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1.
Science ; 369(6507): 1132-1136, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32855341

RESUMO

Naturally occurring proteins vary the precise geometries of structural elements to create distinct shapes optimal for function. We present a computational design method, loop-helix-loop unit combinatorial sampling (LUCS), that mimics nature's ability to create families of proteins with the same overall fold but precisely tunable geometries. Through near-exhaustive sampling of loop-helix-loop elements, LUCS generates highly diverse geometries encompassing those found in nature but also surpassing known structure space. Biophysical characterization showed that 17 (38%) of 45 tested LUCS designs encompassing two different structural topologies were well folded, including 16 with designed non-native geometries. Four experimentally solved structures closely matched the designs. LUCS greatly expands the designable structure space and offers a new paradigm for designing proteins with tunable geometries that may be customizable for novel functions.


Assuntos
Desenho Assistido por Computador , Engenharia de Proteínas/métodos , Dobramento de Proteína , Estrutura Secundária de Proteína
2.
J Theor Biol ; 505: 110425, 2020 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-32735992

RESUMO

The interaction between the angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a pivotal role in virus entry into the host cells. Since recombinant ACE2 protein has been suggested as an anti-SARS-CoV-2 therapeutic agent, this study was conducted to design an ACE2 protein with more desirable properties. In this regard, the amino acids with central roles in enzymatic activity of the ACE2 were substituted. Moreover, saturation mutagenesis at the interaction interface between the ACE2 and RBD was performed to increase their interaction affinity. The best mutations to increase the structural and thermal stability of the ACE2 were also selected based on B factors and mutation effects. The obtained resulted revealed that the Arg273Gln and Thr445Gly mutation have drastically reduced the binding affinity of the angiotensin-II into the active site of ACE2. The Thr27Arg mutation was determined to be the most potent mutation to increase the binding affinity. The Asp427Arg mutation was done to decrease the flexibility of the region with high B factor. The Pro451Met mutation along with the Gly448Trp mutation was predicted to increase the thermodynamic stability and thermostability of the ACE2. The designed therapeutic ACE2 would have no enzymatic activity while it could bear stronger interaction with Spike glycoprotein of the SARS-CoV-2. Moreover, decreased in vivo enzymatic degradation would be anticipated due to increased thermostability. This engineered ACE2 could be exploited as a novel therapeutic agent against COVID-19 after necessary evaluations.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Desenho de Fármacos , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Engenharia de Proteínas/métodos , Substituição de Aminoácidos , Betacoronavirus/genética , Sítios de Ligação , Evolução Molecular Direcionada , Humanos , Pandemias , Peptidil Dipeptidase A/uso terapêutico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo
3.
BMC Bioinformatics ; 21(1): 275, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32611389

RESUMO

BACKGROUND: Protein engineering has many applications for industry, such as the development of new drugs, vaccines, treatment therapies, food, and biofuel production. A common way to engineer a protein is to perform mutations in functionally essential residues to optimize their function. However, the discovery of beneficial mutations for proteins is a complex task, with a time-consuming and high cost for experimental validation. Hence, computational approaches have been used to propose new insights for experiments narrowing the search space and reducing the costs. RESULTS: In this study, we developed Proteus (an acronym for Protein Engineering Supporter), a new algorithm for proposing mutation pairs in a target 3D structure. These suggestions are based on contacts observed in other known structures from Protein Data Bank (PDB). Proteus' basic assumption is that if a non-interacting pair of amino acid residues in the target structure is exchanged to an interacting pair, this could enhance protein stability. This trade is only allowed if the main-chain conformation of the residues involved in the contact is conserved. Furthermore, no steric impediment is expected between the proposed mutations and the surrounding protein atoms. To evaluate Proteus, we performed two case studies with proteins of industrial interests. In the first case study, we evaluated if the mutations suggested by Proteus for four protein structures enhance the number of inter-residue contacts. Our results suggest that most mutations proposed by Proteus increase the number of interactions into the protein. In the second case study, we used Proteus to suggest mutations for a lysozyme protein. Then, we compared Proteus' outcomes to mutations with available experimental evidence reported in the ProTherm database. Four mutations, in which our results agree with the experimental data, were found. This could be initial evidence that changes in the side-chain of some residues do not cause disturbances that harm protein structure stability. CONCLUSION: We believe that Proteus could be used combined with other methods to give new insights into the rational development of engineered proteins. Proteus user-friendly web-based tool is available at < http://proteus.dcc.ufmg.br >.


