RESUMO
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'.
Assuntos
Proteínas de Fluorescência Verde/química , Hidrozoários/metabolismo , Modelos Moleculares , Engenharia de Proteínas , Algoritmos , Motivos de Aminoácidos , Animais , Simulação por Computador , Bases de Dados de Proteínas , Sistemas Inteligentes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Conformação Proteica , Dobramento de Proteína , Redobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Rapid developments in nucleic acid nanotechnology have enabled the rational design and construction of self-assembling DNA and RNA nanostructures that are highly programmable. We recently developed a replicable single-stranded RNA origami (RNA-OG) technology that allows a long RNA molecule to be programmed to self-assemble into nanostructures of various shapes. Here, we show that such RNA-OG is highly stable in serum/plasma, and we thus exploited its immunostimulatory potential. We demonstrated that the RNA-OG stimulates a potent innate response primarily through a Toll-like receptor 3 (TLR3) pathway. In a murine peritoneal metastatic colon cancer model, intraperitoneally injected RNA-OG induced significant tumor retardation or regression by activating NK- and CD8-dependent antitumor immunity and antagonizing the peritoneal immunosuppressive environment. Unlike polyinosinic/polycytidylic acid (PolyIC), a well-known double-stranded RNA analogue, the RNA-OG treatment did not cause a high level of type-I interferons in the blood nor apparent toxicity upon its systemic administration in the animals. This work establishes the function of RNA-OG as a potent line of TLR3 agonists that are safe and effective for cancer immunotherapy.
Assuntos
Imunoterapia , Nanoestruturas , Animais , Fatores Imunológicos , Camundongos , Nanotecnologia , Poli I-CRESUMO
Since the demonstration of nicotine vaccines as a possible therapeutic intervention for the effects of tobacco smoke, extensive effort has been made to enhance nicotine specific immunity. Linker modifications of nicotine haptens have been a focal point for improving the immunogenicity of nicotine, in which the evaluation of these modifications usually relies on in vivo animal models, such as mice, rats or nonhuman primates. Here, we present two in vitro screening strategies to estimate and predict the immunogenic potential of our newly designed nicotine haptens. One utilizes a competition enzyme-linked immunoabsorbent assay (ELISA) to profile the interactions of nicotine haptens or hapten-protein conjugates with nicotine specific antibodies, both polyclonal and monoclonal. Another relies on computational modeling of the interactions between haptens and amino acid residues near the conjugation site of the carrier protein to infer linker-carrier protein conjugation effect on antinicotine antibody response. Using these two in vitro methods, we ranked the haptens with different linkers for their potential as viable vaccine candidates. The ELISA-based hapten ranking was in an agreement with the results obtained by in vivo nicotine pharmacokinetic analysis. A correlation was found between the average binding affinity (IC50) of the haptens to an anti-Nic monoclonal antibody and the average brain nicotine concentration in the immunized mice. The computational modeling of hapten and carrier protein interactions helps exclude conjugates with strong linker-carrier conjugation effects and low in vivo efficacy. The simplicity of these in vitro screening strategies should facilitate the selection and development of more effective nicotine conjugate vaccines. In addition, these data highlight a previously under-appreciated contribution of linkers and hapten-protein conjugations to conjugate vaccine immunogenicity by virtue of their inclusion in the epitope that binds and activates B cells.
Assuntos
Anticorpos/química , Haptenos/química , Nicotina/química , Animais , Anticorpos/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Linfócitos B/metabolismo , Simulação por Computador , Ensaio de Imunoadsorção Enzimática , Haptenos/imunologia , Humanos , Camundongos Endogâmicos BALB C , Nicotina/imunologia , Nicotina/farmacocinética , Estreptavidina/química , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologiaRESUMO
Design of an efficient fingerprint that detects homologous proteins at distant sequence identity has been a great challenge. This paper proposes a strategy to extract an ideal-like fingerprint with high specificity and sensitivity from a group of sequences related to a fold. The approach is devised based on the assumptions that the critical residues for a protein fold may be conserved in three aspects, i.e. sequence, structure, and intramolecular interaction, and embedded in secondary structures. We hypothesized that the residues satisfying such conditions simultaneously may work as an efficient fingerprint. This idea was tested on protein folds of various classes, such as beta-strand rich, alpha + beta proteins and alpha/beta proteins with discrete sequence similarities. The fingerprint for each fold was generated by selecting the overlapped conserved residues (OCR) from the conserved residues obtained using independent three alignment methods, i.e. multiple sequence alignment, structure-based alignment, and alignment based on the interstrand hydrogen-bonds. The OCR fingerprints showed more than 90% detection efficiency for all the folds tested and were identified to be almost the minimal fingerprints composed of only critical residues. This study is expected to provide an important conceptual improvement in the identification or design of ideal fingerprints for a protein fold.
Assuntos
Sequência Conservada/genética , Proteínas/genética , Proteínas/metabolismo , Algoritmos , Sequência de Aminoácidos , Ligação de Hidrogênio , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Sensibilidade e Especificidade , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Modelos Moleculares , Proteínas/química , Simulação por Computador , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Interações Hidrofóbicas e Hidrofílicas , Mutagênese Sítio-Dirigida , Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Estabilidade Proteica , Proteínas/genética , Eletricidade Estática , TermodinâmicaRESUMO
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.
Assuntos
Arginina/genética , Proteínas de Fluorescência Verde/química , Lisina/genética , Motivos de Aminoácidos , Substituição de Aminoácidos , Arginina/química , Detergentes/química , Proteínas de Fluorescência Verde/genética , Meia-Vida , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Lisina/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Desnaturação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Compostos de Amônio Quaternário/química , Dodecilsulfato de Sódio/química , Espectrometria de Fluorescência , Propriedades de Superfície , Ureia/químicaRESUMO
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.
Assuntos
Aminoácidos/química , Proteínas de Fluorescência Verde/química , Engenharia de Proteínas , Deleção de Sequência , Sequência de Aminoácidos , Sítios de Ligação , Fenômenos Biofísicos , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
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.