Abstract
Creating artificial protein families affords new opportunities to explore the determinants of structure and biological function free from many of the constraints of natural selection. We have created an artificial family comprising ˜3,000 P450 heme proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (>73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, is a valuable resource for sequence-structure-function studies. Logistic regression analysis of the multiple sequence alignment identifies key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests possible structural differences between parents CYP102A1 and CYP102A2.
Highlights
Our understanding of how protein sequence relates to structure and function is aided by comparisons of sequences related by evolution [1,2]
We would like to create artificial protein families in order to probe the range of sequence and functional diversity that is compatible with a given structure, free from the constraint of having to function in the narrow context of the host organism
The products of millions of years of divergence and natural selection, protein families contain members that differ at large numbers of amino acids residues
Summary
Our understanding of how protein sequence relates to structure and function is aided by comparisons of sequences related by evolution [1,2]. Having shown this residue to be important to activity in both parents, we chose eight inactive chimeras containing unfavorable 1–8 block combinations to determine whether swapping these positions could ‘‘rescue’’ the activity. Evaluating libraries with randomly sampled crossovers, as was done here, and a recently developed global optimization of recombination sites [47] are both preferred over the SCHEMA profile, which neglects important structural interactions between amino acids distant in the primary sequence Based on this design, three cytochromes P450 were divided into ‘‘building blocks’’ and combinatorially reassembled to yield a library in which 47% of the members fold and correctly bind heme. By this conservative estimate, SCHEMA-guided recombination has increased the frequency of folded chimeras by nearly four orders of magnitude
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