A Soluble, Folded Protein without Charged Amino Acid Residues

Abstract

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.

Effects of protein size, thermodynamic stability, and net charge on cotranslational folding on the ribosome

“…We further note that the f FL profile for the fully uncharged S6 0 variant is similar to that of wildtype S6, demonstrating that a protein that is devoid of charged residues can fold within the exit tunnel. To our knowledge, this is only the second example of efficient folding of a protein completely devoid of charged residues 49 . Strikingly, for the negatively charged variants S6 +9-16 and S6 -16 , high values of f FL persist to much larger L values than for the neutral and positively charged variants, and the peaks in the f FL profiles are broad and indistinct.…”

Glucose oxidase stabilization against thermal inactivation using high hydrostatic pressure and hydrophobic modification

“…Water can also have anomalous characteristics (Biela et al, 2012(Biela et al, , 2013Breiten et al, 2013;Chandler, 2005;Krimmer et al, 2014;Patel et al, 2012), thus its role is difficult to assess. A final comment considers the recent construction and analysis of a protein lacking charges (Hojgaard et al, 2016). This variant protein remains soluble and stable, indicating that electrostatic interactions are not required for its structure-function relationship.…”

“…Aniline modification thermally stabilized GOx, while benzoate modification destabilized the enzyme. A final comment considers the recent construction and analysis of a protein lacking charges (Hojgaard et al, 2016). The activities of the unmodified and modified GOx species were determined by Michaelis-Menten kinetics (Table IV).…”

The solvent-excluded surfaces of water-soluble proteins

“…Previous analyses of the relationship between protein surface and protein-solvent interaction have focused mainly on SAS area and surface charge at residue-level [26,31,54,34]. However, quantitative relationships between SESs and protein solvation and folding remain largely unknown and controversial [55,56,17,57]. For example the past efforts to correlate SAS area with experimentally-determined solubility have only met limited success [9].…”

“…Protein-solvent interaction is believed to contribute largely to the solvation, folding and structure of a water-soluble protein [1,2,3,4,5] and plays an important role in its function such as ligand binding [6]. However it is challenging to quantify such contributions [7,8] using either experimental approach [9,10] or theoretical model [11,12,13,14,15] or molecular dynamic (MD 1 ) simulation [16,17] or structural information [18,19]. For example, due to the difficulty to evaluate protein-solvent interaction it is not clear at present how evolution has optimized the surfaces of naturallyoccurring water-soluble proteins to make them best adapted to aqueous solvent.…”