Arginine to lysine mutations increase the aggregation stability of a single chain variable fragment through unfolded state interactions

James Austerberry, Angela Thistlethwaite, Karl Fisher, Alexander P Golovanov, Alain Pluen, Reza Esfandiary, Christopher F van der Walle, Jim Warwicker, Jeremy P Derrick, Robin Curtis

Research output: Contribution to journalArticlepeer-review

340 Downloads (Pure)


Increased protein solubility is known to correlate with an increase in the proportion of lysine over arginine residues. Previous work has shown that the aggregation propensity of a single-chain variable fragment (scFv) does not correlate with its conformational stability or native-state protein-protein interactions. Here we test the hypothesis that aggregation is driven by the colloidal stability of partially unfolded states, studying the behaviour of scFv mutants harbouring single or multiple site-specific arginine/lysine mutations in denaturing buffers. In 6 M guanidine hydrochloride (GdmCl) or 8 M urea, repulsive protein-protein interactions were measured for the wild-type and lysine enriched (4RK) scFvs on account of weakened short-ranged attractions and increased excluded volume, in contrast to the arginine enriched (7KR) scFv which demonstrated strong reversible association. In 3 M GdmCl, the minimum concentration at which the scFvs were unfolded, the hydrodynamic radius of 4RK remained constant but increased for the wild-type and especially for 7KR. Individually swapping lysine back to arginine in 4KR indicated that the observed aggregation propensity of arginine in denaturing conditions was non-specific. Interestingly, one such swap generated a scFv with especially low aggregation rates under low/high ionic strength and denaturing buffers; molecular modelling identified hydrogen-bonding between the arginine side chain and main chain peptide groups, stabilising the structure. The arginine/lysine ratio is not routinely considered in biopharmaceutical scaffold design, or current amyloid prediction methods. This work therefore suggests a simple method to increase the stability of a biopharmaceutical protein against aggregation.

Original languageEnglish
Early online date17 Jul 2019
Publication statusPublished - 2019


Dive into the research topics of 'Arginine to lysine mutations increase the aggregation stability of a single chain variable fragment through unfolded state interactions'. Together they form a unique fingerprint.

Cite this