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Strong non-ideality effects at low protein concentrations: considerations for elongated proteins

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Yarawsky, Alexander E., Dinu, Vlad, Harding, Stephen E., Herr, Andrew B.·2023·European Biophysics Journal
DOI 10.1007/s00249-023-01648-xPMC PMC10599268

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Abstract

A recent investigation was aimed at obtaining structural information on a highly extended protein via SEC-MALS-SAXS. Significantly broadened elution peaks were observed, reminiscent of a phenomenon known as viscous fingering. This phenomenon is usually observed above 50 mg/mL for proteins like bovine serum albumin (BSA). Interestingly, the highly extended protein (Brpt5.5) showed viscous fingering at concentrations lower than 5 mg/mL. The current study explores this and other non-ideal behavior, emphasizing the presence of these effects at relatively low concentrations for extended proteins. BSA, Brpt5.5, and a truncated form of Brpt5.5 referred to as Brpt1.5 are studied systematically using size-exclusion chromatography (SEC), sedimentation velocity analytical ultracentrifugation (AUC), and viscosity. The viscous fingering effect is quantified using two approaches and is found to correlate well with the intrinsic viscosity of the proteins-Brpt5.5 exhibits the most severe effect and is the most extended protein tested in the study. By AUC, the hydrodynamic non-ideality was measured for each protein via global analysis of a concentration series. Compared to BSA, both Brpt1.5 and Brpt5.5 showed significant non-ideality that could be easily visualized at concentrations at or below 5 mg/mL and 1 mg/mL, respectively. A variety of relationships were examined for their ability to differentiate the proteins by shape using information from AUC and/or viscosity. Furthermore, these relationships were also tested in the context of hydrodynamic modeling. The importance of considering non-ideality when investigating the structure of extended macromolecules is discussed.

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Metadata

Authors
Yarawsky, A. E., Dinu, V., Harding, S. E., & Herr, A. B.
Affiliations
  • Cincinnati Children's Hospital Medical Center (US)
  • University of Nottingham (GB)
Corresponding author
Yarawsky, Alexander E.
ORCID
Venue
European Biophysics Journal
Publisher
Springer Science and Business Media LLC
Published
2023-04-13
Volume
52
Issue
4-5
Pages
427-438
ISSN
  • 0175-7571 (ISSN-L)
  • 1432-1017
DOI
10.1007/s00249-023-01648-x
PMC
PMC10599268
OpenAlex
W4365446685
Cited by
7
References
1
Citations by year
2026: 1 · 2025: 1 · 2024: 2 · 2023: 3
FWCI
0.62
Type
journal article
Language
en
Open access
Licence
Locations
  • European Biophysics Journal (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
National Institute of General Medical Sciences (R01-GM094363)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
Keywords
viscous fingering, low protein concentration, bovine serum albumin, intrinsic viscosity, size exclusion chromatography, hydrodynamic modeling
MeSH terms
Serum Albumin, Bovine, Viscosity, X-Ray Diffraction, Macromolecular Substances, Scattering, Small Angle, Hydrodynamics

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