Purification and biochemical properties of Rac1, 2, 3 and the splice variant Rac1b.
Rac proteins (Rac1, 1b, 2, 3) belong to the GTP-binding proteins (or GTPases) of the Ras superfamily and thus act as molecular switches cycling between an active GTP-bound and an inactive GDP-bound form through nucleotide exchange and hydrolysis. Like most other GTPases, these proteins adopt different conformations depending on the bound nucleotide, the main differences lying in the conformation of two short and flexible loop structures designated as the switch I and switch II region. The three distinct mammalian Rac isoforms, Rac1, 2 and 3, share a very high sequence identity (up to 90%), with Rac1b being an alternative splice variant of Rac1 with a 19 amino acid insertion in vicinity to the switch II region. We have demonstrated that Rac1 and Rac3 are very closely related with respect to their biochemical properties, such as effector interaction, nucleotide binding, and hydrolysis. In contrast, Rac2 displays a slower nucleotide association and is more efficiently activated by the Rac-GEF Tiam1. Modeling and normal mode analysis corroborate the hypothesis that the altered molecular dynamics of Rac2, in particular at the switch I region, may be responsible for different biochemical properties. On the other hand, our structural and biochemical analysis of Rac1b has shown that, compared with Rac1, Rac1b has an accelerated GEF-independent GDP/GTP-exchange and an impaired GTP-hydrolysis, accounting for a self-activating GTPase. This chapter discusses the use of fluorescence spectroscopic methods, allowing real-time monitoring of the interaction of nucleotides, regulators, and effectors with the Rac proteins at submicromolar concentrations and quantification of the kinetic and equilibrium constants.
Haeusler
Lars Christian
LC
Max-Planck Institute for Molecular Physiology, Department of Structural Biology, Dortmund, Germany.
Hemsath
Lars
L
Fiegen
Dennis
D
Blumenstein
Lars
L
Herbrand
Ulrike
U
Stege
Patricia
P
Dvorsky
Radovan
R
Ahmadian
Mohammad Reza
MR
D020558:GTP Phosphohydrolases / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D013050:Spectrometry, Fluorescence / Q000379:methods; D062367:ortho-Aminobenzoates / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D020830:rac1 GTP-Binding Protein / Q000302:isolation & purification; D000097766:RAC2 GTP-Binding Protein
D020558:GTP Phosphohydrolases; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate; D062367:ortho-Aminobenzoates; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C065197:3'-(methylanthraniloyl)-2'-deoxy-guanosine diphosphate