Physicochemical properties of molecules can be linked directly to evolutionary fates of a population in a quantitative and predictive manner.Reversible- and irreversible-folding pathways must be accounted for to accurately determine in vitro kinetic parameters (KM and kcat) at temperatures or conditions in which a significant fraction of free enzyme is unfolded.In vivo population dynamics can be reproduced using in vitro physicochemical measurements within a model that imposes an activity threshold above which there is no added fitness benefit.
In nature, evolution occurs through the continuous adaptation of a population to its environment. The success or failure of organisms during adaptation is based on changes in molecular structure that give rise to changes in fitness that dictate evolutionary fates within a population. Although the conceptual link between genotype, phenotype, and fitness is clear, the ability to relate these complex adaptive landscapes in a quantitative manner remains difficult (Kacser and Burns, 1981; Dykhuizen et al, 1987; Weinreich et al, 2006). Dean and Thornton (2007) coined the term ‘functional synthesis' to capture the synergy between evolutionary and molecular biology to address important questions such as the evolution of complexity. The ‘functional synthesis,' in its most fully realized form, is an integrated systems biology approach to evolutionary dynamics that links physicochemical properties of molecules to evolutionary fates in a quantitative and predictive manner.
Functional synthesis flourishes in an experimental framework that allows investigators to directly link population dynamics (fitness) to changes in molecular function that result from alterations at the nucleotide level. The ‘weak link' approach was developed to tightly couple adaptive changes within the genome to changes in fitness and provide a population-based approach that can be used to examine alterations in function and fitness at the level of atomic structure and function (Counago and Shamoo, 2005; Counago et al, 2006). A homologous recombination strategy was used to replace the chromosomal copy of the essential adenylate kinase gene (adk) of the thermophilic bacterium Geobacillus stearothermophilus with that of the mesophile Bacillus subtilis. Recombinant G. stearothermophilus cells that expressed only B. subtilis adenylate kinase (AKBSUB) were unable to grow at temperatures higher than 55°C because of heat inactivation of the mesophilic enzyme and consequent disruption of adenylate homeostasis (Counago and Shamoo, 2005). Continuously growing populations of bacteria were then subjected to selection at increasing temperatures (from 55 to 70°C) that favor changes in the one gene not adapted for thermostability, adk. During the course of selection, the population was sampled and intermediates of adaptation were observed as mutations to adk. The first mutant to reach fixation was a single mutation AKBSUB Q199R (the glutamine at position 199 replaced with arginine). AKBSUB Q199R was eventually replaced at 62–63°C by five double mutants that arose nearly simultaneously within the population and share AKBSUB Q199R as their progenitor (Figure 4C). Changes to AK activity and thermal stability that resulted from mutation had direct consequences for cellular fitness and, therefore, met our goal for an experimental system that allows us to develop and test models for quantitative molecular evolution. These enzyme activities and stabilities were examined to determine how the mutant populations traversed the adaptive landscape to increased fitness (Counago et al, 2006).
We found that reversible- and irreversible-folding pathways as well as a ‘physiological threshold' above which fitness changes are minimal are necessary to reproduce the in vivo evolutionary fates of the population. Protein-folding parameters must be accounted for to accurately determine in vitro kinetic parameters (KM and kcat) at temperatures in which a significant fraction of free enzyme is unfolded (Scheme I and Equation 1).
Thermostability was assayed using differential scanning calorimetry (DSC) (Figure 4A) and the fraction of unfolded protein (YU) was then extended to accurately predict the extent of stabilization, shift in Tm, in the presence of ligand. The kinetic parameters determined at specific temperatures were then used to construct a temperature-dependent formulation of Equation (1) to model in vitro activity at any given ATP concentration and any temperature (Figure 4B).
Here, we have modeled fitness as a function of in vitro enzyme activity, which is a product of both activity and stability, and the application of a threshold that provides an upper limit on fitness. We hypothesize that an activity threshold exists above which no added fitness benefit is attained (the ‘physiological threshold'). However, as activity falls below this threshold, AK becomes rate limiting and fitness is negatively affected. The experimentally observed rise and fall of mutant alleles is shown in Figure 4C, whereas those predicted from our in vitro model are shown as Figure 4D. This model can successfully reproduce frequencies of mutants in a polymorphic population, including the transient success of three minor mutants and order of disappearance from the population, given only in vitro data and allowing for the activity threshold to be fit to the observed outcomes (Figure 4D). An appealing aspect of our fitness function is that it permits an evaluation of specific and quantitative aspects of protein stability and activity relative to evolutionary fates.
In vivo, diversity within a population is generated by a variety of mechanisms that span single nucleotide changes to genome-wide rearrangements and horizontal gene transfer. However, changes are generated within an organism, it is the physicochemical characteristics of the resulting macromolecules and their resultant changes in the fitness of the organism that are the ‘grist for the mill' of natural selection. Recent work has shown that adaptability can be facilitated by the accumulation of near neutral or even modestly destabilizing mutations that provide more possibilities for success. Chaperones have an important function in buffering biological systems against these destabilizing mutations as well as mistakes in translation that lead to polymorphic populations and have been shown to increase rates of adaptation (Rutherford, 2003; Drummond and Wilke, 2008; Tokuriki and Tawfik, 2009a). Thus, adaptation through protein evolution is circumscribed by protein stability. As most mutational events will be destabilizing (Tokuriki and Tawfik, 2009b), higher mutation rates can lead to decreases in fitness eventually leading to extinction (Zeldovich et al, 2007; Chen and Shakhnovich, 2009). Although our system links the physicochemical properties of adaptive changes that increase stability, the principles apply equally to those changes that might decrease stability of the ensemble either through mutation or translational errors (Drummond and Wilke, 2008). Thus, regardless of how protein diversity is generated, evolutionary dynamics will likely be strongly coupled to stability and function.
Systems biology can offer a great deal of insight into evolution by quantitatively linking complex properties such as protein structure, folding, and function to the fitness of an organism. Although the link between diseases such as Alzheimer's and misfolding is well appreciated, directly showing the importance of protein folding to success in evolution has been more difficult. We show here that predicting success during adaptation can depend critically on enzyme kinetic and folding models. We used a ‘weak link' method to favor mutations to an essential, but maladapted, adenylate kinase gene within a microbial population that resulted in the identification of five mutants that arose nearly simultaneously and competed for success. Physicochemical characterization of these mutants showed that, although steady-state enzyme activity is important, success within the population is critically dependent on resistance to denaturation and aggregation. A fitness function based on in vitro measurements of enzyme activity, reversible and irreversible unfolding, and the physiological context reproduces in vivo evolutionary fates in the population linking organismal adaptation to its physical basis.