Conformational changes at protein-protein interaction followed with an SAW biosensor

The specific interaction between small GTP binding protein Ras and its effector proteins is a typicalexample how cellular signal transduction is executed. The binding properties of the effector Nore1Ato active K-Ras-GppNHp and to inactive K-Ras-GDP were analyzed with a label-free surface acousticwave (SAW) sensor. The measured phase signal was used for the concomitant evaluation of the bindingkinetics. Binding was observed only to the active K-Ras-GppNHp complex. An affinity decrease based onan increase in ionic strength was detected from the calculated K-D's in the range of 6.6 x 10-8M in 100 mMNaCl and of 1.1 x 10(-6) M in 500 mM NaCl. The recorded amplitude signal was used as a measure of theconformational changes. It could be shown that at 100 mM NaCl, the interaction causes an increase inrigidity. At 500 mM NaCl, no increase in rigidity was measured at high concentrations of Nore1A due toreduced binding, suggesting an influence of salt on protein rigidity and hence on the strength of binding. (C) 2014 Elsevier B. V. All rights reserved.