The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules.
Protein Sci
; 29(4): 1018-1034, 2020 04.
Article
en En
| MEDLINE
| ID: mdl-31943488
Every method used to quantify biomolecular interactions has its own strengths and limitations. To quantify protein-DNA binding affinities, nitrocellulose filter binding assays with 32 P-labeled DNA quantify Kd values from 10-12 to 10-8 M but have several technical limitations. Here, we considered the suitability of biolayer interferometry (BLI), which monitors association and dissociation of a soluble macromolecule to an immobilized species; the ratio koff /kon determines Kd . However, for lactose repressor protein (LacI) and an engineered repressor protein ("LLhF") binding immobilized DNA, complicated kinetic curves precluded this analysis. Thus, we determined whether the amplitude of the BLI signal at equilibrium related linearly to the fraction of protein bound to DNA. A key question was the effective concentration of immobilized DNA. Equilibrium titration experiments with DNA concentrations below Kd (equilibrium binding regime) must be analyzed differently than those with DNA near or above Kd (stoichiometric binding regime). For ForteBio streptavidin tips, the most frequent effective DNA concentration was ~2 × 10-9 M. Although variation occurred among different lots of sensor tips, binding events with Kd ≥ 10-8 M should reliably be in the equilibrium binding regime. We also observed effects from multi-valent interactions: Tetrameric LacI bound two immobilized DNAs whereas dimeric LLhF did not. We next used BLI to quantify the amount of inducer sugars required to allosterically diminish protein-DNA binding and to assess the affinity of fructose-1-kinase for the DNA-LLhF complex. Overall, when experimental design corresponded with appropriate data interpretation, BLI was convenient and reliable for monitoring equilibrium titrations and thereby quantifying a variety of binding interactions.
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01-internacional
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MEDLINE
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ADN
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Proteínas
Idioma:
En
Revista:
Protein Sci
Asunto de la revista:
BIOQUIMICA
Año:
2020
Tipo del documento:
Article
Pais de publicación:
Estados Unidos