Abstract
Diversification of protein sequence-structure space is a major concern in protein engineering. Deletion mutagenesis can generate a protein sequence-structure space different from substitution mutagenesis mediated space, but it has not been widely used in protein engineering compared to substitution mutagenesis, because it causes a relatively huge range of structural perturbations of target proteins which often inactivates the proteins. In this study, we demonstrate that, using green fluorescent protein (GFP) as a model system, the drawback of the deletional protein engineering can be overcome by employing the protein structure with high stability. The systematic dissection of N-terminal, C-terminal and internal sequences of GFPs with two different stabilities showed that GFP with high stability (s-GFP), was more tolerant to the elimination of amino acids compared to a GFP with normal stability (n-GFP). The deletion studies of s-GFP enabled us to achieve three interesting variants viz. s-DL4, s-N14, and s-C225, which could not been obtained from n-GFP. The deletion of 191–196 loop sequences led to the variant s-DL4 that was expressed predominantly as insoluble form but mostly active. The s-N14 and s-C225 are the variants without the amino acid residues involving secondary structures around N- and C-terminals of GFP fold respectively, exhibiting comparable biophysical properties of the n-GFP. Structural analysis of the variants through computational modeling study gave a few structural insights that can explain the spectral properties of the variants. Our study suggests that the protein sequence-structure space of deletion mutants can be more efficiently explored by employing the protein structure with higher stability.
Highlights
Protein engineering tools have enabled the generation of valuable proteins with new functions and stabilities and the various fundamental studies on protein structure, function, stability and evolution [1,2]
green fluorescent protein (GFP) Folds Used in this Study Two different GFP sequences, i.e. GFPmut3.1b and GFPhs1, with different stabilities were employed to compare the effect of tolerance to deletion mutation
The GFPhs1 exhibited higher stability and mutational robustness compared to GFPmut3.1b, and their detailed sequences and biophysical properties were described in our previous report [22]
Summary
Protein engineering tools have enabled the generation of valuable proteins with new functions and stabilities and the various fundamental studies on protein structure, function, stability and evolution [1,2]. Deletion mutagenesis is known to induce different structural changes in target proteins from those caused by substitution mutations, which provides important tools for altering protein structures and functions in directions not achievable with substitutions alone [4]. Protein properties such as stability, activity, interface, and size could be changed by the deletion mutagenesis such as N- or C-terminus deletions, loop deletions and random deletions [5,6,7,8,9,10,11]. Despite these potentials of deletion mutagenesis in protein sequence engineering and evolution, the approach has not been popularly used compared to substitution mutagenesis method because deletions lead to relatively large structural perturbations, and so the target proteins are prone to be inactivated [12,13,14]
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