Mean Net Charge of Intrinsically Disordered Proteins: Experimental Determination of Protein Valence by Electrophoretic Mobility Measurements

  • Ana Cristina Sotomayor-Pérez
  • Johanna C. Karst
  • Daniel Ladant
  • Alexandre Chenal
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 896)

Abstract

Under physiological conditions, intrinsically disordered proteins (IDPs) are unfolded, mainly because of their low hydrophobicity and the strong electrostatic repulsion between charged residues of the same sign within the protein. Softwares have been designed to facilitate the computation of the mean net charge of proteins (formally protein valence) from their amino acid sequences. Nevertheless, discrepancies between experimental and computed valence values for several proteins have been reported in the literature. Hence, experimental approaches are required to obtain accurate estimation of protein valence in solution. Moreover, ligand-induced disorder-to-order transition is involved in the folding of numerous IDPs. Some of the ligands are cations or anions, which, upon protein binding, decrease the mean net charge of the protein, favoring its folding via a charge reduction effect. An accurate determination of the mean net charge of protein in both its ligand-free intrinsically disordered state and in its folded, ligand-bound state allows one to estimate the number of ligands bound to the protein in the holo-state. Here, we describe an experimental protocol to determine the mean net charge of protein, from its electrophoretic mobility, its molecular mass and its hydrodynamic radius.

Key words

Mean net charge Protein valence Intrinsically disordered protein IDP Electrophoretic mobility Molecular mass Hydrodynamic radius Static light scattering Quasi-elastic light scattering Analytical ultracentrifugation 

Abbreviations

\( {\mu_e} \)

Electrophoretic mobility, cm2.V−1.s−1 or μm.cm.V−1.s−1 (SI: m2.V−1.s−1)

\( \zeta \)

Zeta potential, V

z

Valence or “mean net charge”

e

Electronic charge, 1.602 × 10−19 coulombs, A.s

U

Applied voltage, V, kg.m2.s−3.A−1

I

Current intensity, A

\( {\varepsilon_0} \)

Vacuum permittivity, 8.854 × 10−12C.V−1.m−1

\( {\varepsilon_r} \)

Relative static permittivity (dielectric constant) of the solvent, 78.54.

\( \varepsilon \)

Buffer permittivity, \( \varepsilon = {\varepsilon_r}{\varepsilon_0} \), C.V−1.m−1

\( {{f} \left/ {{{f_0}}} \right.} \)

Translational frictional ratio of the protein, including shape and hydration parameters

\( f \)

Frictional coefficient of the protein, g.s−1

\( {f_0} \)

Frictional coefficient of an anhydrous sphere of the mass of the protein, g.s−1

Rp

Hydrodynamic radius of the protein, cm

Rb

Hydrodynamic radius of the buffer, cm

R0

Radius of an anhydrous sphere of the mass of the protein, cm

VH

Hydrodynamic volume, cm3

Dt

Translational diffusion coefficient, cm2.s−1

s

Sedimentation coefficient obtained at the temperature of the experiment, Svedberg, 10−13s

\( \bar{\nu } \)

Partial specific volume, cm3.g−1

\( {\eta_s} \)

Viscosity of the solvent, Poise: g.cm−1.s−1

\( \rho \)

Density of the solvent, g.cm−3

M

Molecular mass, g.mol−1

T

Absolute temperature, K

C

Protein concentration, mol.L−1 (M)

\( \delta \)

Time-averaged apparent hydration, \( {g_{{{{\rm{H}}_{{2}}}{\rm{O}}}}} \times g_{\rm{protein}}^{{ - 1}} \)

MCR

Mean count rate

kcps

Kilo-count per second

ZQF

Zeta quality factor

FFR

Fast field reversal

SFR

Slow field reversal

DTS1060C

Malvern electrophoretic mobility cell

DTS1230

Malvern standard for electrophoretic mobility and conductivity

QELS

Quasi-elastic light scattering

kB

Boltzmann’s constant, erg.K−1; (K B: 1.38065 × 10−16 erg.K−1 with erg: g.cm2.s−2 = 10−7 J; 1.38065 × 10−23 J.K−1)

NA

Avogadro’s number, molecules.mol−1

PALS

Phase shift analysis light scattering

κ

Debye length, Inverse screening length, m

ZEN1010

Malvern electrophoretic mobility microcell

Notes

Acknowledgement

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).

