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Carbocations are one of the reaction intermediates formed during several nucleophilic substitutions or elimination reactions. A carbocation is an electron-deficient species with the central carbon atom having six electrons and three bonded atoms. The central carbon in a carbocation is sp2 hybridized with trigonal planar geometry. It has an empty p orbital perpendicular to the plane of the structure that can accept electrons. Thus, carbocations act as strong electrophiles and may react with any...
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When a carbonyl compound is treated with a strong base, the α position gets deprotonated to give a resonance-stabilized intermediate called an enolate. Enolates are ambident nucleophiles because they possess two nucleophilic sites that can attack an electrophile owing to the delocalization of the negative charge between the α carbon and oxygen atoms. When the oxygen atom attacks an electrophile, it is called O-attack, whereas electrophilic attack via the α carbon is known as...
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The carbonyl carbon in an aldehyde or ketone is the site of a nucleophilic attack due to its electron-deficient nature. Depending on the strength of the incoming nucleophile, the reaction occurs via different mechanistic pathways.
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Isolating Free Carbenes, their Mixed Dimers and Organic Radicals
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MicroED Structure of a Protoglobin Reactive Carbene Intermediate.

Emma Danelius1,2, Nicholas J Porter3, Johan Unge1

  • 1Department of Biological Chemistry, University of California, Los Angeles, 615 Charles E. Young Drive South, Los Angeles, California 90095, United States.

Journal of the American Chemical Society
|March 22, 2023
PubMed
Summary
This summary is machine-generated.

Microcrystal electron diffraction (MicroED) determined the structure of an Aeropyrum pernix protoglobin variant. This breakthrough enables detailed studies of protein structures previously beyond reach.

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Area of Science:

  • Structural Biology
  • Biochemistry
  • Crystallography

Background:

  • Microcrystal electron diffraction (MicroED) is a powerful technique for determining molecular structures.
  • While successful for small molecules and peptides, MicroED has had limited application for novel protein structures.
  • Advancements in MicroED technology and protein structure prediction are expanding its capabilities.

Purpose of the Study:

  • To determine the structure of an engineered Aeropyrum pernix protoglobin (ApePgb) variant using MicroED.
  • To utilize an AlphaFold2 model for phasing in the MicroED structure determination.
  • To investigate the structural basis for enhanced carbene transfer activity in the ApePgb variant.

Main Methods:

  • Microcrystal electron diffraction (MicroED) with advanced technology (higher voltage, low-noise detector).
  • Utilized an AlphaFold2 model for phasing.
  • Crystallography and structural analysis of the ApePgb variant and its intermediate.

Main Results:

  • Determined the first MicroED structure of an Aeropyrum pernix protoglobin (ApePgb) variant.
  • Revealed that mutations reorient an alpha helix into a dynamic loop, improving catalytic active site accessibility.
  • Trapped and characterized the reactive iron-carbenoid intermediate in the engineered carbene transfer activity.

Conclusions:

  • Improved MicroED technology and protein structure prediction models enable the study of previously inaccessible protein structures.
  • The determined structure provides insights into how mutations enhance carbene transfer activity.
  • The findings highlight the potential of MicroED for investigating complex biological molecules and their mechanisms.