Assuntos
Proteínas/química , Interface Usuário-Computador , Algoritmos , Bases de Dados de Proteínas , Muramidase/química , Muramidase/genética , Muramidase/metabolismo , Mutagênese , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismo
4.
Life Sci ; 257: 118052, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32634431

RESUMO

AIMS: Granulocyte colony-stimulating factor (G-CSF) is a cytokine that induces proliferation and differentiation of hematopoietic precursor cells and activation of mature neutrophils. G-CSF is overexpressed in several malignant tumors and blocking its binding to the receptor can lead to significant decrease in tumor growth, vascularization and metastasis. Furthermore, targeting G-CSF receptor has shown therapeutic benefit in other diseases such as rheumatoid arthritis, progressive neurodegenerative disorder and uveitis. Camelid single-chain antibodies (nanobodies) have exceptional properties making them appropriate for tumor imaging and therapeutic application. In this study we aim to use the rational design approach to engineer a previously described G-CSF-R targeting nanobody (VHH1), to improve its affinity toward G-CSF-R. MAIN METHODS: We redesigned the complementary determining region 3 (CDR3) domain of the VHH1 nanobody to mimic G-CSF interaction to its receptor and developed five new engineered nanobodies. Binding affinity of the engineered nanobodies was evaluated by ELISA (Enzyme-linked immunosorbent assay) on NFS60 cells. KEY FINDINGS: Enzyme-linked immunosorbent assay (ELISA) confirmed the specificity of the engineered nanobodies and ELISA-based determination of affinity revealed that two of the engineered nanobodies (1c and 5a) bind to G-CSF-R on the surface of NFS60 cells in a dose-dependent manner and with a higher potency compared to the parental nanobody. SIGNIFICANCE: Additional studies are required to better characterize these nanobodies and assess their interaction with G-CSF-R in vitro and in vivo. These newly developed nanobodies could be beneficial in tumor imaging and therapy and make a basis for development of additional engineered nanobodies.


Assuntos
Fator Estimulador de Colônias de Granulócitos/ultraestrutura , Receptores de Fator Estimulador de Colônias de Granulócitos/imunologia , Anticorpos de Domínio Único/imunologia , Anticorpos , Anticorpos Monoclonais/imunologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia de Afinidade/métodos , Ensaio de Imunoadsorção Enzimática , Fator Estimulador de Colônias de Granulócitos/imunologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Neovascularização Patológica/tratamento farmacológico , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única
6.
Nat Commun ; 11(1): 3314, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620861

RESUMO

The amyloid conformation can be adopted by a variety of sequences, but the precise boundaries of amyloid sequence space are still unclear. The currently charted amyloid sequence space is strongly biased towards hydrophobic, beta-sheet prone sequences that form the core of globular proteins and by Q/N/Y rich yeast prions. Here, we took advantage of the increasing amount of high-resolution structural information on amyloid cores currently available in the protein databank to implement a machine learning approach, named Cordax (https://cordax.switchlab.org), that explores amyloid sequence beyond its current boundaries. Clustering by t-Distributed Stochastic Neighbour Embedding (t-SNE) shows how our approach resulted in an expansion away from hydrophobic amyloid sequences towards clusters of lower aliphatic content and higher charge, or regions of helical and disordered propensities. These clusters uncouple amyloid propensity from solubility representing sequence flavours compatible with surface-exposed patches in globular proteins, functional amyloids or sequences associated to liquid-liquid phase transitions.


Assuntos
Algoritmos , Amiloide/química , Proteínas Amiloidogênicas/química , Modelos Químicos , Peptídeos/química , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Amiloidose/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Aprendizado de Máquina , Peptídeos/metabolismo , Conformação Proteica , Engenharia de Proteínas/métodos , Solubilidade
7.
Proc Natl Acad Sci U S A ; 117(28): 16537-16545, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601191

RESUMO

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction of nif genes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein from Azotobacter vinelandii and Klebsiella oxytoca were found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, from K. oxytoca that confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast, Oryza sativa, Nicotiana tabacum, and Arabidopsis thaliana in Escherichia coli, we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.


