Cofactor binding and enzymatic activity in an unevolved superfamily of de novo designed 4-helix bundle proteins

Abstract

To probe the potential for enzymatic activity in unevolved amino acid sequence space, we created a combinatorial library of de novo 4-helix bundle proteins. This collection of novel proteins can be considered an "artificial superfamily" of helical bundles. The superfamily of 102-residue proteins was designed using binary patterning of polar and nonpolar residues, and expressed in Escherichia coli from a library of synthetic genes. Sequences from the library were screened for a range of biological functions including heme binding and peroxidase, esterase, and lipase activities. Proteins exhibiting these functions were purified and characterized biochemically. The majority of de novo proteins from this superfamily bound the heme cofactor, and a sizable fraction of the proteins showed activity significantly above background for at least one of the tested enzymatic activities. Moreover, several of the designed 4-helix bundles proteins showed activity in all of the assays, thereby demonstrating the functional promiscuity of unevolved proteins. These studies reveal that de novo proteins-which have neither been designed for function, nor subjected to evolutionary pressure (either in vivo or in vitro)-can provide rudimentary activities and serve as a "feedstock" for evolution.

Figure 1

Absorption spectra for a representative heme binding assay. Heme incubated in a clarified lysate from cells expressing a 3rd generation de novo protein produces a sharp Soret peak, signifying heme binding. The negative controls are heme incubated in buffer, and heme incubated in a clarified lysate from E. coli cells harboring the empty vector. Both negative controls show broad absorption with no significant peaks. The positive control is purified myoglobin, which yields a sharp Soret peak.

Figure 2

Colorimetric assays in 96-well plates. Each column is a different sample performed in triplicate. (A) Heme binding assay. Column 1 shows heme in buffer, yielding a light green/brown color. Column 2 shows heme incubated in lysates from cells harboring the empty vector. These samples also show a light green/brown color, indicating a low background from endogenous E. coli proteins. Column 3 shows heme incubated in lysates from cells expressing a de novo 4-helix bundle protein from the 3rd generation library. The pink/red color indicates heme binding. (B) Peroxidase activity using ABTS as a colorimetic assay. Column 1 shows the background peroxidase activity for heme in buffer. Column 2 shows the peroxidase activity for heme in lysates from cells containing the empty vector. This represents the peroxidase activity of background E. coli proteins. Column 3 shows the activity for heme added to lysates from cells expressing a 4-helix bundle protein from the 3rd generation library. Significant peroxidase activity produces the dark teal color.

Figure 3

Heme binding screen. Spectra of wells in a 96-well plate containing heme added to de novo proteins in E. coli cell lysates. White boxes represent proteins that do not bind heme, pink boxes represent proteins showing moderate heme binding, and red boxes represent proteins showing high heme binding. Box A1 is heme in buffer alone, and Box B1 is heme in a lysate from cells harboring the empty vector. Boxes C1-H1 are for heme added to previously characterized proteins from the 2nd and 3rd generation libraries. The remaining boxes are randomly chosen protein samples from the 3rd generation library.

Figure 4

Frequency of active proteins in a superfamily of de novo 4-helix bundles. Each bar represents a different protein. Activity levels are ranked from lowest to highest. (A) Frequency of heme binding: The y-axis is the ratio of the absorbance at 412 nm (Soret peak) to the reading at 375 nm (local minimum). Gray bars represent proteins with a ratio ≤1. These samples do not show heme binding. Pink bars represent proteins that show heme binding with a ratio >1, and red bars represent proteins that show high heme binding with a ratio >2. Inset shows a magnification to illustrate individual bars. (B) Frequency of peroxidase activity at a single time point. Gray bars represent proteins that do not show peroxidase activity, light teal bars represent proteins with activity above background, and dark teal bars represent proteins with activity two-fold above background. (C) Frequency of esterase activity at a single time point. Gray bars represent proteins that do not show esterase activity, and purple bars represent proteins with activity above background. (D) Frequency of lipase activity at a single time point. Gray bars represent proteins that do not show activity, and orange bars represent proteins that show activity above background. For panels B–D, the background activity is calculated as the average value of the endogenous E. coli proteins (control cells harboring empty vector) across each 96-well plate plus three standard deviations. Insets in panels B–D show the reactions catalyzed by the de novo proteins.

Figure 5

Expression levels of 96 arbitrarily chosen proteins, and the correlation of expression with activity. (A) Proteins were expressed using auto-induction media, and cell lysates were analyzed by SDS-PAGE. Expression is detected by the presence of a strong band at the bottom of each gel (MW 12 kD), as indicated by the arrow. Each lane shows a different protein sample. The first lane in the top row is buffer, and the second lane shows cells harboring the empty vector showing background expression of endogenous E. coli proteins. (B) The correlation of expression and activity. The top row in each chart shows a graphic representation of the protein gels. A single asterisk (*) represents proteins that express at moderate levels and two asterisks (**) represent proteins that express at high levels. The second row shows the heme binding. Pink represents proteins that bind heme and red represents proteins with high heme binding. The third row shows the peroxidase data with light teal boxes representing proteins that have activity above background and dark teal boxes representing proteins having activity two-fold above background. The fourth row and fifth row shows esterase and lipase data, respectively, where shaded boxes indicate proteins that have activity above background. Vertical columns of colored boxes indicate a given protein exhibits several different activities.

Figure 6

Sequences of proteins chosen for purification and biochemical characterization. Top: Design template for 2nd generation proteins and the amino acid sequences of S824 and S836. Bottom: Design template for 3rd generation proteins and the amino acid sequences of WA20, WA32, T-C8 and T-D10. The sequences follow the binary pattern design with red indicating polar residues and yellow indicating nonpolar residues. Turn sequences and charged residues or glycine residues are highlighted in blue.

Figure 7

Kinetic profiles and rate constants calculated by Michaelis-Menten kinetics: (A) Peroxidase activity showing rate of product formation (oxidized ABTS) versus substrate (H₂O₂) concentration. The negative control includes buffer + heme + ABTS. (B) Esterase activity showing rate of p-nitrophenol formation versus substrate (p-nitrophenyl acetate) concentration. (C) Lipase activity showing rate of p-nitrophenol formation versus substrate (p-nitrophenyl palmitate) concentration.