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Related Concept Videos

¹H NMR: Complex Splitting01:13

¹H NMR: Complex Splitting

A proton M that is coupled to a proton X results in doublet signals for M. However, NMR-active nuclei can be simultaneously coupled to more than one nonequivalent nucleus. When M is coupled to a second proton A, such as in styrene oxide, each peak in the doublet is split into another doublet.
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied first.
¹³C NMR: Distortionless Enhancement by Polarization Transfer (DEPT)01:20

¹³C NMR: Distortionless Enhancement by Polarization Transfer (DEPT)

When proton-coupled carbon-13 spectra are simplified by a broadband proton decoupling technique, structural information about the coupled protons is lost. Distortionless enhancement by polarization transfer (DEPT) is a technique that provides information on the number of hydrogens attached to each carbon in a molecule. While the DEPT experiment utilizes complex pulse sequences, the pulse delay and flip angle are specifically manipulated. The resulting signals have different phases depending on...
Interpreting ¹H NMR Signal Splitting: The (n + 1) Rule01:10

Interpreting ¹H NMR Signal Splitting: The (n + 1) Rule

In the AX proton spin system, proton A can sense the two spin states of a coupled proton X, resulting in a doublet NMR signal with two peaks of equal (1:1) intensity. When proton A is coupled to two equivalent protons (AX2 spin system), the spin states of each X can be aligned with or against the external field, creating three possible scenarios. This results in a 1:2:1  triplet signal, where the central peak corresponds to the chemical shift of A and is twice as large or intense as the others.
pH Regulation in Cells01:28

pH Regulation in Cells

pH plays a critical role in maintaining normal cellular activities. It helps maintain the structure and function of various proteins, dictates the charge on cellular membranes, and is crucial for metabolic reactions inside the cell. Moreover, cells use the energy from the proton motive force to generate ATP.
Cytosolic pH
Under physiological conditions, the cytosolic pH is slightly more acidic than the extracellular pH. However, cells must prevent further acidification of their cytosol to...
Extraction: Effects of pH00:53

Extraction: Effects of pH

Consider a neutral form of an amine, B, with a partition coefficient, K, in a liquid mixture containing organic and aqueous phases. The pH of the aqueous phase affects the charge on acidic and basic solutes, and the charged form is usually more soluble in the aqueous phase. Suppose the conjugate acid form of the amine is soluble only in the aqueous phase while the base form is soluble in both phases. Then the distribution coefficient, D, can be given as the ratio of amine concentration in the...
Phosphate Buffer01:22

Phosphate Buffer

The phosphate buffer system is a critical biological mechanism for maintaining pH stability in the body. This system operates primarily through two components: sodium dihydrogen phosphate (NaH2PO4), which acts as a weak acid, and sodium hydrogen phosphate (Na2HPO4), which serves as a weak base.
Sodium dihydrogen phosphate does not fully dissociate in neutral or acidic solutions. When a strong base, such as sodium hydroxide (NaOH), is introduced into the solution, sodium dihydrogen phosphate...

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Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes
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Functional reassembly of a split PH domain.

Kenji Sugimoto1, Yasuo Mori, Keisuke Makino

  • 1Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan.

Journal of the American Chemical Society
|April 24, 2003
PubMed
Summary

Researchers designed a split pleckstrin homology (PH) domain that functionally reassembles. This split PH domain successfully binds its target molecule, demonstrating functional recovery and potential for native split PH domain studies.

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Area of Science:

  • Molecular Biology
  • Protein Structure and Function

Background:

  • Pleckstrin homology (PH) domains are conserved protein modules crucial for cellular signaling and cytoskeletal organization.
  • The function and ligand-binding capabilities of split PH domains, found in various signaling proteins, require further clarification.

Purpose of the Study:

  • To investigate if a coiled-coil module can mediate the functional reassembly of a designed split pleckstrin homology (PH) domain.
  • To determine if the reassembled split PH domain retains ligand-binding specificity.

Main Methods:

  • Dissecting a well-characterized PH domain from phospholipase Cdelta(1) into N-terminal and C-terminal halves.
  • Tethering coiled-coil modules to each subunit to facilitate reassembly.
  • Utilizing isothermal titration microcalorimetry to assess complex formation and binding affinity.
  • Testing the binding of the reassembled split PH domain to inositol trisphosphate (IP(3)) and L-IP(3).

Main Results:

  • Isothermal titration microcalorimetry confirmed the formation of a thermodynamically stable 1:1 complex between the split PH domain halves via coiled-coil interaction.
  • The reassembled split PH domain exhibited specific binding to IP(3), mirroring the selectivity of the native PLCdelta(1) PH domain.
  • The split PH domain did not bind to L-IP(3), further confirming preserved binding specificity.

Conclusions:

  • A split PH domain can fold into a functional structure upon proximity-induced reassembly mediated by tethered coiled-coil modules.
  • This study provides a model for understanding the reassembly and function of native split PH domains, suggesting they may possess unique roles.
  • The findings imply that reassembly of split PH domains can restore biological activity and ligand-binding specificity.