Details

Autor(en) / Beteiligte

• Nikolaev, Andrey
• Kuzmin, Alexander
• Markeeva, Elena
• Kuznetsova, Elizaveta
• Ryzhykau, Yury L.
• Semenov, Oleg
• Anuchina, Arina
• Remeeva, Alina
• Gushchin, Ivan

Titel

Reengineering of a flavin‐binding fluorescent protein using ProteinMPNN

Ist Teil von

• Protein science, 2024-04, Vol.33 (4), p.e4958-n/a

Ort / Verlag

Hoboken, USA: John Wiley & Sons, Inc

Erscheinungsjahr

2024

Quelle

MEDLINE

Beschreibungen/Notizen

• Recent advances in machine learning techniques have led to development of a number of protein design and engineering approaches. One of them, ProteinMPNN, predicts an amino acid sequence that would fold and match user‐defined backbone structure. Its performance was previously tested for proteins composed of standard amino acids, as well as for peptide‐ and protein‐binding proteins. In this short report, we test whether ProteinMPNN can be used to reengineer a non‐proteinaceous ligand‐binding protein, flavin‐based fluorescent protein CagFbFP. We fixed the native backbone conformation and the identity of 20 amino acids interacting with the chromophore (flavin mononucleotide, FMN) while letting ProteinMPNN predict the rest of the sequence. The software package suggested replacing 36–48 out of the remaining 86 amino acids so that the resulting sequences are 55%–66% identical to the original one. The three designs that we tested experimentally displayed different expression levels, yet all were able to bind FMN and displayed fluorescence, thermal stability, and other properties similar to those of CagFbFP. Our results demonstrate that ProteinMPNN can be used to generate diverging unnatural variants of fluorescent proteins, and, more generally, to reengineer proteins without losing their ligand‐binding capabilities.