Positive Charges Determine the Topology and Functionality of the Transmembrane Domain in the Chloroplastic Outer Envelope Protein Toc34

The chloroplastic outer envelope protein Toc34 is inserted into the membrane by a COOH-terminal membrane anchor domain in the orientation Ncyto-Cin. The insertion is independent of ATP and a cleavable transit sequence. The cytosolic domain of Toc34 does not influence the insertion process and can be replaced by a different hydrophilic reporter peptide. Inversion of the COOH-terminal, 45-residue segment, including the membrane anchor domain (Toc34Cinv), resulted in an inverted topology of the protein, i.e., Nin-Ccyto. A mutual exchange of the charged amino acid residues NH2- and COOH-proximal of the hydrophobic α-helix indicates that a double-positive charge at the cytosolic side of the transmembrane α-helix is the sole determinant for its topology. When the inverted COOH-terminal segment was fused to the chloroplastic precursor of the ribulose-1,5-bisphosphate carboxylase small subunit (pS34Cinv), it engaged the transit sequence–dependent import pathway. The inverted peptide domain of Toc34 functions as a stop transfer signal and is released out of the outer envelope protein translocation machinery into the lipid phase. Simultaneously, the NH2-terminal part of the hybrid precursor remained engaged in the inner envelope protein translocon, which could be reversed by the removal of ATP, demonstrating that only an energy-dependent force but no further ionic interactions kept the precursor in the import machinery.

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Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope.

The Journal of Cell Biology ◽

10.1083/jcb.134.2.315 ◽

1996 ◽

Vol 134 (2) ◽

pp. 315-327 ◽

Cited By ~ 112

Author(s):

Y Ma ◽

A Kouranov ◽

S E LaSala ◽

D J Schnell

Keyword(s):

Outer Membrane ◽

Protein Import ◽

Small Subunit ◽

Chloroplast Envelope ◽

Stable Complex ◽

Nucleoside Triphosphate ◽

Cross Linking ◽

Outer Envelope ◽

Bisphosphate Carboxylase ◽

Precursor Proteins

The interactions of precursor proteins with components of the chloroplast envelope were investigated during the early stages of protein import using a chemical cross-linking strategy. In the absence of energy, two components of the outer envelope import machinery, IAP86 and IAP75, cross-linked to the transit sequence of the precursor to the small subunit of ribulose-1, 5-bisphosphate carboxylase (pS) in a precursor binding assay. In the presence of concentrations of ATP or GTP that support maximal precursor binding to the envelope, cross-linking to the transit sequence occurred predominantly with IAP75 and a previously unidentified 21-kD polypeptide of the inner membrane, indicating that the transit sequence had inserted across the outer membrane. Cross-linking of envelope components to sequences in the mature portion of a second precursor, preferredoxin, was detected in the presence of ATP or GTP, suggesting that sequences distant from the transit sequence were brought into the vicinity of the outer membrane under these conditions. IAP75 and a third import component, IAP34, were coimmunoprecipitated with IAP86 antibodies from solubilized envelope membranes, indicating that these three proteins form a stable complex in the outer membrane. On the basis of these observations, we propose that IAP86 and IAP75 act as components of a multisubunit complex to mediate energy-independent recognition of the transit sequence and subsequent nucleoside triphosphate-induced insertion of the transit sequence across the outer membrane.

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Differential accumulation of plastid preprotein translocon components during spruce (Picea abies L. Karst.) needle development

Biological Chemistry ◽

10.1515/bc.2005.091 ◽

2005 ◽

Vol 386 (8) ◽

pp. 777-783 ◽

Cited By ~ 6

Author(s):

Hrvoje Fulgosi ◽

Hrvoje Lepeduš ◽

Vera Cesar ◽

Nikola Ljubešić

Keyword(s):

Protein Import ◽

Protein Translocation ◽

Early Stage ◽

Small Subunit ◽

Outer Envelope ◽

Receptor Isoforms ◽

Evolutionarily Conserved ◽

Functional Complex ◽

Plastid Protein ◽

Preprotein Translocation

Abstract We demonstrate that basic components of the plastid protein-import apparatus originally found in pea, Toc34, Toc159, and Tic110, are also conserved in evolutionarily younger gymnosperms. We show that multiple isoforms of the preprotein receptor Toc34 differentially accumulate in various stages of needle development, while the amounts of Toc159 drastically decrease during chloroplast morphogenesis. Spruce Toc34 and Toc159 receptors are able to recognise and interact with the angiosperm precursor of the Rubisco small subunit. Young proplastids found in closed buds contain a highly elevated number of protein translocation complexes equipped with only two types of outer envelope receptors, Toc159 and a 30-kDa Toc34-related protein. Photosystem II (PSII) can already be assembled in a fully functional complex at this very early stage of needle development, suggesting that no additional receptor isoforms are needed for translocation of all necessary PSII components. We conclude that the accumulation of evolutionarily conserved plastid preprotein translocation components is differentially regulated during spruce needle development.

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Molecular Docking Analysis of Phytochemical Thymoquinone as a Therapeutic Agent on SARS-Cov-2 Envelope Protein

Biointerface Research in Applied Chemistry ◽

10.33263/briac111.83898401 ◽

2020 ◽

Vol 11 (1) ◽

pp. 8389-8401

Keyword(s):

Molecular Docking ◽

Envelope Protein ◽

Pore Formation ◽

Transmembrane Domain ◽

Binding Free Energy ◽

Three Dimensional ◽

Homology Model ◽

Amino Terminal ◽

E Protein ◽

Α Helix

The sudden outbreak due to severe acute respiratory syndrome coronavirus 2 (SARS- Cov-2) is responsible for causing acute, highly dreadful coronavirus disease (COVID‐19). The pore-forming proteins in the SARS- CoV-2 envelope protein employ amphipathic α-helix for pore formation. The pore openings are essential for the transport of ions, toxins, and viroporin activity. Moreover, there is an insurgence to identify lead compounds to target the novel coronavirus for therapeutic purposes. Therefore in the present study, the SARS CoV-2 envelope protein sequence was analyzed, constructed the three-dimensional homology model, and screened against the bioactive phytochemical Thymoquinone (TQ). Molecular docking was performed between the modeled E protein and TQ to study protein-ligand interactions using ArgusLab 4.0. The investigation revealed that the modeled E protein contains a single α-amphipathic helix identified for the first time across the Amino-terminal region of the transmembrane domain may contribute to pore formation of small membrane proteins. Molecular docking results showed the promising inhibitory potential of the ligand TQ and the binding free energy of the bound complex was found to be -9.01 kcal/mol. The in silico approach has explicitly demonstrated the significant inhibitory effects of the ligand TQ. Therefore it may be used effectively as an antagonist against the SARS- CoV-2 infection owing to its outstanding pharmacological properties.

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Structural and Technological Approach to Reveal the Role of the Lipid Phase in the Formation of Soy Emulsion Gels with Chia Oil

Gels ◽

10.3390/gels7020048 ◽

2021 ◽

Vol 7 (2) ◽

pp. 48

Author(s):

Ana M. Herrero ◽

Claudia Ruiz-Capillas

Keyword(s):

Raman Spectroscopy ◽

Structural Properties ◽

Structural Changes ◽

Protein Secondary Structure ◽

Lipid Phase ◽

Α Helix ◽

Β Sheet ◽

Shed Light ◽

Chia Oil

Considerable attention has been paid to emulsion gels (EGs) in recent years due to their interesting applications in food. The aim of this work is to shed light on the role played by chia oil in the technological and structural properties of EGs made from soy protein isolates (SPI) and alginate. Two systems were studied: oil-free SPI gels (SPI/G) and the corresponding SPI EGs (SPI/EG) that contain chia oil. The proximate composition, technological properties (syneresis, pH, color and texture) and structural properties using Raman spectroscopy were determined for SPI/G and SPI/EG. No noticeable (p > 0.05) syneresis was observed in either sample. The pH values were similar (p > 0.05) for SPI/G and SPI/EG, but their texture and color differed significantly depending on the presence of chia oil. SPI/EG featured significantly lower redness and more lightness and yellowness and exhibited greater puncture and gel strengths than SPI/G. Raman spectroscopy revealed significant changes in the protein secondary structure, i.e., higher (p < 0.05) α-helix and lower (p < 0.05) β-sheet, turn and unordered structures, after the incorporation of chia oil to form the corresponding SPI/EG. Apparently, there is a correlation between these structural changes and the textural modifications observed.

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Nucleotide sequences of two pea cDNA clones encoding the small subunit of ribulose 1,5-bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptide.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)32995-8 ◽

1983 ◽

Vol 258 (3) ◽

pp. 1399-1402 ◽

Cited By ~ 8

Author(s):

G Coruzzi ◽

R Broglie ◽

A Cashmore ◽

N H Chua

Keyword(s):

Chlorophyll A ◽

Small Subunit ◽

Nucleotide Sequences ◽

Cdna Clones ◽

Bisphosphate Carboxylase

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Replacement of a conserved arginine in the assembly domain of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit interferes with holoenzyme formation.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)50055-2 ◽

1992 ◽

Vol 267 (15) ◽

pp. 10576-10582

Author(s):

R Flachmann ◽

H.J. Bohnert

Keyword(s):

Small Subunit ◽

Bisphosphate Carboxylase

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Precursor for the light-harvesting chlorophyll a/b-binding protein synthesized in Escherichia coli blocks import of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)49716-0 ◽

1992 ◽

Vol 267 (20) ◽

pp. 14328-14334

Author(s):

J.E. Oblong ◽

G.K. Lamppa

Keyword(s):

Escherichia Coli ◽

Chlorophyll A ◽

Light Harvesting ◽

Binding Protein ◽

Small Subunit ◽

Bisphosphate Carboxylase

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The key amino acids of E protein involved in early flavivirus infection: viral entry

Virology Journal ◽

10.1186/s12985-021-01611-2 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Tao Hu ◽

Zhen Wu ◽

Shaoxiong Wu ◽

Shun Chen ◽

Anchun Cheng

Keyword(s):

Amino Acids ◽

Conformational Changes ◽

Viral Entry ◽

Envelope Protein ◽

Cell Attachment ◽

Envelope Proteins ◽

Infection Process ◽

Early Infection ◽

Protein Amino Acids ◽

Α Helix

AbstractFlaviviruses are enveloped viruses that infect multiple hosts. Envelope proteins are the outermost proteins in the structure of flaviviruses and mediate viral infection. Studies indicate that flaviviruses mainly use envelope proteins to bind to cell attachment receptors and endocytic receptors for the entry step. Here, we present current findings regarding key envelope protein amino acids that participate in the flavivirus early infection process. Among these sites, most are located in special positions of the protein structure, such as the α-helix in the stem region and the hinge region between domains I and II, motifs that potentially affect the interaction between different domains. Some of these sites are located in positions involved in conformational changes in envelope proteins. In summary, we summarize and discuss the key envelope protein residues that affect the entry process of flaviviruses, including the process of their discovery and the mechanisms that affect early infection.

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