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1.
J Am Chem Soc ; 146(32): 22622-22628, 2024 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-39083370

RESUMEN

Chemical mutagenesis via dehydroalanine (Dha) is a powerful method to tailor protein structure and function, allowing the site-specific installation of post-translational modifications and non-natural functional groups. Despite the impressive versatility of this method, applications have been limited, as products are formed as epimeric mixtures, whereby the modified amino acid is present as both the desired l-configuration and a roughly equal amount of the undesired d-isomer. Here, we describe a simple remedy for this issue: removal of the d-isomer via proteolysis using a d-stereoselective peptidase, alkaline d-peptidase (AD-P). We demonstrate that AD-P can selectively cleave the d-isomer of epimeric residues within histone H3, GFP, Ddx4, and SGTA, allowing the installation of non-natural amino acids with stereochemical control. Given the breadth of modifications that can be introduced via Dha and the simplicity of our method, we believe that stereoselective chemoenzymatic mutagenesis will find broad utility in protein engineering and chemical biology applications.


Asunto(s)
Mutagénesis , Estereoisomerismo , Cinética , Alanina/química , Alanina/análogos & derivados , Ingeniería de Proteínas , Histonas/química , Histonas/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteínas/genética
2.
Microorganisms ; 11(4)2023 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-37110501

RESUMEN

Bacteria use an array of sigma factors to regulate gene expression during different stages of their life cycles. Full-length, atomic-level structures of sigma factors have been challenging to obtain experimentally as a result of their many regions of intrinsic disorder. AlphaFold has now supplied plausible full-length models for most sigma factors. Here we discuss the current understanding of the structures and functions of sigma factors in the model organism, Bacillus subtilis, and present an X-ray crystal structure of a region of B. subtilis SigE, a sigma factor that plays a critical role in the developmental process of spore formation.

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