Assuntos
Proteínas de Bactérias/genética , Expressão Gênica , Klebsiella oxytoca/enzimologia , Nitrogenase/genética , Engenharia de Proteínas/métodos , Biologia Sintética/métodos , Proteínas de Bactérias/metabolismo , Klebsiella oxytoca/genética , Mitocôndrias/enzimologia , Mitocôndrias/genética , Nitrogenase/metabolismo , Plantas/genética , Plantas/metabolismo , Processamento de Proteína Pós-Traducional
8.
Proc Natl Acad Sci U S A ; 117(25): 14110-14118, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32522868

RESUMO

Interleukin-6 (IL-6) family cytokines signal through multimeric receptor complexes, providing unique opportunities to create novel ligand-based therapeutics. The cardiotrophin-like cytokine factor 1 (CLCF1) ligand has been shown to play a role in cancer, osteoporosis, and atherosclerosis. Once bound to ciliary neurotrophic factor receptor (CNTFR), CLCF1 mediates interactions to coreceptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). By increasing CNTFR-mediated binding to these coreceptors we generated a receptor superagonist which surpassed the potency of natural CNTFR ligands in neuronal signaling. Through additional mutations, we generated a receptor antagonist with increased binding to CNTFR but lack of binding to the coreceptors that inhibited tumor progression in murine xenograft models of nonsmall cell lung cancer. These studies further validate the CLCF1-CNTFR signaling axis as a therapeutic target and highlight an approach of engineering cytokine activity through a small number of mutations.


Assuntos
Subunidade alfa do Receptor do Fator Neutrófico Ciliar/agonistas , Citocinas/metabolismo , Engenharia de Proteínas/métodos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/antagonistas & inibidores , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Receptor gp130 de Citocina/metabolismo , Citocinas/química , Citocinas/genética , Humanos , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Ligantes , Neurônios/metabolismo , Ligação Proteica , Ratos , Transdução de Sinais
9.
PLoS Comput Biol ; 16(6): e1007447, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32511232

RESUMO

The K* algorithm provably approximates partition functions for a set of states (e.g., protein, ligand, and protein-ligand complex) to a user-specified accuracy ε. Often, reaching an ε-approximation for a particular set of partition functions takes a prohibitive amount of time and space. To alleviate some of this cost, we introduce two new algorithms into the osprey suite for protein design: fries, a Fast Removal of Inadequately Energied Sequences, and EWAK*, an Energy Window Approximation to K*. fries pre-processes the sequence space to limit a design to only the most stable, energetically favorable sequence possibilities. EWAK* then takes this pruned sequence space as input and, using a user-specified energy window, calculates K* scores using the lowest energy conformations. We expect fries/EWAK* to be most useful in cases where there are many unstable sequences in the design sequence space and when users are satisfied with enumerating the low-energy ensemble of conformations. In combination, these algorithms provably retain calculational accuracy while limiting the input sequence space and the conformations included in each partition function calculation to only the most energetically favorable, effectively reducing runtime while still enriching for desirable sequences. This combined approach led to significant speed-ups compared to the previous state-of-the-art multi-sequence algorithm, BBK*, while maintaining its efficiency and accuracy, which we show across 40 different protein systems and a total of 2,826 protein design problems. Additionally, as a proof of concept, we used these new algorithms to redesign the protein-protein interface (PPI) of the c-Raf-RBD:KRas complex. The Ras-binding domain of the protein kinase c-Raf (c-Raf-RBD) is the tightest known binder of KRas, a protein implicated in difficult-to-treat cancers. fries/EWAK* accurately retrospectively predicted the effect of 41 different sets of mutations in the PPI of the c-Raf-RBD:KRas complex. Notably, these mutations include mutations whose effect had previously been incorrectly predicted using other computational methods. Next, we used fries/EWAK* for prospective design and discovered a novel point mutation that improves binding of c-Raf-RBD to KRas in its active, GTP-bound state (KRasGTP). We combined this new mutation with two previously reported mutations (which were highly-ranked by osprey) to create a new variant of c-Raf-RBD, c-Raf-RBD(RKY). fries/EWAK* in osprey computationally predicted that this new variant binds even more tightly than the previous best-binding variant, c-Raf-RBD(RK). We measured the binding affinity of c-Raf-RBD(RKY) using a bio-layer interferometry (BLI) assay, and found that this new variant exhibits single-digit nanomolar affinity for KRasGTP, confirming the computational predictions made with fries/EWAK*. This new variant binds roughly five times more tightly than the previous best known binder and roughly 36 times more tightly than the design starting point (wild-type c-Raf-RBD). This study steps through the advancement and development of computational protein design by presenting theory, new algorithms, accurate retrospective designs, new prospective designs, and biochemical validation.


Assuntos
Biologia Computacional , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas p21(ras)/química , Algoritmos , Computadores , Humanos , Interferometria , Lectinas/química , Ligantes , Modelos Estatísticos , Linguagens de Programação , Ligação Proteica , Domínios Proteicos , Software
10.
Science ; 368(6492)2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32409444

RESUMO

De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo-designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs.


Assuntos
Anticorpos Neutralizantes/biossíntese , Biologia Computacional/métodos , Epitopos Imunodominantes/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Vacinas contra Vírus Sincicial Respiratório/química , Vírus Sincicial Respiratório Humano/imunologia , Motivos de Aminoácidos , Humanos , Epitopos Imunodominantes/imunologia , Conformação Proteica , Proteínas Recombinantes de Fusão/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia
11.
Proc Natl Acad Sci U S A ; 117(22): 12041-12049, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32424098

RESUMO

Split inteins are privileged molecular scaffolds for the chemical modification of proteins. Though efficient for in vitro applications, these polypeptide ligases have not been utilized for the semisynthesis of proteins in live cells. Here, we biochemically and structurally characterize the naturally split intein VidaL. We show that this split intein, which features the shortest known N-terminal fragment, supports rapid and efficient protein trans-splicing under a range of conditions, enabling semisynthesis of modified proteins both in vitro and in mammalian cells. The utility of this protein engineering system is illustrated through the traceless assembly of multidomain proteins whose biophysical properties render them incompatible with a single expression system, as well as by the semisynthesis of dual posttranslationally modified histone proteins in live cells. We also exploit the domain swapping function of VidaL to effect simultaneous modification and translocation of the nuclear protein HP1α in live cells. Collectively, our studies highlight the VidaL system as a tool for the precise chemical modification of cellular proteins with spatial and temporal control.


Assuntos
Inteínas/fisiologia , Biossíntese de Proteínas/fisiologia , Engenharia de Proteínas/métodos , Processamento de Proteína/fisiologia , Engenharia Celular/métodos
12.
Mol Cell ; 78(5): 824-834.e15, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32325029

RESUMO

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.


Assuntos
Glicosiltransferases/metabolismo , Polissacarídeos/metabolismo , Engenharia de Proteínas/métodos , Vias Biossintéticas , Membrana Celular/metabolismo , Glicosilação , Glicosiltransferases/química , Glicosiltransferases/fisiologia , Células HEK293 , Células Hep G2 , Humanos , Células K562 , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/metabolismo , N-Acetilgalactosaminiltransferases/fisiologia , Polissacarídeos/química , Proteínas/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(16): 8845-8849, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32253306

RESUMO

The genetic incorporation of noncanonical amino acids (ncAAs) into proteins has been realized in bacteria, yeast, and mammalian cells, and recently, in multicellular organisms including plants and animals. However, the addition of new building blocks to the genetic code of tissues from human origin has not yet been achieved. To this end, we report a self-replicating Epstein-Barr virus-based episomal vector for the long-term encoding of ncAAs in human hematopoietic stem cells and reconstitution of this genetically engineered hematopoietic system in mice.


