Selection and structural analysis of de novo proteins from an α3β3 genetic library

Abstract

The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding a3b3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP-based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8-anilino-1-naphthalenesulfonate (ANS) binding assay, and analytical size-exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both a-helix and b-sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten-globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three a-helices and three b-strands as intended by its design. Thus, it would appear that artificially generated a3b3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.

Comparative characterization of random‐sequence proteins consisting of 5, 12, and 20 kinds of amino acids

“…[9][10][11] Jumawid et al have constructed a3b3 de novo proteins through binary combination of simplified hydrophobic (Val, Ile, and Leu) and hydrophilic (Ala, Glu, Lys, and Thr) amino acid sets. 12 These findings all support the view that the full amino acid alphabet set is not essential for protein folding.…”

Amino acid alphabet reduction preserves fold information contained in contact interactions in proteins

“…In a subsequent study, the substitution of a lysine in a position normally occupied by the hydrophobic amino acids (i.e., the PHPKPHP pattern) for the edge strands in a 6‐strand beta sheet protein discouraged oligomerization due to the need for lysine to be water accessible, forming instead monomeric six‐stranded beta‐sheet proteins. An attempt to design a mixed alpha‐beta (α3β3) protein scaffold de novo also utilized binary H/P patterning using the amino acids AEKT for polar positions and ILV for hydrophobic positions. While this alphabet is distinct from that used by Hecht's group, the two groups also occur as distinct subgroups in the optimized 2‐letter alphabet reduction derived in this work (Fig.…”

Artificial proteins from combinatorial approaches

“…Mix of 5-10 amino acids secondary structure modules (a-helix, b-strand, turn) polymerized in 100 residues polypeptides Binary patterning: arrangement of polar and nonpolar residues to favor a-helix or b-sheet α-helix β-strand loop [31,32] [ 34,35] Mix and polymerization of natural structural elements (a-helix, b-sheet, loop) Secondary structure shuffling Secondary structure elements extracted from natural existing proteins [21], [23], [37,38] Four-helix bundle Binary patterning C N [33], [36] Libraries of proteins with internal repeats Symmetric scaffolds:…”

“…A combinatorial approach coupling the use of binary patterning and simplified amino acid alphabet has been used to design a library of reduced diversity but high folding propensity encoding de novo proteins made of three helices and three strands (a3b3) [38]. Folded proteins from the library were screened using a GFP fusion system and were clearly demonstrated to have a large fraction of the sequence with a well-folded unique structure.…”