Targeting motifs and functional parameters governing the assembly of connexins into gap junctions

2000 ◽  
Vol 349 (1) ◽  
pp. 281-287 ◽  
Author(s):  
Patricia E. M. MARTIN ◽  
James STEGGLES ◽  
Claire WILSON ◽  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Lucifer Yellow ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Green Fluorescent

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

scholarly journals Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian Cells

1999 ◽  
Vol 10 (6) ◽  
pp. 2033-2050 ◽  
Author(s):  
Karen Jordan ◽  
Joell L. Solan ◽  
Michel Dominguez ◽  
Michael Sia ◽  
Art Hand ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Dye Transfer ◽  
Green Fluorescent

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

scholarly journals Purification, Characterization, and Gene Analysis of a Chitosanase (ChoA) from Matsuebacter chitosanotabidus3001

1999 ◽  
Vol 181 (21) ◽  
pp. 6642-6649 ◽  
Author(s):  
Jae Kweon Park ◽  
Kumiko Shimono ◽  
Nobuhisa Ochiai ◽  
Kazutaka Shigeru ◽  
Masako Kurita ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fluorescent Protein ◽  
Genomic Library ◽  
Amino Acid Sequences ◽  
Glycol Chitosan ◽  
Gene Encoding ◽  
Reading Frame ◽  
Acid Protein ◽  
Green Fluorescent ◽  
Potential Inhibitors

ABSTRACT The extracellular chitosanase (34,000 M r) produced by a novel gram-negative bacterium Matsuebacter chitosanotabidus 3001 was purified. The optimal pH of this chitosanase was 4.0, and the optimal temperature was between 30 and 40°C. The purified chitosanase was most active on 90% deacetylated colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives. Among potential inhibitors, the purified chitosanase was only inhibited by Ag+. Internal amino acid sequences of the purified chitosanase were obtained. A PCR fragment corresponding to one of these amino acid sequences was then used to screen a genomic library for the entire choA gene encoding chitosanase. Sequencing of the choA gene revealed an open reading frame encoding a 391-amino-acid protein. The N-terminal amino acid sequence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases. The 80-amino-acid excretion signal of ChoA fused to green fluorescent protein was functional in Escherichia coli. Taken together, these results suggest that we have identified a novel, previously unreported chitosanase.

scholarly journals Two Highly Similar Chitinases from Marine Vibrio Species have Different Enzymatic Properties

Marine Drugs ◽  
2020 ◽  
Vol 18 (3) ◽  
pp. 139
Author(s):  
Xinxin He ◽  
Min Yu ◽  
Yanhong Wu ◽  
Lingman Ran ◽  
Weizhi Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Extracellular Enzymes ◽  
Vibrio Harveyi ◽  
Amino Acid Sequences ◽  
Enzymatic Properties ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Marine Vibrio ◽  
Key Sites

Chitinase, as one of the most important extracellular enzymes in the marine environment, has great ecological and applied values. In this study, two chitinases (Chi1557 and Chi4668) with 97.33% amino acid sequences identity were individually found in Vibrio rotiferianus and Vibrio harveyi. They both were encoding by 561 amino acids, but differed in 15 amino acids and showed different enzymatic properties. The optimal temperature and pH ranges were 45–50 °C and pH 5.0–7.0 for Chi1557, while ~50 °C and pH 3.0–6.0 for Chi4668. K+, Mg2+, and EDTA increased the enzymatic activity of Chi4668 significantly, yet these factors were inhibitory to Chi1557. Moreover, Chi1557 degraded colloidal chitin to produce (GlcNAc)2 and minor GlcNAc, whereas Chi4668 produce (GlcNAc)2 with minor (GlcNAc)3 and (GlcNAc)4. The Kcat/Km of Chi4668 was ~4.7 times higher than that of Chi1557, indicating that Chi4668 had stronger catalytic activity than Chi1557. Furthermore, site-directed mutagenesis was performed on Chi1557 focusing on seven conserved amino acid residues of family GH18 chitinases. Chi1557 was almost completely inactive after Glu154, Gln219, Tyr221, or Trp312 was individually mutated, retained ~50% activity after Tyr37 was mutated, and increased two times activity after Asp152 was mutated, indicating that these six amino acids were key sites for Chi1557.