References

  1. 1.
    Delgado AV, Gonzalez-Caballero F, Hunter RJ, Koopal LK, Lyklema J (2005) Measurement and interpretation of electrokinetic phenomena. Pure Appl Chem 77:1753–1805CrossRefGoogle Scholar
  2. 2.
    Abramson HA, Moyer LS, Gorin MH (1942) Electrophoresis of proteins and the chemistry of cell surfaces. Reinhold Publishing Coorporation, New YorkGoogle Scholar
  3. 3.
    Basak SK, Ladisch MR (1995) Correlation of electrophoretic mobilities of proteins and peptides with their physicochemical properties. Anal Biochem 226:51–58PubMedCrossRefGoogle Scholar
  4. 4.
    Sotomayor-Perez AC, Ladant D, Chenal A (2011) Calcium-induced folding of intrinsically disordered Repeat-in-Toxin (RTX) motifs via changes of protein charges and oligomerization states. J Biol Chem 286:16997–17004Google Scholar
  5. 5.
    Henry DC (1931) The cataphoresis of suspended particles. Part 1. The equation of cataphoresis. Proc R Soc Lond A 133:106–129CrossRefGoogle Scholar
  6. 6.
    Debye VP, Hückel E (1924) Bemerkungen zu einem Satze über die kataphorestische Wanderungsgeschwindingkeit suspendierter teilchen. Physikalische Zeitschrift 3Google Scholar
  7. 7.
    Smoluchowski M (1903) Przyczynek do teoryi endosmozy elekrycznej i niektorych zjawisk pokrewnych. Bull Acad Sci CracovieGoogle Scholar
  8. 8.
    Wall S (2010) The history of electrokinetic phenomena. Curr Opin Colloid Interface Sci 15:119–124CrossRefGoogle Scholar
  9. 9.
    Gorin MH (1939) An equilibrium theory of ionic conductance. J Chem Phys 7:405–413CrossRefGoogle Scholar
  10. 10.
    Winzor DJ (2004) Determination of the net charge (valence) of a protein: a fundamental but elusive parameter. Anal Biochem 325:1–20PubMedCrossRefGoogle Scholar
  11. 11.
    Adamson NJ, Reynolds EC (1997) Rules relating electrophoretic mobility, charge and molecular size of peptides and proteins. J Chromatogr 699:133–147CrossRefGoogle Scholar
  12. 12.
    Issaq HJ, Janini GM, Atamna IZ, Muschik GM, Lukszo J (1992) Capillary electrophoresis separation of small peptides: effect of pH, buffer additives, and temperature. J Liq Chrom Relat Tech 15:1129–1142CrossRefGoogle Scholar
  13. 13.
    Walbroehl Y, Jorgenson JW (1989) Capillary zone electrophoresis for the determination of electrophoretic mobilities and diffusion coefficients of proteins. J Microcolumn Sep 1:41–45CrossRefGoogle Scholar
  14. 14.
    Velick SF (1949) The interaction of enzymes with small ions. I. An electrophoretic and equilibrium analysis of ldolase in phosphate and acetate buffers. J Phys Colloid Chem 53:135–149PubMedCrossRefGoogle Scholar
  15. 15.
    Longsworth LG (1941) The influence of pH on the mobility and diffusion of ovalbumin. Ann NY Acad Sci 41:267–285CrossRefGoogle Scholar
  16. 16.
    Ivory CF (1990) Electrophoresis of proteins: batch and continuous methods. In: Flickinger M, Drew S (eds) The encyclopedia of bioprocess technology: fermentation, biocatalysis and bioseparations. Wiley, Chapter 9Google Scholar
  17. 17.
    Rickard EC, Strohl MM, Nielsen RG (1991) Correlation of electrophoretic mobilities from capillary electrophoresis with physicochemical properties of proteins and peptides. Anal Biochem 197:197–207PubMedCrossRefGoogle Scholar
  18. 18.
    Karst JC, Sotomayor Perez AC, 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 AK (eds) Intrinsically Disordered Proteins: Volume I. Experimental TechniquesGoogle Scholar
  19. 19.
    Bourdeau RW, Malito E, Chenal A, Bishop BL, Musch MW, Villereal ML, Chang EB, Mosser EM, Rest RF, Tang WJ (2009) Cellular functions and X-ray structure of anthrolysin O, a cholesterol-dependent cytolysin secreted by Bacillus anthracis. J Biol Chem 284:14645–14656PubMedCrossRefGoogle Scholar
  20. 20.
    Chenal A, Guijarro JI, Raynal B, Delepierre M, Ladant D (2009) RTX calcium binding motifs are intrinsically disordered in the absence of calcium: implication for protein secretion. J Biol Chem 284:1781–1789PubMedCrossRefGoogle Scholar
  21. 21.
    Karst JC, Sotomayor Perez AC, Guijarro JI, Raynal B, Chenal A, Ladant D (2010) Calmodulin-induced conformational and hydrodynamic changes in the catalytic domain of Bordetella pertussis adenylate cyclase toxin. Biochemistry 49:318–328PubMedCrossRefGoogle Scholar
  22. 22.
    Sotomayor Perez AC, Karst JC, Davi M, Guijarro JI, Ladant D, Chenal A (2010) Characterization of the regions involved in the calcium-induced folding of the intrinsically disordered RTX motifs from the Bordetella pertussis adenylate cyclase toxin. J Mol Biol 397:534–549PubMedCrossRefGoogle Scholar
  23. 23.
    Rodbard D, Chrambach A (1971) Estimation of molecular radius, free mobility, and valence using polyacylamide gel electrophoresis. Anal Biochem 40:95–134PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Ana Cristina Sotomayor-Pérez
    • 1
    • 2
  • Johanna C. Karst
    • 1
    • 2
  • Daniel Ladant
    • 1
    • 2
  • Alexandre Chenal
    • 1
    • 2
  1. 1.Unité de Biochimie des Interactions Macromoléculaires, CNRS UMR 3528, Institut PasteurParisFrance
  2. 2.Département de Biologie Structurale et Chimie, CNRS UMR 3528Institut Pasteur, Unité de Biochimie des Interactions MacromoléculairesParisFrance

Personalised recommendations