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ATP Driven Pumps I: An Overview01:27

ATP Driven Pumps I: An Overview

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ATP-driven pumps, also known as transport ATPases, are integral membrane proteins. They have binding sites for ATP located on the membrane's cytosolic side and the ion-conducting domain in the transmembrane region. These pumps use the free energy released from ATP hydrolysis to move the solutes across cell membranes against an electrochemical gradient.
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Primary Active Transport01:47

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In contrast to passive transport, active transport involves a substance being moved through membranes in a direction against its concentration or electrochemical gradient. There are two types of active transport: primary active transport and secondary active transport. Primary active transport utilizes chemical energy from ATP to drive protein pumps that are embedded in the cell membrane. With energy from ATP, the pumps transport ions against their electrochemical gradients—a direction...
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Primary Active Transport01:29

Primary Active Transport

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In contrast to passive transport, active transport involves a substance being moved through membranes in a direction against its concentration or electrochemical gradient. There are two types of active transport: primary active transport and secondary active transport. Primary active transport utilizes chemical energy from ATP to drive protein pumps embedded in the cell membrane. With energy from ATP, the pumps transport ions against their electrochemical gradients—a direction they would...
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Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) is an advanced Nuclear Magnetic Resonance (NMR) technique specifically designed to detect and enhance the signals of low-abundance nuclei, such as carbon-13 and nitrogen-15, in small molecules. The fundamental principle behind INEPT is the transfer of polarization from a more abundant and highly polarizable nucleus, typically hydrogen-1, to the low-abundance nucleus of interest. This process effectively boosts the NMR signal of the...
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ATP Synthase: Mechanism01:48

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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 Driven Pumps II: P-type Pumps01:34

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The P-type pumps are a large family of integral membrane transporter ATPases. They are divided into five major types based on substrate specificity, from I to V.
A typical P-type pump has three cytosolic domains: nucleotide-binding (N), phosphorylation (P), and activator (A) domains. These domains are connected to the membrane-spanning helices by short amino acid segments. ATP hydrolysis and covalent phosphoenzyme intermediate formation are crucial parts of the catalytic cycle. At the highly...
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Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time
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Illuminating High-Affinity ATP Binding to the Sodium-Potassium Pump Using Solid-State NMR Spectroscopy.

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  • 1Department of Chemistry, Lancaster University, Bailrigg, Lancaster LA1 4YB, UK.

Molecules (Basel, Switzerland)
|September 13, 2025
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Summary

Solid-state nuclear magnetic resonance (SSNMR) reveals atomic details of ATP binding to the Na,K-ATPase (NKA) membrane protein. This technique provides unique insights into ATP conformation and its environment within the NKA

Keywords:
Na,K-ATPaseREDORdensity functional theorymagic-angle spinningribosesolid-state NMRtorsional angle

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Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays
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Area of Science:

  • Biophysics and Structural Biology
  • Membrane Protein Structure and Dynamics
  • Biomolecular NMR Spectroscopy

Background:

  • Membrane proteins constitute a significant portion of drug targets, making their structural and functional studies crucial.
  • Small molecules regulate membrane protein function, but their atomic details are often challenging to resolve using traditional methods like X-ray crystallography and cryo-electron microscopy (cryo-EM).
  • Unstable or reactive small molecules, such as enzyme substrates, pose particular difficulties for structural determination.

Purpose of the Study:

  • To develop and apply Solid-state nuclear magnetic resonance (SSNMR) methods for detailed structural and dynamic analysis of small molecule interactions with membrane proteins.
  • To investigate the binding conformation and environment of the ATP substrate within the high-affinity site of the Na,K-ATPase (NKA).
  • To showcase the utility of SSNMR in overcoming limitations of other structural techniques for membrane protein-ligand complexes.

Main Methods:

  • Development and application of advanced SSNMR techniques over two decades.
  • Utilized freeze-trapping to stabilize membrane protein samples in native-like environments.
  • Combined SSNMR measurements in frozen and fluid states with chemical shift analysis (DFT calculations), REDOR dipolar coupling measurements, and proton spin diffusion rate measurements.

Main Results:

  • Provided unprecedented atomic-level details on the molecular conformation of ATP bound to the Na,K-ATPase (NKA).
  • Characterized the local binding environment of ATP within the high-affinity nucleotide site of NKA.
  • Demonstrated the capability of SSNMR to resolve the structure and dynamics of unstable or reactive molecules in their physiological context.

Conclusions:

  • SSNMR is a powerful technique for elucidating the structure, dynamics, and binding interactions of small molecules with membrane proteins, especially when other methods fall short.
  • The study presents a detailed and unique picture of ATP in its high-affinity binding site on the Na,K-ATPase, enabled by innovative SSNMR approaches.
  • The developed SSNMR methods offer a valuable tool for drug discovery and optimization targeting membrane proteins.