Core-Directed Protein Design. I. An Experimental Method for Selecting Stable Proteins from Combinatorial Libraries

Abstract

The design of proteins represents a significant challenge to modern-day structural biology. A major obstacle here is the specification of well-packed hydrophobic cores to drive the folding and stabilization of the target. Computational approaches have been used to alleviate this by testing alternate sequences prior to the production and characterization of a few proteins. Here we present the experimental counterpart of this approach. We selected stable variants from a library of ubiquitin hydrophobic-core mutants as follows. Hexahistidine-tagged proteins were displayed on the surface of phage. These proteinphage were immobilized onto Ni-coated surfaces. The bound fusion-phage were treated with protease to remove unstable or poorly folded proteins. Stable phage fusions were eluted and infected into Escherichia coli, which allowed amplification for further selection, sequencing, or protein expression. Two Ni-derivatized supports were tested: Ni-NTA chips for surface plasmon resonance (SPR) and Ni-NTA agarose beads. SPR had an advantage in that the selection process could be monitored directly. This allowed individual clones and experimental conditions to be tested rapidly prior to preparative panning of the library, which was carried out using Ni-NTA agarose beads. We demonstrate the method by selecting stable core mutants of ubiquitin, the characterization of which is described in the following paper [Finucane, M. D., and Woolfson, D. N. (1999) Biochemistry 38, XXXXX-XXXXX]. As our method selects only on the basis of structure and stability, it will be of use in improving the stabilities and structural specificities of proteins of de novo design, and in establishing rules that link sequence and structure.In de novo protein design attempts are made to construct amino acid sequences that adopt prescribed folds. There are two principal reasons for conducting such studies. First, by designing proteins we test hypotheses of how sequence relates to structure. As sequence data are accumulating much faster than structural information, it is becoming even more critical to determine rules that aid protein structure prediction and modeling. Second, successful protein designs would present routes to new proteins with ranges of structures and functions that extend beyond those found in nature.

Novel folded protein domains generated by combinatorial shuffling of polypeptide segments

“…1A) are not capable of independent folding. We used this region (corresponding to the N-terminal 36-residue fragment) as a template to identify sequences from the E. coli genome that are able to create a folded protease-resistant domain, using proteolysis of phage-displayed proteins as a means of selection (27,36,37).…”

Progress in the development and application of computational methods for probabilistic protein design

“…Sequence features important to folding (and other biological properties) may be explored in a manner decoupled from the evolutionary pressures that determine the sequences of Nature's proteins. Combinatorial protein experiments have been used to identify helical proteins (Rojas et al, 1997;Roy et al, 1997a;Roy et al, 1997b), ubiquitin variants (Finucane et al, 1999), high affinity antibodies (Boder et al, 2000), self-assembled protein monolayers (Xu et al, 2001), proteins with amyloid-like properties (Xu et al, 2001), metal-binding peptides (Case & McLendon, 2000), and stable inter-helical oligomers (Arndt et al, 2000). Several excellent reviews of combinatorial experiments and methodology have appeared recently (Giver & Arnold, 1998;Hoess, 2001;Moffet & Hecht, 2001;Zhao & Arnold, 1997).…”

Combinatorial approaches to protein stability and structure

“…The Hecht group has engineered several generations of four-helix bundles in which each individual position is encoded by a degenerate codon that specifies hydrophobic, hydrophilic or turn residues [12,43]. The resulting polypeptides were then examined for expression and later for well-dispersed 1 H NMR spectra from a On beads or chips (using surface plasmon resonance); incorporation of a specific protease site is often helpful [57][58][59][60] In vitro proteolytic treatment of ribosome-displayed proteins…”

“…However, a number of researchers have shown that proteolysis resistance can be used directly as a marker of foldedness [56]. Woolfson and colleagues fused ubiquitin core variants between phage coat protein pIII and a hexahistidine tag [57]. After binding to a Ni-nitrilotriacetic acid surface, the phage fusions are treated with chymotrypsin and then eluted after washing (Fig.…”