Klein-Seetharaman was born in Germany. She completed her undergraduate training at the University of Cologne, where she earned dual honours in biology and chemistry. After earning her doctorate, she moved to the United States, where she worked in the laboratory of Har Gobind Khorana at the Massachusetts Institute of Technology. Her research considered conformational changes in rhodopsin, the G protein coupled receptor. She was a postdoctoral researcher at MIT with Harald Schwalbe, focusing on nuclear magnetic resonance spectroscopy. After eight months as a postdoc, Klein-Seetharaman moved Carnegie Mellon University where she worked with Raj Reddy in biology. She was eventually appointed to the faculty at Carnegie Mellon.