On the conservative nature of intragenic recombination

Abstract

Intragenic recombination rapidly creates protein sequence diversity compared with random mutation, but little is known about the relative effects of recombination and mutation on protein function. Here, we compare recombination of the distantly related ␤-lactamases PSE-4 and TEM-1 to mutation of PSE-4. We show that, among ␤-lactamase variants containing the same number of amino acid substitutions, variants created by recombination retain function with a significantly higher probability than those generated by random mutagenesis. We present a simple model that accurately captures the differing effects of mutation and recombination in real and simulated proteins with only four parameters: (i) the amino acid sequence distance between parents, (ii) the number of substitutions, (iii) the average probability that random substitutions will preserve function, and (iv) the average probability that substitutions generated by recombination will preserve function. Our results expose a fundamental functional enrichment in regions of protein sequence space accessible by recombination and provide a framework for evaluating whether the relative rates of mutation and recombination observed in nature reflect the underlying imbalance in their effects on protein function. directed evolution ͉ mutagenesis ͉ neutrality ͉ lattice proteins ͉ site-directed recombination A major goal in understanding the molecular basis of evolution is to quantitatively describe how effectively mutation and recombination traverse protein sequence space to create new functional proteins (1). Protein sequence distance (measured by counting the number of amino acid substitutions, m, separating two sequences) is a fundamental metric of evolutionary rate and relationships (2), diversity of structure and function (3), and a key variable in protein engineering (4, 5), whereas mutation and recombination are its biochemical cause. Genetic studies (6, 7) and algorithmic inferences from biological sequence data (8-10) have revealed that recombination can occur preferentially within coding sequences, at times with a higher frequency than mutation (11, 12). When sequences encoding divergent but related proteins recombine, large distances may be traveled in sequence space relative to random mutation (13-16) without disturbing function and͞or structure. However, a complete understanding of the underlying relative efficiency of mutation and recombination in accessing nearby or distant regions of sequence space cannot be gained from genomic sequences because these become available only after natural selection has acted.Laboratory (17) and in silico (18) evolution experiments, in contrast, can be used to quantitatively differentiate the effects of mutation or recombination on protein structure and function. By screening or selecting libraries of proteins for retention of parental function and determining the sequences of functional and nonfunctional proteins, one can determine how the retention of function or structure depends on m, the sequence distance. This type of ana...

Biophysics of protein evolution and evolutionary protein biophysics

“…An initial study showed that crossover of two encoding sequences is an effective way of producing a new encoding sequence, suggesting that local sequence patterns are important for determining whether the full protein sequence can fold to a unique structure [324]. This theoretical prediction is consistent with subsequent experiments on b-lactamase indicating that for a given number of amino acid substitutions, recombined variants are much more likely to retain function than variants generated by random point mutations [339]. In another simulation study, evolutionary dynamics that admits both point mutations and recombination leads to a much higher concentration of population in the prototype sequence than if evolution proceeds via point mutations alone, suggesting a significant role of recombination in the prototype-like behaviours of natural proteins [152].…”

Navigating the protein fitness landscape with Gaussian processes

“…Chimeric protein libraries are particularly desirable training sets because they uniformly sample a massive combinatorial space of mutations. In addition, the sequences within chimera libraries have a high probability of functioning (35) and display significant functional diversity (16,36).…”

Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins

“…Although less diverse than the maximally informative chimeras, the single-block swap chimeras still contain on average 15 mutations compared with the closest parent. This is a significant amount of diversity to introduce while still maintaining localization, given that even a single mutation can destroy a protein's ability to fold or function (22).…”

“…SCHEMA recombination offers a systematic method for modular, rational diversity generation that conserves the protein's native structure and function but allows for large changes in sequence (20)(21)(22). SCHEMA divides structurally similar parent proteins into blocks that, when recombined, minimize the libraryaverage disruption of tertiary protein structure (10).…”