Natural history as a predictor of protein evolvability.

Natural selection generally produces specific and efficient enzymes. In contrast, directed evolution experiments usually produce enzyme variants with broadened substrate specificity or enhanced catalytic promiscuity. Some proteins may be more evolvable than others, but few workers consider this problem when choosing starting points for laboratory evolution. Here, we review the variables associated with enzyme evolvability, namely promiscuity and mutational robustness. We present a qualitative model of adaptive evolution and recommend that protein engineers exploit their knowledge of natural history to identify evolvable wild-type proteins. Three examples of 'generalist' proteins that evolved in the laboratory into 'specialists' are described to illustrate the practical utility of this point.

HIV protease-activated molecular switches based on beta-glucuronidase and alkaline phosphatase.

Our long-term goal is to direct the evolution of novel protease variants. To this end we have engineered a new type of protease-activated reporter enzyme. Many protease-activated enzymes evolved in nature, but the introduction of novel regulatory mechanisms into normally unregulated enzymes poses a difficult design challenge. Random Elongation Mutagenesis [1] was used to fuse the p6 peptide, which is recognized and cleaved by HIV protease, and twelve random sequence amino acids to the C-termini of beta-glucuronidase (GUS) and alkaline phosphatase (AP). The resulting GUS-p6-(NNN)12 and AP-p6-(NNN)12 libraries were expressed in E. coli and screened for clones that were inactivated by the C-terminal extension (tail). The inactivated clones were co-expressed with HIV protease, and those that were re-activated were isolated. The AP and GUS activities of the most responsive clones were each >3.5-fold higher when co-expressed with HIV protease, and this activation is correlated with in vivo proteolysis. It should be possible to generalize this strategy to different reporter enzymes, different target proteases, and perhaps to other types of protein-modifying enzymes.

Diversification and specialization of HIV protease function during in vitro evolution.

Our goal is to understand how enzymes adapt to utilize novel substrates. We and others have shown that directed evolution tends to generate enzyme variants with broadened substrate specificity. Broad-specificity enzymes are generally deleterious to living cells, so this observed trend might be an artifact of the most commonly employed high throughput screens. Here, we demonstrate a more natural and effective screening strategy for directed evolution. The gene encoding model enzyme HIV protease was randomly mutated, and the resulting library was expressed in Escherichia coli cells to eliminate cytotoxic broad-specificity variants. The surviving variants were screened for clones with activity against a reporter enzyme. The wild-type human immunodeficiency virus type I protease (HIV PR) is cytotoxic and exhibits no detectable activity in reactions with beta-galactosidase (BGAL). In contrast, the selected variants were nontoxic and exhibited greater activity and specificity against BGAL than did the wild-type HIV PR in reactions with any substrate. A single round of whole gene random mutagenesis and conventional high-throughput screening does not usually effect complete inversions of substrate specificity. This suggests that a combination of positive and purifying selection engenders more rapid adaptation than positive selection alone.

Rational design of p53, an intrinsically unstructured protein, for the fabrication of novel molecular sensors.

The dominant paradigm of protein engineering is structure-based site-directed mutagenesis. This rational approach is generally more effective for the engineering of local properties, such as substrate specificity, than global ones such as allostery. Previous workers have modified normally unregulated reporter enzymes, including beta-galactosidase, alkaline phosphatase, and beta-lactamase, so that the engineered versions are activated (up to 4-fold) by monoclonal antibodies. A reporter that could easily be "reprogrammed" for the facile detection of novel effectors (binding or modifying activities) would be useful in high throughput screens for directed evolution or drug discovery. Here we describe a straightforward and general solution to this potentially difficult design problem. The transcription factor p53 is normally regulated by a variety of post-translational modifications. The insertion of peptides into intrinsically unstructured domains of p53 generated variants that were activated up to 100-fold by novel effectors (proteases or antibodies). An engineered p53 was incorporated into an existing high throughput screen for the detection of human immunodeficiency virus protease, an arbitrarily chosen novel effector. These results suggest that the molecular recognition properties of intrinsically unstructured proteins are relatively easy to engineer and that the absence of crystal structures should not deter the rational engineering of this class of proteins.