The Citrate Carrier CitS Probed by Single-Molecule Fluorescence Spectroscopy

Kästner, Christopher N.; Prummer, Michael; Sick, Beate; Renn, Alois; Wild, Urs P.; Dimroth, Peter

doi:10.1016/s0006-3495(03)74974-1

Cited by 24 publications

(21 citation statements)

References 47 publications

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“…We will review here several cases where this approach has been used very successfully, putting a special emphasis on ET. 192 The proteins were immobilized on Ni-NTA-treated surfaces via an Nterminal His-tag and visualized using a stage-scanning confocal setup. Only one mutant kept its activity after labeling, corresponding to an intracytoplasmic site of attachment for the fluorophore.…”

Section: 94mentioning

confidence: 99%

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

Michalet¹,

Weiss²,

Jaeger³

2006

ChemInform

125

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Section: 94mentioning

confidence: 99%

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

Michalet¹,

Weiss²,

Jaeger³

2006

ChemInform

125

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“…The copurification of tagged and untagged CitS was taken as evidence for an oligomeric structure of the protein (see "Quartenary Structure" in "STRUCTURE" below) (108). Purified CitS was used for mechanistic studies following reconstitution in proteoliposomes (56,109) and for singlemolecule fluorescence spectroscopy studies (57).…”

Section: Purification and Reconstitutionmentioning

confidence: 99%

“…The enzyme solubilized in Triton X-100 was shown to run as a dimer on blue native PAGE (57,150). Purification of BAD-tagged CitS produced from a recombinant plasmid strongly suggested the dimeric structure of the protein (see "Purification and Reconstitution" in "PROTEINS" above) (108).…”

Section: Quaternary Structurementioning

confidence: 99%

“…Finally, strong evidence for the existence of the homodimeric association state of CitS was provided by singlemolecule fluorescence spectroscopy of purified CitS (57). Labeling of purified CitS at the position of cysteine residue Cys394 in region Xa with two different fluorophores (Alexa Fluor 546 and 568) produced a population of CitS dimers with both fluorophores attached.…”

Section: Quaternary Structurementioning

confidence: 99%

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The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism

Sobczak

Lolkema

2005

Microbiol Mol Biol Rev

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SUMMARY The 2-hydroxycarboxylate transporter family is a family of secondary transporters found exclusively in the bacterial kingdom. They function in the metabolism of the di- and tricarboxylates malate and citrate, mostly in fermentative pathways involving decarboxylation of malate or oxaloacetate. These pathways are found in the class Bacillales of the low-CG gram-positive bacteria and in the gamma subdivision of the Proteobacteria. The pathways have evolved into a remarkable diversity in terms of the combinations of enzymes and transporters that built the pathways and of energy conservation mechanisms. The transporter family includes H+ and Na+ symporters and precursor/product exchangers. The proteins consist of a bundle of 11 transmembrane helices formed from two homologous domains containing five transmembrane segments each, plus one additional segment at the N terminus. The two domains have opposite orientations in the membrane and contain a pore-loop or reentrant loop structure between the fourth and fifth transmembrane segments. The two pore-loops enter the membrane from opposite sides and are believed to be part of the translocation site. The binding site is located asymmetrically in the membrane, close to the interface of membrane and cytoplasm. The binding site in the translocation pore is believed to be alternatively exposed to the internal and external media. The proposed structure of the 2HCT transporters is different from any known structure of a membrane protein and represents a new structural class of secondary transporters.

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“…There are, for example, transporters which transport exclusively uncomplexed citrate of which the CitS transporter of Klebsiella pneumoniae is an example. This protein was shown to exclusively recognize Hcitrate 2À (Kästner et al, 2003). Other transporters recognize citrate metal complexes with a defined metal specificity.…”

Section: Discussionmentioning

confidence: 99%

Physiologically relevant divalent cations modulate citrate recognition by the McpS chemoreceptor

Lacal

García-Fontana

Callejo-García

et al. 2011

J of Molecular Recognition

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The McpS chemoreceptor of Pseudomonas putida KT2440 recognizes six different tricarboxylic acid (TCA) cycle intermediates. However, the magnitude of the chemotactic response towards these compounds differs largely, which has led to distinguish between strong attractants (malate, succinate, fumarate, oxaloacetate) and weak attractants (citrate, isocitrate). Citrate is abundantly present in plant tissues and root exudates and can serve as the only carbon source for growth. Citrate is known to form complexes with divalent cations which are also abundantly present in natural habitats of this bacterium. We have used isothermal titration calorimetry to study the formation of citrate-metal ion complexes. In all cases binding was entropy driven but significant differences in affinity were observed ranging from K(D)=157 µM (for Mg(2+)) to 3 µM (for Ni(2+)). Complex formation occurred over a range of pH and ionic strength. The ligand binding domain of McpS (McpS-LBD) was found to bind free citrate, but not complexes with physiologically relevant Mg(2+) and Ca(2+). In contrast, complexes with divalent cations which are present as trace elements (Co(2+), Cd(2+) and Ni(2+)) were recognized by McpS-LBD. This discrimination differs from other citrate sensing proteins. These results are discussed in the context of the three dimensional structure of free citrate and its complex with Mg(2+). Chemotaxis assays using P. putida revealed that taxis towards the strong attractant malate is strongly reduced in the presence of free citrate. However, this reduction is much less important in the presence of citrate-Mg(2+) complexes. The physiological relevance of these findings is discussed.

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The Citrate Carrier CitS Probed by Single-Molecule Fluorescence Spectroscopy

Cited by 24 publications

References 47 publications

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism

Physiologically relevant divalent cations modulate citrate recognition by the McpS chemoreceptor

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