Assuntos
Aminoácidos/genética , Diferenciação Celular/genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/fisiologia , Engenharia de Proteínas/métodos , Animais , Sangue Fetal/citologia , Técnicas de Transferência de Genes , Código Genético , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Herpesvirus Humano 4/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Plasmídeos/genética , Cultura Primária de Células/métodos , Transfecção/métodos , Quimeras de Transplante , Transplante Heterólogo/métodos
14.
Nat Chem Biol ; 16(7): 725-730, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32284602

RESUMO

Anti-CRISPR (Acr) proteins are powerful tools to control CRISPR-Cas technologies. However, the available Acr repertoire is limited to naturally occurring variants. Here, we applied structure-based design on AcrIIC1, a broad-spectrum CRISPR-Cas9 inhibitor, to improve its efficacy on different targets. We first show that inserting exogenous protein domains into a selected AcrIIC1 surface site dramatically enhances inhibition of Neisseria meningitidis (Nme)Cas9. Then, applying structure-guided design to the Cas9-binding surface, we converted AcrIIC1 into AcrIIC1X, a potent inhibitor of the Staphylococcus aureus (Sau)Cas9, an orthologue widely applied for in vivo genome editing. Finally, to demonstrate the utility of AcrIIC1X for genome engineering applications, we implemented a hepatocyte-specific SauCas9 ON-switch by placing AcrIIC1X expression under regulation of microRNA-122. Our work introduces designer Acrs as important biotechnological tools and provides an innovative strategy to safeguard CRISPR technologies.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , MicroRNAs/genética , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Genoma Humano , Células HEK293 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , MicroRNAs/metabolismo , Modelos Moleculares , Mutagênese Insercional , Neisseria meningitidis/enzimologia , Neisseria meningitidis/genética , Plasmídeos/química , Plasmídeos/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , RNA Guia/genética , RNA Guia/metabolismo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética
15.
Nat Commun ; 11(1): 1858, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313034

RESUMO

Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.


Assuntos
Bactérias/metabolismo , Engenharia de Proteínas/métodos , Ribossomos/química , Biologia Sintética/métodos , Alelos , Sistema Livre de Células , Cristalografia por Raios X , Modelos Moleculares , Modelos Teóricos , Conformação Molecular , Mutação , Peptídeos/química , Peptidil Transferases/química , Plasmídeos/genética , Biossíntese de Proteínas , Proteoma , RNA Mensageiro/genética , RNA Ribossômico/genética , RNA Ribossômico 23S/genética , Thermus thermophilus/química
16.
Nat Commun ; 11(1): 1529, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251274

RESUMO

Inteins are protein segments capable of joining adjacent residues via a peptide bond. In this process known as protein splicing, the intein itself is not present in the final sequence, thus achieving scarless peptide ligation. Here, we assess the splicing activity of 34 inteins (both uncharacterized and known) using a rapid split fluorescent reporter characterization platform, and establish a library of 15 mutually orthogonal split inteins for in vivo applications, 10 of which can be simultaneously used in vitro. We show that orthogonal split inteins can be coupled to multiple split transcription factors to implement complex logic circuits in living organisms, and that they can also be used for the in vitro seamless assembly of large repetitive proteins with biotechnological relevance. Our work demonstrates the versatility and vast potential of an expanded library of orthogonal split inteins for their use in the fields of synthetic biology and protein engineering.


Assuntos
Biotecnologia/métodos , Biblioteca Gênica , Inteínas/genética , Engenharia de Proteínas/métodos , Processamento de Proteína , Clonagem Molecular , Estudos de Viabilidade , Fluorescência , Genes Reporter/genética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Peptídeos , Plasmídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição/genética , Transformação Bacteriana
17.
Proc Natl Acad Sci U S A ; 117(16): 8870-8875, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32245816

RESUMO

The ability to precisely design large proteins with diverse shapes would enable applications ranging from the design of protein binders that wrap around their target to the positioning of multiple functional sites in specified orientations. We describe a protein backbone design method for generating a wide range of rigid fusions between helix-containing proteins and use it to design 75,000 structurally unique junctions between monomeric and homo-oligomeric de novo designed and ankyrin repeat proteins (RPs). Of the junction designs that were experimentally characterized, 82% have circular dichroism and solution small-angle X-ray scattering profiles consistent with the design models and are stable at 95 °C. Crystal structures of four designed junctions were in close agreement with the design models with rmsds ranging from 0.9 to 1.6 Å. Electron microscopic images of extended tetrameric structures and ∼10-nm-diameter "L" and "V" shapes generated using the junctions are close to the design models, demonstrating the control the rigid junctions provide for protein shape sculpting over multiple nanometer length scales.