scholarly journals Nature-inspired Circular-economy Recycling (NaCRe) for Proteins: Proof of Concept

2020 ◽  
Author(s):  
Simone Giaveri ◽  
Adeline M. Schmitt ◽  
Laura Roset Julià ◽  
Anna Murello ◽  
Laure Menin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Circular Economy ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Raw Materials ◽  
Free Cell ◽  
Translation System ◽  
Natural Polymers ◽  
Green Fluorescent

AbstractIn 2070, 1012 Kg of polymer-based materials (i.e. plastics) might be produced yearly, posing one of the greatest challenges that humanity has to face. Even though natural polymers, such as proteins and nucleic acids, are more abundant than synthetic ones, they are sustainable. The key property of such natural polymers is that they are sequence-defined. This allows for recycling to start with depolymerization into monomers, and end in the re-assembly of new polymers of arbitrarily different sequence. This process breaks a common recycling paradigm that a material is recycled only into itself. An organism digests proteins into amino acids. These are re-assembled into new proteins whose identity depends on the cell’s needs at the time of protein synthesis. Here we show that the process described above is achievable extra-cellularly. Specifically, we depolymerized a mixture of different peptides and/or proteins into their amino acid constituents and used these amino acids to synthesize fluorescent proteins using an amino acid-free cell-free transcription-translation system. We were successful in recycling proteins with high relevance in materials engineering (β-lactoglobulin films, used for water filtration, or silk fibroin solutions) into a biotechnologically relevant protein (green fluorescent protein). The potential long-term impact of this approach to recycling lies in its compatibility with circular-economy models where raw materials remain in use as long as possible, thus reducing the burden on the planet.

Structural and spectrophotometric investigation of two unnatural amino-acid altered chromophores in the superfolder green fluorescent protein

2021 ◽  
Vol 77 (8) ◽  
Author(s):  
Gregory M. Olenginski ◽  
Juliana Piacentini ◽  
Darcy R. Harris ◽  
Nicolette A. Runko ◽  
Brianna M. Papoutsis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Fluorescent Protein ◽  
Photophysical Properties ◽  
Unnatural Amino Acid ◽  
Green Fluorescent ◽  
Absorbance Band ◽  
Protein Constructs

The spectrophotometric properties of the green fluorescent protein (GFP) result from the post-translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of the β-barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group. The structures and spectrophotometric properties of these superfolder GFP (sfGFP) variants with the unnatural amino acids (UAAs) 4-nitro-L-phenylalanine or 4-cyano-L-phenylalanine were explored. Notably, the characteristic 487 nm absorbance band of wild-type (wt) sfGFP is absent in both unnatural amino-acid-containing protein constructs (Tyr66pNO2Phe-sfGFP and Tyr66pCNPhe-sfGFP). Consequently, neither Tyr66pNO2Phe-sfGFP nor Tyr66pCNPhe-sfGFP exhibited the characteristic emission of wt sfGFP centered at 511 nm when excited at 487 nm. Tyr66pNO2Phe-sfGFP appeared orange due to an absorbance band centered at 406 nm that was not present in wt sfGFP, while Tyr66pCNPhe-sfGFP appeared colorless with an absorbance band centered at 365 nm. Mass spectrometry and X-ray crystallography confirmed the presence of a fully formed chromophore and no significant structural changes in either of these UAA-containing protein constructs, signaling that the change in the observed photophysical properties of the proteins is the result of the presence of the UAA in the chromophore.

