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Nonspecific shielding of unfavorable electrostatic intramolecular interactions in the erythropoietin native-state increase conformational stability and limit non-native aggregation.

ABSTRACT: Previous equilibrium and kinetic folding studies of the glycoprotein erythropoietin indicate that sodium chloride increases the conformational stability of this therapeutically important cytokine, ostensibly by stabilizing the native-state [Banks DD, (2011) The Effect of Glycosylation on the Folding Kinetics of Erythropoietin. J Mol Biol 412:536-550]. The focus of the current report is to determine the underlying cause of the salt dependent increase in erythropoietin conformational stability and to understand if it has any impact on aggregation, an instability that remains a challenge to the biotech industry in maintaining the efficacy and shelf-life of protein therapeutics. Isothermal urea denaturation experiments conducted at numerous temperatures in the absence and presence of sodium chloride indicated that salt stabilizes erythropoietin primarily by increasing the difference in enthalpy between the native and unfolded sates. This result, and the finding that the salt induced increases in erythropoietin melting temperatures were independent of the identity of the salt cation and anion, indicates that salt likely increases the conformational stability of erythropoietin at neutral pH by nonspecific shielding of unfavorable electrostatic interaction(s) in the native-state. The addition of salt (even low concentrations of the strong chaotrope salt guanidinium hydrochloride) also exponentially decreased the initial rate of soluble erythropoietin non-native aggregation at 37 °C storage.

SUBMITTER: Banks DD

PROVIDER: S-EPMC4420528 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Nonspecific shielding of unfavorable electrostatic intramolecular interactions in the erythropoietin native-state increase conformational stability and limit non-native aggregation.

Banks Douglas D DD

Protein science : a publication of the Protein Society 20150411 5

Previous equilibrium and kinetic folding studies of the glycoprotein erythropoietin indicate that sodium chloride increases the conformational stability of this therapeutically important cytokine, ostensibly by stabilizing the native-state [Banks DD, (2011) The Effect of Glycosylation on the Folding Kinetics of Erythropoietin. J Mol Biol 412:536-550]. The focus of the current report is to determine the underlying cause of the salt dependent increase in erythropoietin conformational stability and ...[more]

PMID: 25628168

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Project description:Glycinamide ribonucleotide transformylase (GART) undergoes a pH-dependent coil-helix transition with pK(a) approximately 7. An alpha-helix is formed at high pH spanning 8 residues of a 21-residue-long loop, comprising the segment Thr120-His121-Arg122-Gln123-Ala124-Leu125-Glu126-Asn127. To understand the electrostatic nature of this loop-helix, called the activation loop-helix, which leads to the formation and stability of the alpha-helix, pK(a) values of all ionizable residues of GART have been calculated, using Poisson-Boltzmann electrostatic calculations and crystallographic data. Crystallographic structures of high and low pH E70A GART have been used in our analysis. Low pK(a) values of 5.3, 5.3, 3.9, 1.7, and 4.7 have been calculated for five functionally important histidines, His108, His119, His121, His132, and His137, respectively, using the high pH E70A GART structure. Ten theoretical single and double mutants of the high pH E70A structure have been constructed to identify pairwise interactions of ionizable residues, which have aided in elucidating the multiplicity of electrostatic interactions of the activation loop-helix, and the impact of the activation helix on the catalytic site. Based on our pK(a) calculations and structural data, we propose that: (1) His121 forms a molecular switch for the coil-helix transition of the activation helix, depending on its protonation state; (2) a strong electrostatic interaction between His132 and His121 is observed, which can be of stabilizing or destabilizing nature for the activation helix, depending on the relative orientation and protonation states of the rings of His121 and His132; (3) electrostatic interactions involving His119 and Arg122 play a role in the stability of the activation helix; and (4) the activation helix contains the helix-promoting sequence Arg122-Gln123-Ala124-Leu125-Glu126, but its alignment relative to the N and C termini of the helix is not optimal, and is possibly of a destabilizing nature. Finally, we provide electrostatic evidence that the formation and closure of the activation helix create a hydrophobic environment for catalytic-site residue His108, to facilitate catalysis.

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Nonspecific shielding of unfavorable electrostatic intramolecular interactions in the erythropoietin native-state increase conformational stability and limit non-native aggregation.

Publications

Nonspecific shielding of unfavorable electrostatic intramolecular interactions in the erythropoietin native-state increase conformational stability and limit non-native aggregation.

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