Abstract
Size exclusion chromatography coupled online to a Tetra Detector Array in combination with analytical ultracentrifugation (or with quasi-elastic light scattering) is a useful methodology to characterize hydrodynamic properties of macromolecules, including intrinsically disordered proteins. The time-averaged apparent hydration and the shape factor of proteins can be estimated from the measured parameters (molecular mass, intrinsic viscosity, hydrodynamic radius) by these techniques. Here we describe in detail this methodology and its application to characterize hydrodynamic and conformational changes in proteins.
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Abbreviations
- M :
-
Molecular mass, g/mol
- T :
-
Absolute temperature, K
- C :
-
Protein concentration, mol/L (M)
- \( {\hbox{d}}n/{\hbox{d}}c \) :
-
Refractive index increment, mL/g
- \( {\hbox{d}}A/{\hbox{d}}c \) :
-
Absorbance increment, L/g cm
- \( \left[ \eta \right] \) :
-
Intrinsic viscosity, mL/g
- \( \nu \) :
-
Hydrodynamic shape function, viscosity increment, Simha–Saito shape factor, unitless
- \( \delta \) :
-
Time-averaged apparent hydration, \( {{\hbox{g}}_{{{{\rm{H}}_{{2}}}{\rm{O}}}}}/{\hbox{g}}_{\rm{protein}} \)
- \( {{f} \left/ {{{f_0}}} \right.} \) :
-
Translational frictional ratio of the protein, including shape and hydration parameters
- \( f \) :
-
Frictional coefficient of the protein, g/s
- \( {f_0} \) :
-
Frictional coefficient of an anhydrous sphere of the mass of the protein, g/s
- R H :
-
Hydrodynamic radius of the protein, cm
- R 0 :
-
Radius of an anhydrous sphere of the mass of the protein, cm
- V H :
-
Hydrodynamic volume calculated from the R H, cm3
- D t :
-
Translational diffusion coefficient, cm2/s
- s :
-
Sedimentation coefficient obtained at the temperature of the experiment, Svedberg, 10−13s
- \( \eta \) :
-
Viscosity of the solvent, Poise: g/cm s
- \( \rho \) :
-
Density of the solvent, g/mL
- k B :
-
Boltzmann’s constant, erg/K (K B: 1.38065 × 10−16 erg/K, with erg: g cm2/s2 = 10−7 J 1.38065 × 10−23 J/K)
- N A :
-
Avogadro’s number, molecules/mol
- \( \bar{\nu } \) :
-
Partial specific volume, mL/g
- \( a/b \) :
-
Axial ratio of ellipsoid
- RALS :
-
Right Angle Light-Scattering
- LALS :
-
Low Angle Light-Scattering
- IP :
-
Internal Pressure
- DP :
-
Differential Pressure
- UV :
-
Ultraviolet absorption
- RI:
-
Refractive Index
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Acknowledgements
This work was supported by the Institut Pasteur (Grant PTR374), the Centre National de la Recherche Scientifique (CNRS UMR 3528), and the Agence Nationale de la Recherche, programme Jeunes Chercheurs (ANR, grant ANR-09-JCJC-0012).
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Karst, J.C., Sotomayor-Pérez, A.C., Ladant, D., Chenal, A. (2012). Estimation of Intrinsically Disordered Protein Shape and Time-Averaged Apparent Hydration in Native Conditions by a Combination of Hydrodynamic Methods. In: Uversky, V., Dunker, A. (eds) Intrinsically Disordered Protein Analysis. Methods in Molecular Biology, vol 896. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-3704-8_10
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DOI: https://doi.org/10.1007/978-1-4614-3704-8_10
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