Dynamic transitions in the translocated phloem filament protein

2000 ◽  
Vol 27 (9) ◽  
pp. 733 ◽  
Author(s):  
Kirsten Leineweber ◽  
Alexander Schulz ◽  
Gary A. Thompson
Keyword(s):  
Disulfide Bonds ◽  
Assimilate Transport ◽  
Phloem Sap ◽  
Folding Intermediates ◽  
P Protein ◽  
Molecular Masses ◽  
P Proteins ◽  
The Stability ◽  
Filament Protein ◽  
Higher Sensitivity

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 Recent evidence suggests that the P-proteins of Cucurbita maxima exist in at least two structural states: large polymers that are immobilized in individual sieve elements and small polymers or individual subunits that are translocated over long distances. We investigated variation in the structure of the phloem filament protein (phloem protein 1 or PP1) to determine the translocated form of the protein and its relationship to the polymerized state. It was demonstrated that the stability, folding state and assembly of the phloem filament protein rely on distinct intramolecular disulfide bonds. Acid trapping experiments combined with intergeneric grafts revealed that the phloem filament protein is translocated as an 88 kDa globular protein. By altering the pH of the collection buffer (pH 2–10), four individual conformational isoforms of PP1 with molecular masses of 81, 83, 85 and 88 kDa were consistently observed. The 81 kDa isoform represents the totally reduced phloem filament protein, the 83 and 85 kDa isoforms folding intermediates, and the 88 kDa its native soluble translocated form. The 83 and 85 kDa folding intermediates are susceptible to aggregation causing the gelation and formation of P-protein filaments in oxidized phloem sap. In contrast to the 88 kDa globular transport form, the 81, 83 and 85 kDa isoforms possibly exhibit lower stability, and therefore a higher sensitivity to proteolytic digestion.

Folding of peptides and proteins: role of disulfide bonds, recent developments

2013 ◽  
Vol 4 (6) ◽  
pp. 597-604 ◽  
Author(s):  
Yuji Hidaka ