Rheostats and Toggle Switches for Modulating Protein Function

Abstract

The millions of protein sequences generated by genomics are expected to transform protein engineering and personalized medicine. To achieve these goals, tools for predicting outcomes of amino acid changes must be improved. Currently, advances are hampered by insufficient experimental data about nonconserved amino acid positions. Since the property “nonconserved” is identified using a sequence alignment, we designed experiments to recapitulate that context: Mutagenesis and functional characterization was carried out in 15 LacI/GalR homologs (rows) at 12 nonconserved positions (columns). Multiple substitutions were made at each position, to reveal how various amino acids of a nonconserved column were tolerated in each protein row. Results showed that amino acid preferences of nonconserved positions were highly context-dependent, had few correlations with physico-chemical similarities, and were not predictable from their occurrence in natural LacI/GalR sequences. Further, unlike the “toggle switch” behaviors of conserved positions, substitutions at nonconserved positions could be rank-ordered to show a “rheostatic”, progressive effect on function that spanned several orders of magnitude. Comparisons to various sequence analyses suggested that conserved and strongly co-evolving positions act as functional toggles, whereas other important, nonconserved positions serve as rheostats for modifying protein function. Both the presence of rheostat positions and the sequence analysis strategy appear to be generalizable to other protein families and should be considered when engineering protein modifications or predicting the impact of protein polymorphisms.

RheoScale: A tool to aggregate and quantify experimentally determined substitution outcomes for multiple variants at individual protein positions

“…To develop the RheoScale calculator, we used both simulated data (Figure and ) and experimental data previously published for the LacI/GalR paralogs (Meinhardt et al., ). We further tested the calculator on published data for pyruvate kinase, an enzyme for which multiple experimental functional parameters are available (Ishwar et al., ; Tang et al., ), and three TIM barrel orthologs that were characterized using deep mutational scanning (Chan et al., ).…”

“…Both of these expected scenarios tempt investigators to extrapolate the outcome from one variant (or a few) to the overall role of the position being substituted (neutral or toggle). However, this inference is not appropriate for a third class of protein positions: We observed this class in an experimental study of select nonconserved positions in LacI/GalR homologs (Meinhardt & Swint‐Kruse, ; Meinhardt et al., ; Tungtur, Egan, & Swint‐Kruse, ; Tungtur, Meinhardt, & Swint‐Kruse, ; Tungtur, Parente, & Swint‐Kruse, ). Many substitutions did alter protein function which indicated that, despite their nonconservation, these positions were important (and thus, not neutral).…”

“…Surprisingly, the outcomes did not correlate with either the common biochemical properties of amino acids (e.g., hydrophobicity, charge, etc.) or with the evolutionary frequencies observed for each type of amino acid (Meinhardt et al., ).…”

“…Example score calculations using data from a study of variants in LacI/GalR homologs (Meinhardt et al., ). (A) Representative experimental data are shown for 9 amino acid variants at position 58 in the dimeric version of LacI (gray bars).…”

AlloRep: A Repository of Sequence, Structural and Mutagenesis Data for the LacI/GalR Transcription Regulators

“…Another ~1100 variants are from in vivo and biochemical studies of LacI/GalR chimeras [21,26–29]. The remaining variants are from biochemical studies of LacI, PurR, GalR, CytR, FruR and CcpA.…”