Addition of negatively charged residues can reverse the decrease in the solubility of an acidic protein caused by an artificially introduced non-polar surface patch

Yagi, Sota; Akanuma, Satoshi; Yamagishi, Akihiko

doi:10.1016/j.bbapap.2013.12.011

Cited by 10 publications

(5 citation statements)

References 39 publications

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“…The solubility of EXG:CBM MQMW was significantly affected by removing the positive N-terminus (Figure ). This appears to be in conflict with both the observation of the negligible effect of positive surface area on solubility and other studies showing that the positively charged amino and guanidino groups of lysine and arginine residues, respectively, are not able to increase the solubility of hydrophobic patches in proteins. , On the other hand, acEXG:CBM MQMW has a very low net charge (−1 at pH 7), and lysine residues have been shown to increase the solubility of proteins with a low or positive net charge. , Further, it is possible that the small number of charges unmasks effects that are difficult to study in proteins with a greater number of charged residues. The observation that the solubility of all EXG:CBM variants was affected by the presence of ammonium sulfate (Figure ) supports the current view that ammonium sulfate precipitation does not work by screening electrostatic protein interactions but rather by excluding water from the protein surface .…”

Section: Resultsmentioning

confidence: 78%

“…This appears to be in conflict with both the observation of the negligible effect of positive surface area on solubility 16 and other studies showing that the positively charged amino and guanidino groups of lysine and arginine residues, respectively, are not able to increase the solubility of hydrophobic patches in proteins. 47,48 On the other hand, acEXG:CBM MQMW has a very low net charge (−1 at pH 7), and lysine residues have been shown to increase the solubility of proteins with a low or positive net charge. 14,15 Further, it is possible that the small number of charges unmasks effects that are difficult to study in proteins with a greater number of charged residues.…”

Section: Biochemistrymentioning

confidence: 99%

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A Soluble, Folded Protein without Charged Amino Acid Residues

et al. 2016

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Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find that the protein shows a surprising resilience toward extremes of pH, demonstrating stability and function (cellulose binding) in the pH range from 2 to 11. To ask whether the four charged residues present were required for these properties of this protein, we altered them to nontitratable ones. Remarkably, this chargeless protein is produced reasonably well in Escherichia coli, retains its stable three-dimensional structure, and is still capable of strong cellulose binding. To further deprive this protein of charges, we removed the N-terminal charge by acetylation and studied the protein at pH 2, where the C-terminus is effectively protonated. Under these conditions, the protein retains its function and proved to be both soluble and have a reversible folding-unfolding transition. To the best of our knowledge, this is the first time a soluble, functional protein with no titratable side chains has been produced.

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Section: Resultsmentioning

confidence: 78%

Section: Biochemistrymentioning

confidence: 99%

A Soluble, Folded Protein without Charged Amino Acid Residues

et al. 2016

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“…If the protein net charge is close to zero, an anisotropic charge distribution can cause protein self association [13,14,71,99], which has been correlated with increased aggregation propensity [55,80]. To avoid increasing protein charge anisotropy, charged mutations should carry the same sign as the net charge on the corresponding protein scaffold [31,100]. Deconvoluting between these competing charge effects has led to confusion over whether negatively charged mutations are more effective than positively charged mutations [44,89].…”

Section: Introductionmentioning

confidence: 99%

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Austerberry

Dajani

Panova

et al. 2017

European Journal of Pharmaceutics and Biopharmaceutics

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a b s t r a c tThe aggregation propensities for a series of single-chain variable fragment (scFv) mutant proteins containing supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating 'sticky' surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses interactions between partially folded states under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using conformational stability and native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations.

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“…Supporting this view is the fact that the A11 antibody, which specifically recognizes amyloid oligomers, was developed by forcing Ab to form a micelle-like structure (35). In addition, hydrophobicity correlates strongly with aggregation propensity (36,37), suggesting that most amyloid-forming molecules will contain a stretch of hydrophobic amino acids sufficiently long to form a polymer micelle. This implies that the amyloid phase diagram often contains both ordered and disordered oligomers in addition to the fibril state.…”

Section: Discussionmentioning

confidence: 99%

Thermodynamics of Huntingtin Aggregation

Phan

Schmit

2020

Biophysical Journal

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Amyloid aggregates are found in many neurodegenerative diseases, including Huntington's, Alzheimer's, and prion diseases. The precise role of the aggregates in disease progression has been difficult to elucidate because of the diversity of aggregated states they can adopt. Here, we study the formation of fibrils and oligomers by exon 1 of huntingtin protein. We show that the oligomer states are consistent with polymer micelles that are limited in size by the stretching entropy of the polyglutamine region. The model shows how the sequences flanking the amyloid core modulate aggregation behavior. The N17 region promotes aggregation through weakly attractive interactions, whereas the C38 tail opposes aggregation via steric repulsion. We also show that the energetics of cross-b stacking by polyglutamine would produce fibrils with many alignment defects, but minor perturbations from the flanking sequences are sufficient to reduce the defects to the level observed in experiment. We conclude with a discussion of the implications of this model for other amyloid-forming molecules.

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Addition of negatively charged residues can reverse the decrease in the solubility of an acidic protein caused by an artificially introduced non-polar surface patch

Cited by 10 publications

References 39 publications

A Soluble, Folded Protein without Charged Amino Acid Residues

A Soluble, Folded Protein without Charged Amino Acid Residues

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Thermodynamics of Huntingtin Aggregation

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