Reengineering of a flavin-binding fluorescent protein using ProteinMPNN.

Publisher: Cold Spring Harbor Laboratory Press Country of Publication: United States NLM ID: 9211750 Publication Model: Print Cited Medium: Internet ISSN: 1469-896X (Electronic) Linking ISSN: 09618368 NLM ISO Abbreviation: Protein Sci Subsets: MEDLINE

Publication: 2001- : Woodbury, NY : Cold Spring Harbor Laboratory Press
Original Publication: New York, N.Y. : Cambridge University Press, c1992-

Flavin Mononucleotide*/chemistry ; Proteins*; Ligands ; Flavins/chemistry ; Amino Acids

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.
(© 2024 The Protein Society.)

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Russian Science Foundation; Ministry of Science and Higher Education of the Russian Federation

Keywords: flavin; fluorescent protein; ligand; machine learning; protein engineering

0 (Ligands)
7N464URE7E (Flavin Mononucleotide)
0 (Proteins)
0 (Flavins)
0 (Amino Acids)

Date Created: 20240319 Date Completed: 20240320 Latest Revision: 20250320

20250320

PMC10949330

10.1002/pro.4958

38501498