Complementary Role of Co- and Post-Translational Events in <i>De Novo</i> Protein Biogenesis

Addabbo, Rayna M.; Dalphin, Matthew D.; Mecha, Miranda F.; Liu, Yue; Staikos, Alexios; Guzman-Luna, Valeria; Cavagnero, Silvia

doi:10.1021/acs.jpcb.0c03039

Cited by 14 publications

(79 citation statements)

References 117 publications

(304 reference statements)

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“…The analogous behavior observed for these mutants in E. coli and correlation of misfolding effects with phylogenetic conservation is consistent with a selective pressure to avoid misfolding in vivo and with growing evidence that kinetic factors affect stable protein expression in cells (Fig. 3K) ( 20, 26, 60, 61 ). The strong dependence of activity on temperature and Zn 2+ concentration present during expression suggests that mutations promoting formation of the inactive state may disrupt co-translational folding ( 20, 62 ).…”

Section: Discussionsupporting

confidence: 81%

“…These results strongly support the presence of persistent non-equilibrium folding effects ( Fig. 3D) (20,21,23). A second prediction of the misfolding model was also met by our data: PafA variants with WT activity but with different misfolded fractions (i.e., variants falling along the diagonal blue dashed line in Fig.…”

Section: Many Mutations Reduce Catalysis By Altering Foldingsupporting

confidence: 87%

“…3K) ( 20, 26, 60, 61 ). The strong dependence of activity on temperature and Zn 2+ concentration present during expression suggests that mutations promoting formation of the inactive state may disrupt co-translational folding ( 20, 62 ). We speculate that highly stable proteins like PafA and other secreted enzymes may be more prone to forming long-lived kinetically-trapped states.…”

Section: Discussionmentioning

confidence: 99%

“…We therefore considered and tested an alternate model in which inactive enzyme resulted from a non-equilibrium process-i.e., the formation of long-lived misfolded protein during expression ( Fig. 3D) (20). We varied the temperature and Zn 2+ concentration present during folding, as temperature is known to affect folding efficiency (21,22), and as PafA binds multiple Zn 2+ ions during the folding process.…”

Section: Many Mutations Reduce Catalysis By Altering Foldingmentioning

confidence: 99%

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Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Markin¹,

Mokhtari²,

Sunden³

et al. 2020

Preprint

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Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK, a microfluidic platform for high-throughput expression, purification, and characterization of >1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA, we performed >670,000 reactions to determine >5000 kinetic and physical constants for multiple substrates and inhibitors. These constants allowed us to uncover extensive kinetic partitioning to a misfolded state and isolate catalytic effects, revealing spatially contiguous “regions” of residues linked to particular aspects of function. These regions included active-site proximal residues but also extended to the enzyme surface, providing a map of underlying architecture that could not be derived from existing approaches. HT-MEK, using direct and coupled fluorescent assays, has future applications to a wide variety of problems ranging from understanding molecular mechanisms to medicine to engineering and design.One Sentence SummaryHT-MEK, a microfluidic platform for high-throughput, quantitative biochemistry, reveals enzyme architectures shaping function.

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Section: Discussionsupporting

confidence: 81%

Section: Many Mutations Reduce Catalysis By Altering Foldingsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Many Mutations Reduce Catalysis By Altering Foldingmentioning

confidence: 99%

See 2 more Smart Citations

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Markin¹,

Mokhtari²,

Sunden³

et al. 2020

Preprint

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show abstract

“…While it is possible that the absolute amount of soluble, misfolded protein may decrease in the cellular context they are unlikely to be entirely eliminated, and such a result would not change our molecular explanation for this phenomenon. It has been observed that even when a protein is synthesized by the ribosome in the presence of a chaperone it still populates states that remain soluble and non-functionalthus, vectorial synthesis does not eliminate these subpopulations 66 . Furthermore, the aforementioned studies of the influence of synonymous codons on protein function in cells are consistent with these subpopulations existing in vivo.…”

Section: Discussionmentioning

confidence: 99%

Subpopulations of soluble, misfolded proteins commonly bypass chaperones: How it happens at the molecular level

Halder

Nissley

Sitarik

et al. 2021

Preprint

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Subpopulations of soluble, misfolded proteins can bypass chaperones within cells. The scope of this phenomenon and the lifetimes of these states have not been experimentally quantified, and how such misfolding happens at the molecular level is poorly understood. We address the first issue through a meta-analysis of the experimental literature. We find that in all quantitative protein refolding-function studies, there is always a subpopulation of soluble but misfolded and less-functional protein that does not fold in the presence of one or more chaperones. This subpopulation ranges from 8% to 50% of the soluble protein molecules in solution. Fitting the experimental time traces to a kinetic model, we find these chaperone bypassing misfolded states take months or longer to fold and function in the presence of different chaperones. We next addressed how, at the molecular level, some misfolded proteins can evade chaperones by simulating six different proteins interacting with E. coli GroEL and HtpG chaperones when those proteins are in folded, unfolded, or long-lived, soluble, misfolded states. We observe that both chaperones strongly bind the unfolded state and weakly bind the folded and misfolded states to a similar degree. Thus, these chaperones cannot distinguish between the folded and long-lived misfolded states of these proteins. A structural analysis reveals the misfolded states are highly similar to the native state having a similar size, amount of exposed hydrophobic surface area, and level of tertiary structure formation. These results demonstrate that in vitro it is common for appreciable subpopulations of proteins to remain misfolded, soluble, and evade the refolding action of chaperones for very long times. Further, these results suggest that this happens because these misfolded subpopulations are near-native and therefore interact with chaperones to a similar extent as properly folded proteins. More broadly, these results indicate a mechanism in which long time scale changes in protein structure and function can persist in cells because some proteins non-native states can bypass components of the proteostasis machinery.

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Distribution and solvent exposure of Hsp70 chaperone binding sites across the Escherichia coli proteome

2023

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Many proteins must interact with molecular chaperones to achieve their native state in the cell. Yet, how chaperone binding‐site characteristics affect the folding process is poorly understood. The ubiquitous Hsp70 chaperone system prevents client‐protein aggregation by holding unfolded conformations and by unfolding misfolded states. Hsp70 binding sites of client proteins comprise a nonpolar core surrounded by positively charged residues. However, a detailed analysis of Hsp70 binding sites on a proteome‐wide scale is still lacking. Further, it is not known whether proteins undergo some degree of folding while chaperone bound. Here, we begin to address the above questions by identifying Hsp70 binding sites in 2258 Escherichia coli (E. coli) proteins. We find that most proteins bear at least one Hsp70 binding site and that the number of Hsp70 binding sites is directly proportional to protein size. Aggregation propensity upon release from the ribosome correlates with number of Hsp70 binding sites only in the case of large proteins. Interestingly, Hsp70 binding sites are more solvent‐exposed than other nonpolar sites, in protein native states. Our findings show that the majority of E. coli proteins are systematically enabled to interact with Hsp70 even if this interaction only takes place during a fraction of the protein lifetime. In addition, our data suggest that some conformational sampling may take place within Hsp70‐bound states, due to the solvent exposure of some chaperone binding sites in native proteins. In all, we propose that Hsp70‐chaperone‐binding traits have evolved to favor Hsp70‐assisted protein folding devoid of aggregation.

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Complementary Role of Co- and Post-Translational Events in De Novo Protein Biogenesis

Cited by 14 publications

References 117 publications

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Subpopulations of soluble, misfolded proteins commonly bypass chaperones: How it happens at the molecular level

Distribution and solvent exposure of Hsp70 chaperone binding sites across the Escherichia coli proteome

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