Protein-binding Assays in Biological Liquids Using Microscale Thermophoresis

Overview

Journal Nat Commun

Specialty Biology

Date 2010 Oct 29

PMID 20981028

Citations 467

Authors

Christoph J Wienken

Philipp Baaske

Ulrich Rothbauer

Dieter Braun

Stefan Duhr

Affiliations

Soon will be listed here.

Abstract

Protein interactions inside the human body are expected to differ from the situation in vitro. This is crucial when investigating protein functions or developing new drugs. In this study, we present a sample-efficient, free-solution method, termed microscale thermophoresis, that is capable of analysing interactions of proteins or small molecules in biological liquids such as blood serum or cell lysate. The technique is based on the thermophoresis of molecules, which provides information about molecule size, charge and hydration shell. We validated the method using immunologically relevant systems including human interferon gamma and the interaction of calmodulin with calcium. The affinity of the small-molecule inhibitor quercetin to its kinase PKA was determined in buffer and human serum, revealing a 400-fold reduced affinity in serum. This information about the influence of the biological matrix may allow to make more reliable conclusions on protein functionality, and may facilitate more efficient drug development.

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References

Duhr S, Braun D . Thermophoretic depletion follows Boltzmann distribution. Phys Rev Lett. 2006; 96(16):168301. DOI: 10.1103/PhysRevLett.96.168301. View

Dufour C, Dangles O . Flavonoid-serum albumin complexation: determination of binding constants and binding sites by fluorescence spectroscopy. Biochim Biophys Acta. 2005; 1721(1-3):164-73. DOI: 10.1016/j.bbagen.2004.10.013. View

Schoenborn J, Wilson C . Regulation of interferon-gamma during innate and adaptive immune responses. Adv Immunol. 2007; 96:41-101. DOI: 10.1016/S0065-2776(07)96002-2. View

Project E, Friedman R, Nachliel E, Gutman M . A molecular dynamics study of the effect of Ca2+ removal on calmodulin structure. Biophys J. 2006; 90(11):3842-50. PMC: 1459500. DOI: 10.1529/biophysj.105.077792. View

Latham J, Markov D, Sorensen H, Bornhop D . Photobiotin surface chemistry improves label-free interferometric sensing of biochemical interactions. Angew Chem Int Ed Engl. 2005; 45(6):955-8. DOI: 10.1002/anie.200502418. View

Vigolo D, Buzzaccaro S, Piazza R . Thermophoresis and thermoelectricity in surfactant solutions. Langmuir. 2010; 26(11):7792-801. DOI: 10.1021/la904588s. View

Reineck P, Wienken C, Braun D . Thermophoresis of single stranded DNA. Electrophoresis. 2010; 31(2):279-86. DOI: 10.1002/elps.200900505. View

Wurger A . Transport in charged colloids driven by thermoelectricity. Phys Rev Lett. 2008; 101(10):108302. DOI: 10.1103/PhysRevLett.101.108302. View

Duhr S, Braun D . Why molecules move along a temperature gradient. Proc Natl Acad Sci U S A. 2006; 103(52):19678-82. PMC: 1750914. DOI: 10.1073/pnas.0603873103. View

10.

Dhont J, Wiegand S, Duhr S, Braun D . Thermodiffusion of charged colloids: single-particle diffusion. Langmuir. 2007; 23(4):1674-83. DOI: 10.1021/la062184m. View

11.

Rauch J, Kohler W . Diffusion and thermal diffusion of semidilute to concentrated solutions of polystyrene in toluene in the vicinity of the glass transition. Phys Rev Lett. 2002; 88(18):185901. DOI: 10.1103/PhysRevLett.88.185901. View

12.

WALKER E, Pacold M, Perisic O, Stephens L, Hawkins P, Wymann M . Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol Cell. 2000; 6(4):909-19. DOI: 10.1016/s1097-2765(05)00089-4. View

13.

Jecklin M, Schauer S, Dumelin C, Zenobi R . Label-free determination of protein-ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry. J Mol Recognit. 2009; 22(4):319-29. DOI: 10.1002/jmr.951. View

14.

Fagerstam L, Frostell A, Karlsson R, Kullman M, Larsson A, Malmqvist M . Detection of antigen-antibody interactions by surface plasmon resonance. Application to epitope mapping. J Mol Recognit. 1990; 3(5-6):208-14. DOI: 10.1002/jmr.300030507. View

15.

Baaske P, Wienken C, Reineck P, Duhr S, Braun D . Optical thermophoresis for quantifying the buffer dependence of aptamer binding. Angew Chem Int Ed Engl. 2010; 49(12):2238-41. DOI: 10.1002/anie.200903998. View

16.

Swillens S . Interpretation of binding curves obtained with high receptor concentrations: practical aid for computer analysis. Mol Pharmacol. 1995; 47(6):1197-203. View

17.

Wurger A . Thermophoresis in colloidal suspensions driven by Marangoni forces. Phys Rev Lett. 2007; 98(13):138301. DOI: 10.1103/PhysRevLett.98.138301. View

18.

Ebbinghaus S, Dhar A, McDonald J, Gruebele M . Protein folding stability and dynamics imaged in a living cell. Nat Methods. 2010; 7(4):319-23. DOI: 10.1038/nmeth.1435. View

19.

Bornhop D, Latham J, Kussrow A, Markov D, Jones R, Sorensen H . Free-solution, label-free molecular interactions studied by back-scattering interferometry. Science. 2007; 317(5845):1732-6. DOI: 10.1126/science.1146559. View

20.

Duhr S, Arduini S, Braun D . Thermophoresis of DNA determined by microfluidic fluorescence. Eur Phys J E Soft Matter. 2004; 15(3):277-86. DOI: 10.1140/epje/i2004-10073-5. View