During reconstitution, membrane proteins are randomly inserted into liposomes according to Poisson distribution statistics. When the protein to lipid ratios in the reconstitution mixture are varied ...systematically, the characteristics of this statistical capture permit inferences about the proteins themselves, such as the number of subunits that assemble into a single functional unit. This chapter describes the Poisson distribution as applied to the reconstitution of membrane proteins into proteoliposomes and focuses on an application whereby this statistical behavior is used to determine the number of ion channel subunits that assemble into a functional pore. Practical considerations for performing these experiments are emphasized. Harnessing Poisson dilution statistics provides a function-based method to determine ion channel oligomerization, complementing other biophysical, biochemical, or structural approaches.
Fluorinated organic chemicals, such as per- and polyfluorinated alkyl substances (PFAS) and fluorinated pesticides, are both broadly useful and unusually long-lived. To combat problems related to the ...accumulation of these compounds, microbial PFAS and organofluorine degradation and biosynthesis of less-fluorinated replacement chemicals are under intense study. Both efforts are undermined by the substantial toxicity of fluoride, an anion that powerfully inhibits metabolism. Microorganisms have contended with environmental mineral fluoride over evolutionary time, evolving a suite of detoxification mechanisms. In this perspective, we synthesize emerging ideas on microbial defluorination/fluorination and fluoride resistance mechanisms and identify best approaches for bioengineering new approaches for degrading and making organofluorine compounds.
Most riboswitches are metabolite-binding RNA structures located in bacterial messenger RNAs where they control gene expression. We have discovered a riboswitch class in many bacterial and archaeal ...species whose members are selectively triggered by fluoride but reject other small anions, including chloride. These fluoride riboswitches activate expression of genes that encode putative fluoride transporters, enzymes that are known to be inhibited by fluoride, and additional proteins of unknown function. Our findings indicate that most organisms are naturally exposed to toxic levels of fluoride and that many species use fluoride-sensing RNAs to control the expression of proteins that alleviate the deleterious effects of this anion.
To contend with hazards posed by environmental fluoride, microorganisms export this anion through F(-)-specific ion channels of the Fluc family. Since the recent discovery of Fluc channels, numerous ...idiosyncratic features of these proteins have been unearthed, including strong selectivity for F(-) over Cl(-) and dual-topology dimeric assembly. To understand the chemical basis for F(-) permeation and how the antiparallel subunits convene to form a F(-)-selective pore, here we solve the crystal structures of two bacterial Fluc homologues in complex with three different monobody inhibitors, with and without F(-) present, to a maximum resolution of 2.1 Å. The structures reveal a surprising 'double-barrelled' channel architecture in which two F(-) ion pathways span the membrane, and the dual-topology arrangement includes a centrally coordinated cation, most likely Na(+). F(-) selectivity is proposed to arise from the very narrow pores and an unusual anion coordination that exploits the quadrupolar edges of conserved phenylalanine rings.
By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. ...A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported membrane electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter Gdx-Clo in complex with substrates to a maximum resolution of 2.3 Å. These structures confirm the family's extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the membrane for the hydrophobic substituents of transported drug-like cations.
Fluoride ion (F(-)) is a ubiquitous environmental threat to microorganisms, which have evolved a family of highly selective "Fluc" F(-) channels that export this inhibitory anion from their ...cytoplasm. It is unclear, however, how a thermodynamically passive mechanism like an ion channel can protect against high concentrations of external F(-). We monitored external F(-) concentrations in Escherichia coli suspensions and showed that, in bacteria lacking Fluc, F(-) accumulates when the external medium is acidified, as a predicted function of the transmembrane pH gradient. This weak acid accumulation effect, which results from the high pKa (3.4) and membrane permeability of HF, is abolished by Fluc channels. We also found that, although bacterial growth is inhibited by high concentrations of F(-), bacteria can withstand cytoplasmic F(-) at levels a hundred times higher than those that inhibit proliferation, resuming growth when the F(-) challenge is removed.
The small multidrug resistance (SMR) family of membrane proteins is prominent because of its rare dual topology architecture, simplicity, and small size. Its best studied member, EmrE, is an ...important model system in several fields related to membrane protein biology, from evolution to mechanism. But despite decades of work on these multidrug transporters, the native function of the SMR family has remained a mystery, and many highly similar SMR homologs do not transport drugs at all. Here we establish that representative SMR proteins, selected from each of the major clades in the phylogeny, function as guanidinium ion exporters. Drug-exporting SMRs are all clustered in a single minority clade. Using membrane transport experiments, we show that these guanidinium exporters, which we term Gdx, are very selective for guanidinium and strictly and stoichiometrically couple its export with the import of two protons. These findings draw important mechanistic distinctions with the notably promiscuous and weakly coupled drug exporters like EmrE.
Membrane Exporters of Fluoride Ion McIlwain, Benjamin C; Ruprecht, Michal T; Stockbridge, Randy B
Annual review of biochemistry,
06/2021, Letnik:
90, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Microorganisms contend with numerous and unusual chemical threats and have evolved a catalog of resistance mechanisms in response. One particularly ancient, pernicious threat is posed by fluoride ion ...(F
−
), a common xenobiotic in natural environments that causes broad-spectrum harm to metabolic pathways. This review focuses on advances in the last ten years toward understanding the microbial response to cytoplasmic accumulation of F
−
, with a special emphasis on the structure and mechanisms of the proteins that microbes use to export fluoride: the CLC
F
family of F
−
H
+
antiporters and the Fluc FEX family of F
−
channels.
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•Principles of bacterial membrane physiology.•Molecular determinants of bacterial K+ channels and transporters.•Molecular physiology of bacterial potassium homeostasis.
Potassium ion ...homeostasis is essential for bacterial survival, playing roles in osmoregulation, pH homeostasis, regulation of protein synthesis, enzyme activation, membrane potential adjustment and electrical signaling. To accomplish such diverse physiological tasks, it is not surprising that a single bacterium typically encodes several potassium uptake and release systems. To understand the role each individual protein fulfills and how these proteins work in concert, it is important to identify the molecular details of their function. One needs to understand whether the systems transport ions actively or passively, and what mechanisms or ligands lead to the activation or inactivation of individual systems. Combining mechanistic information with knowledge about the physiology under different stress situations, such as osmostress, pH stress or nutrient limitation, one can identify the task of each system and deduce how they are coordinated with each other. By reviewing the general principles of bacterial membrane physiology and describing the molecular architecture and function of several bacterial K+-transporting systems, we aim to provide a framework for microbiologists studying bacterial potassium homeostasis and the many K+-translocating systems that are still poorly understood.
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