Rapid mapping of protein functional epitopes by combinatorial alanine scanning

Abstract

A combinatorial alanine-scanning strategy was used to determine simultaneously the functional contributions of 19 side chains buried at the interface between human growth hormone and the extracellular domain of its receptor. A phage-displayed protein library was constructed in which the 19 side chains were preferentially allowed to vary only as the wild type or alanine. The library pool was subjected to binding selections to isolate functional clones, and DNA sequencing was used to determine the alanine͞ wild-type ratio at each varied position. This ratio was used to calculate the effect of each alanine substitution as a change in free energy relative to that of wild type. Only seven side chains contribute significantly to the binding interaction, and these conserved residues form a compact cluster in the human growth hormone tertiary structure. The results were in excellent agreement with free energy data previously determined by conventional alanine-scanning mutagenesis and suggest that this technology should be useful for analyzing functional epitopes in proteins.

Rational disruption of the oligomerization of the mini‐ferritin E. coli DPS through protein‐protein interface mutation

“…9,10 Alanine shaving, where individual side changes are conceptually shaved to a methyl residuum and where the stabilities of the resulting mutants are determined with respect to wild type, is the most common method to identify hot spot residues. [11][12][13] This study uses alanine shaving to identify hot spots in a mini-ferritin nano-cage protein.…”

Novel Bcl-2 Homology-3 Domain-like Sequences Identified from Screening Randomized Peptide Libraries for Inhibitors of the Pro-survival Bcl-2 Proteins

“…Each library was subjected to three rounds of panning versus purified Bcl-x L or M2 anti-FLAG antibody (Sigma), then approximately 50 individual positive-binding clones were sequenced as described (20,21). To calculate the functional ratio, the ratio of wild-type clones:mutant clones for each possible codon in the Bcl-x L selection was divided by the corresponding ratio from the anti-FLAG selection (19,22). The relative affinity (IC 50 ) of selected clones was determined by phage competition enzyme-linked immunosorbent assay as described previously (16).…”

“…To investigate this hypothesis, we performed a shotgun alanine scan on the BimBH3 sequence, which, unlike site-specific alanine scanning, allows the importance of a range of residues across a peptide sequence to be probed simultaneously (19,22,37). To achieve this we displayed a 26-residue sequence encompassing a FLAG-tagged BimBH3 domain on the surface of M13 phage particles as a fusion to the minor coat protein gene 3.…”

“…To achieve this we displayed a 26-residue sequence encompassing a FLAG-tagged BimBH3 domain on the surface of M13 phage particles as a fusion to the minor coat protein gene 3. Two libraries were then created in which either residues 1-13 (Library 1) or 14 -26 (Library 2) were all simultaneously mutated using oligonucleotides with degenerate codons possessing limited diversity such that each codon could only encode the wild-type residue at that position, alanine (x1), or depending on the codon employed, up to two other residues (x2 and x3; see supplemental Table 1) (19). These libraries were then panned against Bcl-x L as well as the anti-FLAG antibody to allow a "function ratio" to be calculated as described under "Experimental Procedures."…”

“…These libraries were then panned against Bcl-x L as well as the anti-FLAG antibody to allow a "function ratio" to be calculated as described under "Experimental Procedures." Function ratios significantly greater than or less than 1 indicate a deleterious or beneficial mutation, respectively (19,22,37).…”