Abstract
The accurate characterization of proteins in both their native and denatured states is essential to effectively understand protein function, folding and stability. As a proof of concept, a micro rheological method is applied, based on the characterization of thermal fluctuations of a micro cantilever immersed in a bovine serum albumin solution, to assess changes in the viscosity associated with modifications in the protein’s structure under the denaturant effect of urea. Through modeling the power spectrum density of the cantilever’s fluctuations over a broad frequency band, it is possible to implement a fitting procedure to accurately determine the viscosity of the fluid, even at low volumes. Increases in viscosity during the denaturant process are identified using the assumption that the protein is a hard sphere, with a hydrodynamic radius that increases during unfolding. This is modeled accordingly through the Einstein-Batchelor formula. The Einstein-Batchelor formula estimates are verified through dynamic light scattering, which measures the hydrodynamic radius of proteins. Thus, this methodology is proven to be suitable for the study of protein folding in samples of small size at vanishing shear stresses.
Highlights
Rheological measurements are recognized as suitable for in the assessment of unfolding phenomena [1], as the hydrodynamic volume of a protein at sufficiently high concentrations can have a significant impact on the viscosity of a protein solution
The first data set, obtained with zero urea concentration, shows minimal variation in solution viscosity with Bovine serum albumin (BSA) concentration, indicating that the protein remained in a folded state, regardless of BSA concentration
Viscosity shows a linear dependency on BSA concentration, which is characteristic of low φ
Summary
Rheological measurements are recognized as suitable for in the assessment of unfolding phenomena [1], as the hydrodynamic volume of a protein at sufficiently high concentrations can have a significant impact on the viscosity of a protein solution. That apply relatively high shearing, can promote artificial losses in protein structure, resulting in biased measurements. These effects have been found to be more prominent in proteins with high content of alpha helix domains [2, 3] whereas they are dramatically reduced for the cytochrome c protein [4]. Due to their principle of functioning, classical rheometers require large sample volumes at sufficiently high protein concentrations, which may increase the cost of an assay and make it difficult to detect minute variations in viscosity in small samples. A wide variety of modern rheometers, such as micro fluidity
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