A Soluble, Folded Protein without Charged Amino Acid Residues.
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.
Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen , DK-2200 Copenhagen N, Denmark.
Casper
C
Højgaard
Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen , DK-2200 Copenhagen N, Denmark.
Christian
C
Kofoed
Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen , DK-2200 Copenhagen N, Denmark.
Roall
R
Espersen
Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen , DK-2200 Copenhagen N, Denmark.
Kristoffer Enøe
KE
Johansson
Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen , DK-2200 Copenhagen N, Denmark.
Mara
M
Villa
Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen , DK-2200 Copenhagen N, Denmark.
Martin
M
Willemoës
Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen , DK-2200 Copenhagen N, Denmark.
Kresten
K
Lindorff-Larsen
Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen , DK-2200 Copenhagen N, Denmark.
Kaare
K
Teilum
Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen , DK-2200 Copenhagen N, Denmark.
Jakob R
JR
Winther
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D040143:Cellulomonas; D002482:Cellulose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012995:Solubility
D000596:Amino Acids; D001426:Bacterial Proteins; D002482:Cellulose