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

  • External authors:
  • Angela Thistlethwaite
  • Karl Fisher
  • Reza Esfandiary
  • Christopher F van der Walle
  • Jim Warwicker

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Arginine to lysine mutations increase the aggregation stability of a single chain variable fragment through unfolded state interactions. / Austerberry, James; Thistlethwaite, Angela; Fisher, Karl; Golovanov, Alexander P; Pluen, Alain; Esfandiary, Reza; van der Walle, Christopher F; Warwicker, Jim; Derrick, Jeremy P; Curtis, Robin.

In: Biochemistry, 2019.

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@article{7f686d73de5a476aa1602c8362df4389,
title = "Arginine to lysine mutations increase the aggregation stability of a single chain variable fragment through unfolded state interactions",
abstract = "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.",
author = "James Austerberry and Angela Thistlethwaite and Karl Fisher and Golovanov, {Alexander P} and Alain Pluen and Reza Esfandiary and {van der Walle}, {Christopher F} and Jim Warwicker and Derrick, {Jeremy P} and Robin Curtis",
year = "2019",
doi = "10.1021/acs.biochem.9b00367",
language = "English",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",

}

RIS

TY - JOUR

T1 - Arginine to lysine mutations increase the aggregation stability of a single chain variable fragment through unfolded state interactions

AU - Austerberry, James

AU - Thistlethwaite, Angela

AU - Fisher, Karl

AU - Golovanov, Alexander P

AU - Pluen, Alain

AU - Esfandiary, Reza

AU - van der Walle, Christopher F

AU - Warwicker, Jim

AU - Derrick, Jeremy P

AU - Curtis, Robin

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

U2 - 10.1021/acs.biochem.9b00367

DO - 10.1021/acs.biochem.9b00367

M3 - Article

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

ER -