How synonymous mutations alter enzyme structure and function over long timescales

Jiang, Yang; Neti, Syam Sundar; Sitarik, Ian; Pradhan, Priya; To, Philip; Xia, Yingzi; Fried, Stephen D.; Booker, Squire J.; O’Brien, Edward P.

doi:10.1038/s41557-022-01091-z

Cited by 46 publications

(94 citation statements)

References 46 publications

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“…Notably, our simulation results for the refolding kinetics of the RD1 protein (see Fig 4b ) show that the notion of a misfolded long-lived intermediate, resembling the native structure (high fraction of native contacts), but without the native entanglement, can be carried over from large proteins (hundreds of residues) in the cotranslational context [43,44] to the folding of an already fully synthesized small natively entangled protein. The long-lived intermediate IT acts indeed as a kinetic trap, slowing down the folding time by one order of magnitude (see Table 1).…”

Section: Discussionmentioning

confidence: 83%

“…Different entangled motifs could indeed originate already in the early stages of the folding process, with a low fraction of native contacts, to be then possibly "frozen" when the fraction of native contacts is increased. It will be interesting to study whether and how the kinetic partitioning between the many differently entangled misfolded subpopulations observed in cotranslational folding simulations [43,44] can arise through differently entangled unfolded configurations/subpopulations. The entanglement parameter ⟨G ′ ⟩ introduced here, should be an essential indicator for characterizing the partitioning of states.…”

Section: Discussionmentioning

confidence: 99%

“…Such misfolded species were characterized as long-lived kinetic traps, native-like in several respects, with their misfolding due to a change in the entanglement properties of the native structure. Moreover, the shift in the competition between differently entangled misfolded subpopulations, along cotranslational folding pathways, was shown to determine the functional impact of synonymous mutations [44].…”

Section: Introductionmentioning

confidence: 99%

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Folding kinetics of an entangled protein

Salicari

Baiesi

Orlandini

et al. 2023

Preprint

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The possibility of the protein backbone adopting lasso-like entangled motifs has attracted increasing attention. After discovering the surprising abundance of natively entangled single-domain proteins, it was shown that misfolded entangled subpopulations might become thermosensitive or escape the homeostasis network just after translation. To investigate the role of entanglement in shaping folding kinetics, we introduce a novel indicator and analyze simulations of a coarse-grained, structure-based model for two small single-domain proteins. The model recapitulates the well-known two-state folding mechanism of a non-entangled SH3 domain. However, despite its small size, a natively entangled antifreeze RD1 protein displays a rich refolding behavior, populating two distinct kinetic intermediates: a short-lived, entangled, near-unfolded state and a long-lived, non-entangled, near-native state. The former directs refolding along a fast pathway, whereas the latter is a kinetic trap, consistently with known experimental evidence of two different characteristic times. Upon trapping, the natively entangled loop forms without being threaded by the N-terminal residues. After trapping, the native entangled structure emerges by either backtracking to the unfolded state or threading through the already formed but not yet entangled loop. Along the fast pathway, the earlier the native contacts form, the more their formation time may fluctuate. Trapping does not occur because the native contacts at the closure of the lasso-like loop form after those involved in the N-terminal thread, confirming previous predictions. Despite this, entanglement may appear already in unfolded configurations. Remarkably, a long-lived, near-native intermediate, with non-native entanglement properties, recalls what was observed in cotranslational folding.

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Section: Discussionmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Folding kinetics of an entangled protein

Salicari

Baiesi

Orlandini

et al. 2023

Preprint

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“…The presence of silent mutations leading to codons with lower usages was previously reported to increase the ability of protein secretion tags to export proteins extracellularly by 1.6 folds [ 32 ]. It was recently reported to affect mutational fitness in eukaryotes [ 61 ] or in some studied cases, the folding of microbial proteins [ 62 ].…”

Section: Figurementioning

confidence: 99%

Engineering Ag43 Signal Peptides with Bacterial Display and Selection

Koh

Tay

Gan

2022

MPs

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Protein display, secretion, and export in prokaryotes are essential for utilizing microbial systems as engineered living materials, medicines, biocatalysts, and protein factories. To select for improved signal peptides for Escherichia coli protein display, we utilized error-prone polymerase chain reaction (epPCR) coupled with single-cell sorting and microplate titer to generate, select, and detect improved Ag43 signal peptides. Through just three rounds of mutagenesis and selection using green fluorescence from the 56 kDa sfGFP-beta-lactamase, we isolated clones that modestly increased surface display from 1.4- to 3-fold as detected by the microplate plate-reader and native SDS-PAGE assays. To establish that the functional protein was displayed extracellularly, we trypsinized the bacterial cells to release the surface displayed proteins for analysis. This workflow demonstrated a fast and high-throughput method leveraging epPCR and single-cell sorting to augment bacterial surface display rapidly that could be applied to other bacterial proteins.

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“…However, these tools fail to account for long-range dependencies and complex codon usage patterns that arise in natural DNA sequences and do not reliably produce high expression CDSs [26][27][28]. For example, existing codon optimization tools may yield suboptimal DNA sequences that transcribe well in the host, but impede proper folding of the recombinant protein during translation [29]. Alternatively, existing tools may yield sequences that enable proper folding but limit the stability and expression level of the transcribed mRNA [30][31][32][33][34], resulting in diminished yields of functional, soluble protein.…”

Section: Introductionmentioning

confidence: 99%

Deep learning-based codon optimization with large-scale synonymous variant datasets enables generalized tunable protein expression

Constant

Gutierrez

Sastry

et al. 2023

Preprint

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Increasing recombinant protein expression is of broad interest in industrial biotechnology, synthetic biology, and basic research. Codon optimization is an important step in heterologous gene expression that can have dramatic effects on protein expression level. Several codon optimization strategies have been developed to enhance expression, but these are largely based on bulk usage of highly frequent codons in the host genome, and can produce unreliable results. Here, we develop deep contextual language models that learn the codon usage rules from natural protein coding sequences across members of theEnterobacteralesorder. We then fine-tune these models with over 150,000 functional expression measurements of synonymous coding sequences from three proteins to predict expression inE. coli. We find that our models recapitulate natural context- specific patterns of codon usage and can accurately predict expression levels across synonymous sequences. Finally, we show that expression predictions can generalize across proteins unseen during training, allowing forin silicodesign of gene sequences for optimal expression. Our approach provides a novel and reliable method for tuning gene expression with many potential applications in biotechnology and biomanufacturing.

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How synonymous mutations alter enzyme structure and function over long timescales

Cited by 46 publications

References 46 publications

Folding kinetics of an entangled protein

Folding kinetics of an entangled protein

Engineering Ag43 Signal Peptides with Bacterial Display and Selection

Deep learning-based codon optimization with large-scale synonymous variant datasets enables generalized tunable protein expression

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