Searching sequence space for protein catalysts

Abstract

Genetic selection was used to explore the probability of finding enzymes in protein sequence space. Large degenerate libraries were prepared by replacing all secondary structure units in a dimeric, helical bundle chorismate mutase with simple binarypatterned modules based on a limited set of four polar and four nonpolar residues. Two-stage in vivo selection yielded catalytically active variants possessing biophysical and kinetic properties typical of the natural enzyme even though Ϸ80% of the protein originates from the simplified modules and >90% of the protein consists of only eight different amino acids. This study provides a quantitative assessment of the number of sequences compatible with a given fold and implicates previously unidentified residues needed to form a functional active site. Given the extremely low incidence of enzymes in completely unbiased libraries, strategies that combine chemical information with genetic selection, like the one used here, may be generally useful in designing novel protein scaffolds with tailored activities. Despite recent progress on the de novo design of structurally defined proteins (1-4), creation of stable scaffolds with tailored enzymatic activities remains an unrealized challenge. Not only is our understanding of the relationship between sequence, structure, and function incomplete, but the requirement for catalysis imposes severe constraints on design. Misplacement of catalytic residues by even a few tenths of an angstrom can mean the difference between full activity and none at all.Direct selection of catalysts from pools of fully randomized polypeptides is a conceivable alternative to de novo design, requiring no foreknowledge of structure or mechanism. An analogous approach has yielded RNA catalysts for a variety of chemical reactions (5). However, a 100-residue protein has 20 100 (1.3 ϫ 10 130 ) possible sequences. Even a library with the mass of the Earth itself-5.98 ϫ 10 27 g-would comprise at most 3.3 ϫ 10 47 different sequences, or a miniscule fraction of such diversity. Unless protein catalysts are unexpectedly abundant and evenly distributed in sequence space, such a strategy will clearly be impractical.Combination of these two approaches represents a potentially attainable middle ground. For example, basic structural information, such as the sequence preferences of helices and sheets or the tendency of hydrophobic residues to be buried in the protein interior, might be used to design focused libraries from which catalysts could be selected with reasonable probability. In fact, binary patterning of polar and nonpolar amino acids (6-8) has been used to generate four-helix bundle proteins (9) that exhibit some native-like properties, including protease resistance and cooperative unfolding (10, 11).Here we show that a combinatorial approach that couples modular design and selection can successfully reproduce a known catalytic activity with an unnatural sequence based on a severely restricted set of building blocks. Specifically, we have constructed large bi...

An Active Enzyme Constructed from a 9-Amino Acid Alphabet

“…In a previous study, we exploited selection in vivo to replace all secondary structure units in an AroQ chorismate mutase (CM) 2 with simple binary-patterned modules of 4 polar and 4 apolar residues (9). However, the total number of amino acid types present in the resulting CMs was 14 since the highly conserved active site amino acids and the loop residues were held constant in the original design.…”

Directed evolution of protein enzymes using nonhomologous random recombination

“…Although active-site residues are expected to be intolerant to substitution, in principle, it is possible for secondary structure elements to be exchanged without loss of function when they play similar structural roles in both contexts and do not form precise and crucial interactions with neighboring residues. Consistent with this hypothesis, libraries of sequences matching only the hydrophobic pattern of the wild-type MjCM have been found to result in functional variants (6). However, fumarase substitutions within predicted helical regions of CM genes were prevalent in libraries only before functional selection.…”

Structural determinants of protein folding

“…The concept of a minimized alphabet for protein folding was subsequently extended to the design of a small b-sheeted protein, the SH3 domain, in which five amino acids (isoleucine, lysine, glutamate, alanine, and glycine) were sufficient for coding the 53-residue domain [218]. In a more recent article by Taylor et al, it was demonstrated that the enzyme AroQ chorismate mutase could be simplified to binary modules of four polar and four nonpolar residues [219]. It was also subsequently demonstrated that a fully functional variant of the enzyme could be constructed with a mere nine-letter amino acid alphabet [220].…”