Novel determinant of PKC-ε anchoring at cardiac Z-lines

2005 ◽  
Vol 289 (5) ◽  
pp. H1941-H1950 ◽  
Author(s):  
Seth L. Robia ◽  
Misuk Kang ◽  
Jeffery W. Walker
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Cardiac Myocyte ◽  
Fluorescent Protein ◽  
Cardiac Cells ◽  
Protein Protein Interactions ◽  
Multiple Points ◽  
Myocyte Function ◽  
Green Fluorescent

The Z-line represents a critical link between the transverse tubule network and cytoskeleton of cardiac cells with a role in anchoring structural proteins, ion channels, and signaling molecules. Protein kinase C-ε (PKC-ε) regulates cardiac excitability, cardioprotection, and growth, possibly as a consequence of translocation to the Z-line/T tubule region. To investigate the mechanism of PKC-ε translocation, fragments of its NH2-terminal 144-amino acid variable domain, εV1, were fused with green fluorescent protein and evaluated by quantitative Fourier image analysis of decorated myocytes. Deletion of 23 amino acids from the NH2-terminus of εV1, including an EAVSLKPT motif important for binding to a receptor for activated C kinase (RACK2), reduced but did not abolish Z-line binding. Further deletions of up to 84 amino acids from the NH2-terminus of εV1 also did not prevent Z-line decoration. However, deletions of residues 85–144 from the COOH-terminus strongly reduced Z-line binding. COOH-terminal deletions caused 2.5-fold greater loss of binding energy (ΔΔG) than did NH2-terminal deletions. Synthetic peptides derived from these regions modulated εV1 binding and cardiac myocyte function, but also revealed considerable heterogeneity within populations of adult cardiac myocytes. The COOH-terminal subdomain important for Z-line anchoring maps to a surface in the εV1 crystal structure that complements the eight-amino acid RACK2 binding site and two previously identified membrane docking motifs. PKC-ε anchoring at the cardiac Z-line/T tubule appears to rely on multiple points of contact probably involving protein-lipid and protein-protein interactions.

scholarly journals Functional Regions of the Pseudomonas aeruginosa Cytotoxin ExoU

2005 ◽  
Vol 73 (1) ◽  
pp. 573-582 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Alan R. Hauser
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Phospholipase Activity ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a potent patatin-like phospholipase, causes rapid cell death following its injection into host cells by the Pseudomonas aeruginosa type III secretion system. To better define regions of ExoU required for cytotoxicity, transposon-based linker insertion mutagenesis followed by site-directed mutagenesis of individual residues was employed by using a Saccharomyces cerevisiae model system. Random insertion of five amino acids identified multiple regions within ExoU that are required for cell killing. Five regions were chosen for further characterization: three corresponded to the oxyanion hole, hydrolase motif, and catalytic aspartate motif of the patatin-like domain within the N-terminal half of ExoU; one corresponded to an uncharacterized part of the patatin-like domain; and one corresponded to a region near the C terminus. Specific individual amino acid substitutions in each of the four N-terminal regions prevented killing of yeast and significantly reduced phospholipase activity. Whereas five amino acid insertions in the fifth region near the C terminus markedly reduced cytotoxicity and phospholipase activity, substitution of individual amino acids did not abolish either activity. To determine whether each of the five identified regions of ExoU was also essential for cytotoxicity in human cells, representative mutant forms of ExoU fused to green fluorescent protein were expressed in HeLa cells. These variants of ExoU were readily visualized and caused minimal cytotoxicity to HeLa cells, while wild-type ExoU fused to green fluorescent protein induced significant cell lysis and no detectable fluorescence. Thus, a minimum of five regions, including one which is well removed from the patatin-like domain, are required for the cytotoxicity and phospholipase activity of ExoU.

scholarly journals Molecular Cloning and Bioinformatics Analysis of DQA Gene from Mink (Neovison vison)

