Redesigning the type II' ß-turn in green fluorescent protein to type I': implications for folding kinetics and stability.

Both Type I' and Type II' ß-turns have the same sense of the ß-turn twist that is compatible with the ß-sheet twist. They occur predominantly in two residue ß-hairpins, but the occurrence of Type I' ß-turns is two times higher than Type II' ß-turns. This suggests that Type I' ß-turns may be more stable than Type II' ß-turns, and Type I' ß-turn sequence and structure can be more favorable for protein folding than Type II' ß-turns. Here, we redesigned the native Type II' ß-turn in GFP to Type I' ß-turn, and investigated its effect on protein folding and stability. The Type I' ß-turns were designed based on the statistical analysis of residues in natural Type I' ß-turns. The substitution of the native "GD" sequence of i+1 and i+2 residues with Type I' preferred "(N/D)G" sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' ß-turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' ß-turns in natural ß-hairpins can be further optimized by converting the sequence to Type I'.

A variant of green fluorescent protein exclusively deposited to active intracellular inclusion bodies.

BACKGROUND: Inclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Recently, some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science and nanotechnology. RESULTS: In this study, we characterized the IBs of DL4, a deletion variant of green fluorescent protein which forms active intracellular aggregates. The DL4 proteins expressed in Escherichia coli were exclusively deposited to IBs, and the IBs were estimated to be mostly composed of active proteins. The spectral properties and quantum yield of the DL4 variant in the active IBs were almost same with those of its native protein. Refolding and stability studies revealed that the deletion mutation in DL4 didn't affect the folding efficiency of the protein, but destabilized its structure. Analyses specific for amyloid-like structures informed that the inner architecture of DL4 IBs might be amorphous rather than well-organized. The diameter of fluorescent DL4 IBs could be decreased up to 100-200 nm by reducing the expression time of the protein in vivo. CONCLUSIONS: To our knowledge, DL4 is the first GFP variant that folds correctly but aggregates exclusively in vivo without any self-aggregating/assembling tags. The fluorescent DL4 IBs have potentials to be used as fluorescent biomaterials. This study also suggests that biologically active IBs can be achieved through engineering a target protein itself.

Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library.

DNA polymerases the key enzymes for several biotechnological applications. Obviously, nature has not evolved these enzymes to be compatible with applications in biotechnology. Thus, engineering of a natural scaffold of DNA polymerases may lead to enzymes improved for several applications. Here, we investigated a two-step approach for the design and construction of a combinatorial library of mutants of KlenTaq DNA polymerase. First, we selected amino acid sites for saturation mutagenesis that interact with the primer/template strands or are evolutionarily conserved. From this library, we identified mutations that little interfere with DNA polymerase activity. Next, these functionally active mutants were combined randomly to construct a second library with enriched sequence diversity. We reasoned that the combination of mutants that have minuscule effect on enzyme activity and thermostability, will result in entities that have an increased mutation load but still retain activity. Besides activity and thermostability, we screened the library for entities with two distinct properties. Indeed, we identified two different KlenTaq DNA polymerase variants that either exhibit increased mismatch extension discrimination or increased reverse transcription PCR activity, respectively.

Protegrin-1 cytotoxicity towards mammalian cells positively correlates with the magnitude of conformational changes of the unfolded form upon cell interaction.

Porcine protegrin-1 (PG-1) is a broad-spectrum antimicrobial peptide (AMP) with potent antimicrobial activities. We produced recombinant PG-1 and evaluated its cytotoxicity toward various types of mammalian cell lines, including embryonic fibroblasts, retinal cells, embryonic kidney cells, neuroblastoma cells, alveolar macrophage cells, and neutrophils. The sensitivity of the different mammalian cells to cytotoxic damage induced by PG-1 differed significantly among the cell types, with retinal neuron cells and neutrophils being the most significantly affected. A circular dichroism analysis showed there was a precise correlation between conformational changes in PG-1 and the magnitude of cytotoxicity among the various cell type. Subsequently, a green fluorescent protein (GFP) penetration assay using positively charged GFPs indicated there was a close correlation between the degree of penetration of charged GFP into cells and the magnitude of PG-1 cytotoxicity. Furthermore, we also showed that inhibition of the synthesis of anionic sulphated proteoglycans on the cell surface decreases the cytotoxic damage induced by PG-1 treatment. Taken together, the observed cytotoxicity of PG-1 towards different membrane surfaces is highly driven by the membrane's anionic properties. Our results reveal a possible mechanism underlying cell-type dependent differences in cytotoxicity of AMPs, such as PG-1, toward mammalian cells.

Modulation of protein stability and aggregation properties by surface charge engineering.

An attempt to alter protein surface charges through traditional protein engineering approaches often affects the native protein structure significantly and induces misfolding. This limitation is a major hindrance in modulating protein properties through surface charge variations. In this study, as a strategy to overcome such a limitation, we attempted to co-introduce stabilizing mutations that can neutralize the destabilizing effect of protein surface charge variation. Two sets of rational mutations were designed; one to increase the number of surface charged amino acids and the other to decrease the number of surface charged amino acids by mutating surface polar uncharged amino acids and charged amino acids, respectively. These two sets of mutations were introduced into Green Fluorescent Protein (GFP) together with or without stabilizing mutations. The co-introduction of stabilizing mutations along with mutations for surface charge modification allowed us to obtain functionally active protein variants (s-GFP(+15-17) and s-GFP(+5-6)). When the protein properties such as fluorescent activity, folding rate and kinetic stability were assessed, we found the possibility that the protein stability can be modulated independently of activity and folding by engineering protein surface charges. The aggregation properties of GFP could also be altered through the surface charge engineering.

A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein.

Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering.

Deletional protein engineering based on stable fold.

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.

Conjugation of proteins by installing BIO-orthogonally reactive groups at their N-termini.

N-terminal site-specific modification of a protein has many advantages over methods targeting internal positions, but it is not easy to install reactive groups onto a protein in an N-terminal specific manner. We here report a strategy to incorporate amino acid analogues specifically in the N-terminus of a protein in vivo and demonstrate it by preparing green fluorescent protein (GFP) having bio-orthogonally reactive groups at its N-terminus. In the first step, GFP was engineered to be a foldable, internal methionine-free sequence via the semi-rational mutagenesis of five internal methionine residues and the introduction of mutations for GFP folding enhancement. In the second step, the N-terminus of the engineered protein was modified in vivo with bio-orthogonally functional groups by reassigning functional methionine surrogates such as L-homopropargylglycine and L-azidohomoalanine into the first methionine codon of the engineered internal methionine-free GFP. The N-terminal specific incorporation of unnatural amino acids was confirmed by ESI-MS analysis and the incorporation did not affect significantly the specific activity, refolding rate and folding robustness of the protein. The two proteins which have alkyne or azide groups at their N-termini were conjugated each other by bio-orthogonal Cu(I)-catalyzed click chemistry. The strategy used in this study is expected to facilitate bio-conjugation applications of proteins such as N-terminal specific glycosylation, labeling of fluorescent dyes, and immobilization on solid surfaces.