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Related Concept Videos

ATP Synthase: Mechanism01:48

ATP Synthase: Mechanism

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In animals, the mitochondrial F1F0 ATP synthase is the key protein that synthesizes ATP molecules through a complex catalytic mechanism. While the nuclear genome encodes the majority of ATP synthase subunits, the mitochondrial genome encodes some of the enzyme's most critical components. The formation of this multi-subunit enzyme is a complex multi-step process regulated at the level of transcription, translation, and assembly. Defects in one or more of these steps can result in decreased...
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ATP Synthase: Structure01:18

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ATP synthase or ATPase is among the most conserved proteins found in bacteria, mammals, and plants. This enzyme can catalyze a forward reaction in response to the electrochemical gradient, producing ATP from ADP and inorganic phosphate. ATP synthase can also work in a reverse direction by hydrolyzing ATP and generating an electrochemical gradient. Different forms of ATP synthases have evolved special features to meet the specific demands of the cell. Based on their specific feature, ATP...
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Post-translational Translocation of Proteins to the RER01:27

Post-translational Translocation of Proteins to the RER

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A sizable fraction of proteins destined for ER are first synthesized in the cell cytosol and then transported across the ER membrane–a process called post-translational translocation. Similar to cotranslationally translocated proteins, these proteins also use the Sec translocon complex to enter the ER lumen.
Targeting proteins to the ER
Hsp40 and Hsp70 chaperone molecules bind the translated proteins in the cytosol to prevent their folding. The chaperone binding helps to keep the signal...
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ABC Transporters: Exporter01:31

ABC Transporters: Exporter

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ATP-binding cassette or ABC transporter is the largest superfamily of integral membrane proteins. The transporters have transmembrane-binding domains (TMDs) and nucleotide-binding domains (NBDs). The TMDs are specific to their substrates, whereas the NBDs are similar to engines that complete ATP hydrolysis to complete the substrate transport. They can be full transporters consisting of two TMDs and NBDs, half transporters with one TMD and NBD, while some encoded with a single TMD or NBD are...
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The ADP/ATP Carrier Protein01:42

The ADP/ATP Carrier Protein

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ADP/ATP carrier or AAC protein is the most abundant carrier protein in the inner mitochondrial membrane. It transports large quantities of ADP and ATP, equivalent to the average human body weight, every day. Among other transporters, ACC protein is one of the best-studied members of the mitochondrial carrier protein family. The ADP/ATP carrier protein comprises two transmembrane helices connected to a loop and a single alpha-helix on the matrix side. It switches between two conformational...
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Energy to Drive Translocation01:37

Energy to Drive Translocation

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Mitochondrial protein import is powered by two distinct energy sources: ATP hydrolysis and electrochemical potential across the inner membrane. Newly synthesized precursors are bound by cytosolic chaperones of the Hsp70 family, which guide them to the import receptors on the mitochondrial surface. Utilizing the energy of ATP hydrolysis, Hsp70 chaperones transfer these precursors to the TOM receptors on the mitochondrial outer membrane.
Generally, polypeptides are unfolded by two distinct...
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Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
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ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter.

Friedrich Finkenwirth1, Michael Sippach2, Heidi Landmesser3

  • 1From the Institut für Biologie/Mikrobiologie and.

The Journal of Biological Chemistry
|May 21, 2015
PubMed
Summary

Energy-coupling factor (ECF) transporters utilize ATP binding and hydrolysis to facilitate vitamin and ion transport. This study reveals the catalytic cycle of a biotin transporter, showing ATP binding drives substrate capture and hydrolysis releases it.

Keywords:
ABC transporterATPECF transporterbiotincross-linkingelectron paramagnetic resonance (EPR)fluorescencelipid bilayer nanodiscsvitamin uptake

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Energy-coupling factor (ECF) transporters are crucial for importing vitamins and metal ions in prokaryotes.
  • These transporters comprise ATP-binding cassette (ABC) ATPases, a substrate-specific S component, and a T component.

Purpose of the Study:

  • To elucidate the catalytic mechanism of ECF transporters by studying a reconstituted bacterial biotin transporter.
  • To investigate the roles of ATP binding/hydrolysis and protein-protein interactions in transporter function.

Main Methods:

  • Reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs.
  • Biochemical assays including ATPase activity measurements, cross-linking of variants, and substrate capture assays.
  • Biophysical techniques such as site-specific spin labeling and EPR spectroscopy, and fluorescence spectroscopy.

Main Results:

  • ATP binding, not biotin, modulates ATPase activity, with hydrolysis triggering substrate release.
  • Closure and reopening of the ATPase dimer (BioM2) correlate with ATP binding and hydrolysis.
  • Structural and functional evidence confirmed the role of hydrophobic residues in S/T unit interactions, suggesting reorientation during catalysis.
  • A toppling mechanism for the transporter was proposed based on fluorescence data.

Conclusions:

  • This study provides the first experimental insights into the catalytic cycle of an ECF transporter within a lipid environment.
  • The findings clarify the dynamic conformational changes and molecular interactions governing ECF transporter function.