2019 ◽  
Vol 20 (5) ◽  
pp. 1037
Author(s):  
Zhaobin Fan ◽  
Houfeng Zhang ◽  
Min Rong ◽  
Dongmei Meng ◽  
Zhenxing Yu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mechanisms ◽  
Signal Sequence ◽  
Amino Acid Sequences ◽  
Neovison Vison ◽  
Random Coil ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Full Length Sequence

In the present study, we cloned, sequenced, and explored the structural and functional characteristics of the major histocompatibility complex (MHC)-DQA gene from mink (Neovison vison) for the first time. The full-length sequence of DQA gene was 1147-bp-long, contained a coding region of 768-bp, which was predicted to encoding 255 amino acid residues. The comparison between DQA from mink (Neovison vison) and other MHC-DQA molecules from different animal species showed that nucleotide and encoded amino acid sequences of the mink DQA gene exhibited high similarity with the ferret (Mustela pulourius furo). Phylogenetic analysis revealed that mink (Neovison vison) DQA is grouped with that of ferret (Mustela pulourius furo). The cloned sequence contained a 23-amino acid NH2-terminal signal sequence with the signal peptide cutting site located in amino acids 23–24, and had three Asn-Xaa-Ser/Thr sequons. Three cysteine residues were also identified (Cys-85, Cys-121, and Cys-138). The 218 to 240 amino acids were predicted to be the transmembrane domains. The prediction of the secondary structure revealed three α-helixes and fourteen β-sheets in Neovison vison DQA protein, while random coil was a major pattern. In this study, the whole CDS sequence of Neovison vison DQA gene was successfully cloned, which was valuable for exploring the function and antiviral molecular mechanisms underlying the molecule. The findings of the present study have laid the foundation for the disease resistance and breeding of mink.

scholarly journals An Acidic Cluster of Human Cytomegalovirus UL99 Tegument Protein Is Required for Trafficking and Function

2004 ◽  
Vol 78 (3) ◽  
pp. 1488-1502 ◽  
Author(s):  
Thomas R. Jones ◽  
Shi-Wu Lee
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Fluorescent Protein ◽  
Amino Acid Sequences ◽  
Cytoplasmic Vacuole ◽  
Viral Growth ◽  
Reading Frame ◽  
Tegument Proteins ◽  
Green Fluorescent ◽  
And Function

ABSTRACT The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.

scholarly journals Comprehensive mutagenesis to identify amino acid residues contributing to the difference in thermostability between two originally thermostable ancestral proteins

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258821
Author(s):  
Satoshi Akanuma ◽  
Minako Yamaguchi ◽  
Akihiko Yamagishi
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rational Design ◽  
Synergistic Effects ◽  
Amino Acid Sequences ◽  
Amino Acid Substitutions ◽  
Amino Acid Residues ◽  
Identify Amino Acid ◽  
The Difference ◽  
The Stability

Further improvement of the thermostability of inherently thermostable proteins is an attractive challenge because more thermostable proteins are industrially more useful and serve as better scaffolds for protein engineering. To establish guidelines that can be applied for the rational design of hyperthermostable proteins, we compared the amino acid sequences of two ancestral nucleoside diphosphate kinases, Arc1 and Bac1, reconstructed in our previous study. Although Bac1 is a thermostable protein whose unfolding temperature is around 100°C, Arc1 is much more thermostable with an unfolding temperature of 114°C. However, only 12 out of 139 amino acids are different between the two sequences. In this study, one or a combination of amino acid(s) in Bac1 was/were substituted by a residue(s) found in Arc1 at the same position(s). The best mutant, which contained three amino acid substitutions (S108D, G116A and L120P substitutions), showed an unfolding temperature more than 10°C higher than that of Bac1. Furthermore, a combination of the other nine amino acid substitutions also led to improved thermostability of Bac1, although the effects of individual substitutions were small. Therefore, not only the sum of the contributions of individual amino acids, but also the synergistic effects of multiple amino acids are deeply involved in the stability of a hyperthermostable protein. Such insights will be helpful for future rational design of hyperthermostable proteins.

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