in excellent agreement with the values reported in the literature for partition to DMPC membranes in the liquid disordered phase [10], as well as to the lipid bilayer of inside-out vesicles of Lactococcus lactis [44]. A maximal value of K P is observed at pH = 10, in agreement with the higher abundance of the neutral form, predicted in literature studies [12,45,46] and also by the MarvinSketch software [24]. The affinity for the POPC bilayers is essentially unaffected by the pH when the latter varies between 7 and 11, although it is predicted that at pH = 7, the most abundant species is the monocation.…”

Section: Partition To Popc Luvssupporting

confidence: 89%

Interaction of Hoechst 33342 with POPC Membranes at Different pH Values

et al. 2023

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Hoechst 33342 (H33342) is a fluorescent probe that is commonly used to stain the DNA of living cells. To do so, it needs to interact with and permeate through cell membranes, despite its high overall charge at physiological pH values. In this work, we address the effect of pH in the association of H33342 with lipid bilayers using a combined experimental and computational approach. The partition of H33342 to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid membranes was experimentally quantified using fluorescence spectroscopy and isothermal titration calorimetry (ITC) measurements. Quantum chemical calculations were performed to select the most stable isomer of H33342 for the overall charges 0, +1, and +2, expected to predominate across the 5 < pH < 10 range. The interaction of these isomers with POPC bilayers was then studied by both unrestrained and umbrella sampling molecular dynamics (MD) simulations. Both experimental results and computational free energy profiles indicate that the partition coefficient of H33342 displays a small variation over a wide pH range, not exceeding one order of magnitude. The enthalpy variation upon partition to the membrane suggests efficient hydrogen bonding between the probe and the lipid, namely, for the protonated +2 form, which was confirmed in the MD simulation studies. The relatively high lipophilicity obtained for the charged species contrasts with the decrease in their general hydrophobicity as estimated from octanol/water partition. This highlights the distinction between lipophilicity and hydrophobicity, as well as the importance of considering the association with lipid bilayers when predicting the affinity for biomembranes.

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Section: Partition To Popc Luvssupporting

confidence: 89%

Interaction of Hoechst 33342 with POPC Membranes at Different pH Values

et al. 2023

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“…Hoescht 33342 has a logP of 4.5, consistent with a mildly hydrophobic substrate, yet contains three ionizable groups: the tetrahydropyrazine amine (pKa 7.87), and two benzoimidazole groups with pKa’s of 5.02 and 5.81 22 . At pH 7, the tetrahydropyrazine amine is likely to be protonated, resulting in an overall charge of +1 for Hoechst 33342 23 .…”

Section: Resultsmentioning

confidence: 99%

The net electrostatic potential and hydration of ABCG2 affect substrate transport

Gose,

Aitken,

Wang

et al. 2023

Nat Commun

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ABCG2 is a medically important ATP-binding cassette transporter with crucial roles in the absorption and distribution of chemically-diverse toxins and drugs, reducing the cellular accumulation of chemotherapeutic drugs to facilitate multidrug resistance in cancer. ABCG2’s capacity to transport both hydrophilic and hydrophobic compounds is not well understood. Here we assess the molecular basis for substrate discrimination by the binding pocket. Substitution of a phylogenetically-conserved polar residue, N436, to alanine in the binding pocket of human ABCG2 permits only hydrophobic substrate transport, revealing the unique role of N436 as a discriminator. Molecular dynamics simulations show that this alanine substitution alters the electrostatic potential of the binding pocket favoring hydration of the transport pore. This change affects the contact with substrates and inhibitors, abrogating hydrophilic compound transport while retaining the transport of hydrophobic compounds. The N436 residue is also required for optimal transport inhibition of ABCG2, as many inhibitors are functionally impaired by this ABCG2 mutation. Overall, these findings have biomedical implications, broadly extending our understanding of substrate and inhibitor interactions.

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“…First, two-photon absorption (TPA) of Hoechst would add non-specific background to the SRS signals, which, after spectral decomposition and normalization, could artificially increase the lipid density and decrease the protein density. Second, most Hoechst molecules carry a positive charge at the intracellular pH ( Swain et al, 2020 ). When Hoechst localizes to the nucleoplasm, their counter ions can increase the osmotic pressure inside the nuclear envelope and thus decrease nuclear mass density.…”

Section: Resultsmentioning

confidence: 99%

The uniformity and stability of cellular mass density in mammalian cell culture

Liu

Kirschner

2022

Front. Cell Dev. Biol.

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Cell dry mass is principally determined by the sum of biosynthesis and degradation. Measurable change in dry mass occurs on a time scale of hours. By contrast, cell volume can change in minutes by altering the osmotic conditions. How changes in dry mass and volume are coupled is a fundamental question in cell size control. If cell volume were proportional to cell dry mass during growth, the cell would always maintain the same cellular mass density, defined as cell dry mass dividing by cell volume. The accuracy and stability against perturbation of this proportionality has never been stringently tested. Normalized Raman Imaging (NoRI), can measure both protein and lipid dry mass density directly. Using this new technique, we have been able to investigate the stability of mass density in response to pharmaceutical and physiological perturbations in three cultured mammalian cell lines. We find a remarkably narrow mass density distribution within cells, that is, significantly tighter than the variability of mass or volume distribution. The measured mass density is independent of the cell cycle. We find that mass density can be modulated directly by extracellular osmolytes or by disruptions of the cytoskeleton. Yet, mass density is surprisingly resistant to pharmacological perturbations of protein synthesis or protein degradation, suggesting there must be some form of feedback control to maintain the homeostasis of mass density when mass is altered. By contrast, physiological perturbations such as starvation or senescence induce significant shifts in mass density. We have begun to shed light on how and why cell mass density remains fixed against some perturbations and yet is sensitive during transitions in physiological state.

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Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP

Cited by 8 publications

References 37 publications

Interaction of Hoechst 33342 with POPC Membranes at Different pH Values

Interaction of Hoechst 33342 with POPC Membranes at Different pH Values

The net electrostatic potential and hydration of ABCG2 affect substrate transport

The uniformity and stability of cellular mass density in mammalian cell culture

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