Assuntos
Modelos Moleculares , Engenharia de Proteínas/métodos , Proteínas/ultraestrutura , Sequências Repetitivas de Aminoácidos/genética , Dicroísmo Circular , Microscopia Eletrônica , Biblioteca de Peptídeos , Conformação Proteica em alfa-Hélice/genética , Dobramento de Proteína , Proteínas/química , Proteínas/genética , Espalhamento a Baixo Ângulo , Difração de Raios X
18.
Nat Chem Biol ; 16(4): 379-382, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32198493

RESUMO

Tyrosine sulfation is an important post-translational modification found in higher eukaryotes. Here we report an engineered tyrosyl-tRNA synthetase/tRNA pair that co-translationally incorporates O-sulfotyrosine in response to UAG codons in Escherichia coli and mammalian cells. This platform enables recombinant expression of eukaryotic proteins homogeneously sulfated at chosen sites, which was demonstrated by expressing human heparin cofactor II in mammalian cells in different states of sulfation.


Assuntos
Engenharia de Proteínas/métodos , Somatomedinas/química , Tirosina/análogos & derivados , Animais , Códon de Terminação/metabolismo , Escherichia coli/metabolismo , Código Genético , Cofator II da Heparina/metabolismo , Humanos , Mamíferos , Processamento de Proteína Pós-Traducional , Proteínas/química , Tirosina/química , Tirosina-tRNA Ligase/metabolismo
19.
Proc Natl Acad Sci U S A ; 117(11): 5791-5800, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123106

RESUMO

Targeted degradation approaches such as proteolysis targeting chimeras (PROTACs) offer new ways to address disease through tackling challenging targets and with greater potency, efficacy, and specificity over traditional approaches. However, identification of high-affinity ligands to serve as PROTAC starting points remains challenging. As a complementary approach, we describe a class of molecules termed biological PROTACs (bioPROTACs)-engineered intracellular proteins consisting of a target-binding domain directly fused to an E3 ubiquitin ligase. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. We then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA) to elicit rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Overall, bioPROTACs are powerful tools for interrogating degradation approaches, target biology, and potentially for making therapeutic impacts.


Assuntos
Antígeno Nuclear de Célula em Proliferação/metabolismo , Engenharia de Proteínas/métodos , Proteólise , Ubiquitina-Proteína Ligases/genética , Sítios de Ligação , Células HEK293 , Humanos , Terapia de Alvo Molecular/métodos , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(11): 5733-5740, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123103

RESUMO

The field of chemical modification of proteins has been dominated by random modification of lysines or more site-specific labeling of cysteines, each with attendant challenges. Recently, we have developed oxaziridine chemistry for highly selective modification of methionine called redox-activated chemical tagging (ReACT) but have not broadly tested the molecular parameters for efficient and stable protein modification. Here we systematically scanned methionines throughout one of the most popular antibody scaffolds, trastuzumab, used for antibody engineering and drug conjugation. We tested the expression, reactivities, and stabilities of 123 single engineered methionines distributed over the surface of the antibody when reacted with oxaziridine. We found uniformly high expression for these mutants and excellent reaction efficiencies with a panel of oxaziridines. Remarkably, the stability to hydrolysis of the sulfimide varied more than 10-fold depending on temperature and the site of the engineered methionine. Interestingly, the most stable and reactive sites were those that were partially buried, presumably because of their reduced access to water. There was also a 10-fold variation in stability depending on the nature of the oxaziridine, which was determined to be inversely correlated with the electrophilic nature of the sulfimide. Importantly, the stabilities of the best analogs were sufficient to support their use as antibody drug conjugates and potent in a breast cancer mouse xenograft model over a month. These studies provide key parameters for broad application of ReACT for efficient, stable, and site-specific antibody and protein bioconjugation to native or engineered methionines.


Assuntos
Aziridinas/análise , Imunoconjugados/química , Metionina/análise , Animais , Antineoplásicos/normas , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Feminino , Humanos , Imunoconjugados/genética , Imunoconjugados/imunologia , Camundongos , Camundongos Nus , Engenharia de Proteínas/métodos , Estabilidade Proteica
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