Directed Evolution: An Overview 320 Analysis of Protein-Protein Interactions 322 Introducing Disorder to Study Natural and Evolutionary Mechanisms 324 Examples of IDPs Studied Using Protein Engineering/Directed Evolution 325

Increased Rigidity of Protein Domains Results in Increased Protein Stability 325

An increase in the proportion of intrinsically disordered proteins (IDPs) concomitant with an increase in organism complexity (Tompa, Dosztanyi et al. 2006; Ward, Sodhi et al. 2004) suggests an important role for disorder in evolution. Uncovering details regarding the origin and changes that occurred in the evolution of IDPs may shed light on the evolution of more ordered proteins and the rise of complex protein functions in humans. Moreover, such information will provide insight to the role of disorder, IDP functionality, and their mechanisms of action at the molecular level. Such information for IDPs, however, is dicult to obtain using traditional biochemical and biophysical methods. Directed evolution can mimic such combinatorial processes that occur in natural evolution. Pairing directed evolution with detailed analyses of the biophysical properties of IDPs, therefore, could bring new understanding to protein disorder and evolution, as well as indicate novel strategies for engineering proteins for biotechnological and therapeutic purposes. In this chapter, we provide an overview of recent research concerning the natural evolution of IDPs, as well as advances in the use of directed evolution methods to gain insight to IDP function.