ABSTRACT
In this chapter, we will describe recent findings on the correlation between stability and the soluble, functional expression of proteins in cells. We will then discuss how stability influences the evolutionary dynamics of enzyme function. Finally, we will point out strategies to increase the evolvability of enzymes (i.e., the ability of an enzyme to acquire new functions, such as altered or enhanced catalytic activities, and thereby improve our ability to generate new biocatalysts). 4.2 Protein Stability and the Evolution of
Protein stability is broadly defined and often ambiguous. Thermodynamic stability (DGU-N: the difference in free energy
between the unfolded and native states) is often used to describe the relationship between protein expression and stability in evolution (Bloom and Arnold, 2009; Bershtein et al., 2006; DePristo et al., 2005; Tokuriki and Tawfik, 2009; Zeldovich et al., 2007). In the present chapter, it is also used to describe the evolutionary dynamics of enzymes (Fig. 4.1a). However, the thermodynamic stability model may only apply to certain kind of proteins, such as small proteins that fold quickly (Sanchez-Ruiz, 2010). Protein folding within the cellular milieu is a very complicated event and the simple thermodynamic model does not take into account the full complexity of the relationship between stability and enzyme fitness. Indeed, recent examples have shown that thermodynamic stability does not necessarily correlate with the protein abundance in the cell, or with organismal fitness (Bershtein et al., 2012; Bershtein et al., 2013; Diaz et al., 2011; Wyganowski et al., 2013). For example, the biophysical analysis of over 30 variants of bacterial phosphotriesterase (PTE) showed no correlation between in vitro thermal or chemical stability and soluble expression level in the cell, although, the stability of some intermediate folding states can correlate with in vivo expression levels (Wyganowski et al., 2013). These observations suggest that kinetic stability, which relates to the energy levels of folding intermediates and controls the accessibility of pathways leading to aggregation or degradation, plays a significant role in determining the level of functional protein, particularly for larger and more complex proteins (Dobson 2003; Hartl and Hayer-Hartl, 2009; Sanchez-Ruiz, 2010). Unlike thermodynamic stability, kinetic stability is difficult to quantify as the energy levels of many different intermediates in a complex network of possible folding pathways can influence it, and subsequently, the fate of the protein (Fig. 4.1b). Thus, the relative importance of kinetic stability versus thermodynamic stability in protein folding still remains unclear. However, as most biophysical studies to date have focused on “well-behaved,” relatively small proteins, it is likely that thermody-namically driven folding is overrepresented in the literature. In the future, we may discover that the kinetic stability model is actually more applicable for a greater number of proteins.In any case, whether in vivo expression is determined by thermodynamic or kinetic stability, the concentration of soluble and functional enzyme [E]0 is directly related to its fitness. Therefore,
modulating stability in enzyme engineering has to focus on maintaining or increasing [E]0. It is crucial to determine whether a target enzyme folds properly or not during in vivo expression, as this will influence which experimental strategies will be chosen to improve stability and thereby, the evolvability of the targeted enzyme.
