Abstract
Protein quantification is essential in a great variety of biochemical assays, yet the inherent systematic errors associated with the concentration determination of intrinsically disordered proteins (IDPs) using classical methods are hardly appreciated. Routinely used assays for protein quantification, such as the Bradford assay or ultraviolet absorbance at 280 nm, usually seriously misestimate the concentrations of IDPs due to their distinct and variable amino acid composition. Therefore, dependable method(s) have to be worked out/adopted for this task. By comparison to elemental analysis as the gold standard, we show through the example of four globular proteins and nine IDPs that the ninhydrin assay and the commercial QubitTM Protein Assay provide reliable data on IDP quantity. However, as IDPs can show extreme variation in amino acid composition and physical features not necessarily covered by our examples, even these techniques should only be used for IDPs following standardization. The far-reaching implications of these simple observations are demonstrated through two examples: (i) circular dichroism spectrum deconvolution, and (ii) receptor-ligand affinity determination. These actual comparative examples illustrate the potential errors that can be incorporated into the biophysical parameters of IDPs, due to systematic misestimation of their concentration. This leads to inaccurate description of IDP functions.
Highlights
Exact determination of protein concentrations is central to modeling in biochemistry, enzymology, molecular biophysics and practically all branches of molecular life sciences
Each protein (Tables 1, 2) was prepared in a solution of about 10 mg/ml, extensively dialyzed against phosphate-buffered saline (PBS) 0.5X supplemented with 0.5 mM Tris(2-carboxyethyl)phosphine HCl (TCEP) and its purity was checked on an overloaded SDSPAGE to visualize the majority of the polypeptidic contaminants (Figure 1)
The concentration as determined by the Bradford assay is overestimated by 20% for globular proteins, whereas in the case of intrinsically disordered proteins (IDPs) it is underestimated by 63% on average (Figure 3)
Summary
Exact determination of protein concentrations is central to modeling in biochemistry, enzymology, molecular biophysics and practically all branches of molecular life sciences. IDPs do not fold into a well-defined 3D structure, because of their distinct amino acid composition: they are unusually enriched in Gln, Gly, Ser, Lys, Glu, and Pro (disorder-promoting) and depleted in Trp, Tyr, Phe, Cys, Ile, Leu, and Val (order-promoting) amino acids (Romero et al, 2001) Due to this compositional bias, IDPs typically have low molar extinction coefficient at 280 nm and weak binding of CBB, which relies primarily on hydrophobic interactions with aromatic residues, and electrostatic interaction (between its anionic form) with basic amino acid residues, of the protein (Weist et al, 2008). Their concentration is often underestimated to a great extent, which is further affected by contaminating macromolecules, causing an often unnoticed error that propagates into their quantitative parameters (Szollosi et al, 2007; Georgiou et al, 2008)
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