title: Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions.
abstract: Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science.
authors.affiliation: Systems Biophysics and Functional Nanosystems, Ludwig-Maximilians-Universität München, Amalienstrasse 54, 80799 Munich, Germany.
authors.forename: Susanne A I
authors.initials: SA
authors.lastname: Seidel
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Dijkman
authors.forename: Wendy A
authors.initials: WA
authors.lastname: Lea
authors.forename: Geert
authors.initials: G
authors.lastname: van den Bogaart
authors.forename: Moran
authors.initials: M
authors.lastname: Jerabek-Willemsen
authors.forename: Ana
authors.initials: A
authors.lastname: Lazic
authors.forename: Jeremiah S
authors.initials: JS
authors.lastname: Joseph
authors.forename: Prakash
authors.initials: P
authors.lastname: Srinivasan
authors.forename: Philipp
authors.initials: P
authors.lastname: Baaske
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
authors.forename: Ilia
authors.initials: I
authors.lastname: Katritch
authors.forename: Fernando A
authors.initials: FA
authors.lastname: Melo
authors.forename: John E
authors.initials: JE
authors.lastname: Ladbury
authors.forename: Gideon
authors.initials: G
authors.lastname: Schreiber
authors.forename: Anthony
authors.initials: A
authors.lastname: Watts
authors.forename: Dieter
authors.initials: D
authors.lastname: Braun
authors.forename: Stefan
authors.initials: S
authors.lastname: Duhr
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D019281:Dimerization; D051380:GRB2 Adaptor Protein / Q000737:chemistry; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry; D007834:Lasers; D008968:Molecular Conformation; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D015800:Protozoan Proteins / Q000737:chemistry; D051381:Rats; D043705:Receptor, Adenosine A2A / Q000737:chemistry; D018028:Receptors, Neurotensin / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature; D013816:Thermodynamics; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry
chemical_list: D051380:GRB2 Adaptor Protein; D011506:Proteins; D015800:Protozoan Proteins; D043705:Receptor, Adenosine A2A; D018028:Receptors, Neurotensin; D065093:beta-Lactamase Inhibitors; C114036:neurotensin type 1 receptor; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D001618:beta-Lactamases