Folding without glutamine: Proteome-scale removal of a canonical amino acid
- 1Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México
Abstract
All known life uses a canonical alphabet of 20 amino acids, yet which residues are structurally essential remains untested at proteome scale. Isoleucine was recently eliminated from ribosomal proteins in Escherichia coli, establishing that amino acid alphabet reduction is feasible. Here we ask whether an entire amino acid can be eliminated from a proteome, and develop a computational framework to identify the best target. Using two independent approaches — ProteinMPNN structure-based probabilities and multiple sequence alignment conservation — we define “keep-score” as the fraction of positions where the native residue is optimal. Both methods converge on an unexpected candidate: glutamine has the lowest keep-score, with proline and glycine the highest — counter to substitution-matrix intuition. Glutamine’s low keep-score is not explained by a single obvious substitute; glutamate is preferred in only 14% of replacements. Removing this target proteome-wide, we replaced every glutamine across 4,148 proteins with ProteinMPNN-optimal alternatives and modeled the resulting sequences with AlphaFold2. The recoded proteome maintained native-like structures: 85.5% of models exceeded a TMscore of 0.8, indistinguishable from the 85.9% baseline for rebuilt native sequences. Orthogonal validation — ESMFold predictions, Rosetta energy calculations, and conformational ensemble comparisons — revealed no evidence of systematic misfolding attributable to glutamine removal. As a consistency check, a glutamine-free TEM-1 β-lactamase designed with ProteinMPNN conferred ampicillin resistance in E. coli, indicating fold preservation can extend to biological function. Evolutionary evidence further supports these findings: 24% of E. coli proteins have glutamine-free orthologs in prokaryotic genomes, and glutamine ranks lowest in keep-score in Saccharomyces cerevisiae. These results show that an entire amino acid can be removed from a proteome with folds broadly preserved, establishing keep-score as a general framework and glutamine as the primary candidate for recoding. Crucially, this reflects a computational analysis of structural replaceability, not a demonstration of biological dispensability. Author summary: All life builds its proteins from the same 20 amino acids. But are all 20 truly necessary, or could some be removed without destroying protein structure? Recent work showed this is possible: a bacterium was engineered to make key proteins without one amino acid. But which one would be easiest to remove from an entire organism? We developed a computational framework to answer this for every protein in Escherichia coli. Using an AI model that predicts which amino acids fit a protein’s three-dimensional shape, together with evolutionary conservation data, we ranked all 20 by how often each is the best choice at its position. Both independently singled out glutamine as the most readily removable and the best single target — a surprise, because no single alternative dominates its replacement; instead, many amino acids can fill in depending on structural context. Rebuilding the proteome without glutamine yielded predicted structures nearly identical to the originals. A computationally designed glutamine-free enzyme, tested against its native counterpart, retained full activity, showing that fold preservation can translate to biological function. Our framework applies to any organism, offering a rational strategy for choosing which amino acid to target when building cells with simplified genetic codes.
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BibTeX
@article{garciarrubio2026folding,
title = {{Folding without glutamine: Proteome-scale removal of a canonical amino acid}},
author = {Alejandro Garciarrubio and Blanca Ramos-Cerrillo and Enrique Morett and Lorenzo Segovia},
journal = {PLOS Computational Biology},
year = {2026},
month = nov,
doi = {10.1371/journal.pcbi.1014931},
url = {https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1014931},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Folding without glutamine: Proteome-scale removal of a canonical amino acid
AU - Alejandro Garciarrubio
AU - Blanca Ramos-Cerrillo
AU - Enrique Morett
AU - Lorenzo Segovia
PY - 2026
DA - 2026/11/03
JO - PLOS Computational Biology
DO - 10.1371/journal.pcbi.1014931
UR - https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1014931
AB - All known life uses a canonical alphabet of 20 amino acids, yet which residues are structurally essential remains untested at proteome scale. Isoleucine was recently eliminated from ribosomal proteins in Escherichia coli, establishing that amino acid alphabet reduction is feasible. Here we ask whether an entire amino acid can be eliminated from a proteome, and develop a computational framework to identify the best target. Using two independent approaches — ProteinMPNN structure-based probabilities and multiple sequence alignment conservation — we define “keep-score” as the fraction of positions where the native residue is optimal. Both methods converge on an unexpected candidate: glutamine has the lowest keep-score, with proline and glycine the highest — counter to substitution-matrix intuition. Glutamine’s low keep-score is not explained by a single obvious substitute; glutamate is preferred in only 14% of replacements. Removing this target proteome-wide, we replaced every glutamine across 4,148 proteins with ProteinMPNN-optimal alternatives and modeled the resulting sequences with AlphaFold2. The recoded proteome maintained native-like structures: 85.5% of models exceeded a TMscore of 0.8, indistinguishable from the 85.9% baseline for rebuilt native sequences. Orthogonal validation — ESMFold predictions, Rosetta energy calculations, and conformational ensemble comparisons — revealed no evidence of systematic misfolding attributable to glutamine removal. As a consistency check, a glutamine-free TEM-1 β-lactamase designed with ProteinMPNN conferred ampicillin resistance in E. coli, indicating fold preservation can extend to biological function. Evolutionary evidence further supports these findings: 24% of E. coli proteins have glutamine-free orthologs in prokaryotic genomes, and glutamine ranks lowest in keep-score in Saccharomyces cerevisiae. These results show that an entire amino acid can be removed from a proteome with folds broadly preserved, establishing keep-score as a general framework and glutamine as the primary candidate for recoding. Crucially, this reflects a computational analysis of structural replaceability, not a demonstration of biological dispensability. Author summary: All life builds its proteins from the same 20 amino acids. But are all 20 truly necessary, or could some be removed without destroying protein structure? Recent work showed this is possible: a bacterium was engineered to make key proteins without one amino acid. But which one would be easiest to remove from an entire organism? We developed a computational framework to answer this for every protein in Escherichia coli. Using an AI model that predicts which amino acids fit a protein’s three-dimensional shape, together with evolutionary conservation data, we ranked all 20 by how often each is the best choice at its position. Both independently singled out glutamine as the most readily removable and the best single target — a surprise, because no single alternative dominates its replacement; instead, many amino acids can fill in depending on structural context. Rebuilding the proteome without glutamine yielded predicted structures nearly identical to the originals. A computationally designed glutamine-free enzyme, tested against its native counterpart, retained full activity, showing that fold preservation can translate to biological function. Our framework applies to any organism, offering a rational strategy for choosing which amino acid to target when building cells with simplified genetic codes.
SN - 1553-7358
ER -