Hitoshi Toyota

Experimental rugged fitness landscape in protein sequence space.

The fitness landscape in sequence space determines the process of biomolecular evolution. To plot the fitness landscape of protein function, we carried out in vitro molecular evolution beginning with a defective fd phage carrying a random polypeptide of 139 amino acids in place of the g3p minor coat protein D2 domain, which is essential for phage infection. After 20 cycles of random substitution at sites 12–130 of the initial random polypeptide and selection for infectivity, the selected phage showed a 1.7×104-fold increase in infectivity, defined as the number of infected cells per ml of phage suspension. Fitness was defined as the logarithm of infectivity, and we analyzed (1) the dependence of stationary fitness on library size, which increased gradually, and (2) the time course of changes in fitness in transitional phases, based on an original theory regarding the evolutionary dynamics in Kauffmans n-k fitness landscape model. In the landscape model, single mutations at single sites among n sites affect the contribution of k other sites to fitness. Based on the results of these analyses, k was estimated to be 18–24. According to the estimated parameters, the landscape was plotted as a smooth surface up to a relative fitness of 0.4 of the global peak, whereas the landscape had a highly rugged surface with many local peaks above this relative fitness value. Based on the landscapes of these two different surfaces, it appears possible for adaptive walks with only random substitutions to climb with relative ease up to the middle region of the fitness landscape from any primordial or random sequence, whereas an enormous range of sequence diversity is required to climb further up the rugged surface above the middle region.

How selection affects phenotypic fluctuation

The large degree of phenotypic fluctuation among isogenic cells highlighted by recent studies on stochastic gene expression confers fitness on some individuals through a ‘bet‐hedging’ strategy, when faced with different selective environments. Under a single selective environment, the fluctuation may be suppressed through evolution, as it prevents maintenance of individuals around the fittest state and/or function. However, as fluctuation can increase phenotypic diversity, similar to mutation, it may contribute to the survival of individuals even under a single selective environment. To discuss whether the fluctuation increases over the course of evolution, cycles of mutation and selection for higher GFP fluorescence were carried out in Escherichia coli. Mutant genotypes possessing broad GFP fluorescence distributions with low average values emerged under strong selection pressure. These ‘broad mutants’ appeared independently on the phylogenetic tree and increased fluctuations in GFP fluorescence were attributable to the variance in mRNA abundance. In addition to the average phenotypic change by genetic mutation, the observed increase in phenotypic fluctuation acts as an evolutionary strategy to produce an extreme phenotype under severe selective environments.

Emergence of Polyproline II-Like Structure at Early Stages of Experimental Evolution from Random Polypeptides

To examine whether a primordial functional protein at the early stages of evolution has structural features, we carried out experimental evolution consisting of 25 cycles (generations) of mutation and selection toward DNA-binding function using a random-sequence polypeptide of 139 amino acid residues with no secondary structure as the initial sequence. In each generation, 16 clones were sampled arbitrarily for sequence analysis, and a phylogenetic tree was constructed. Polypeptide evolution proceeded from the initial point on branch I in 2 main directions of branches II and III. The initial and 2 evolved polypeptides (one at the 24th generation on branch III and the other at the 25th generation on branch II) were purified to examine their functional and structural properties. Although binding of the initial polypeptide to the target DNA was not detected by surface plasmon resonance measurements, the 2 evolved polypeptides bound to the DNA with dissociation constants of 1.6 and 1.0 microM, respectively, indicating an increase in affinity during the experimental evolution. Circular dichroism spectra of the evolved polypeptides, but not of the initial polypeptide, showed features characteristic of the polyproline II (PPII)-like structure, a left-handed helical structure commonly found in natural proteins, suggesting that the structure emerged through the experimental evolution. The same structural feature was found in another experimental evolution toward catalytic activity. These results demonstrate that the PPII-like structure is one of the common features that could have appeared in the early evolutionary stages of primordial functional protein.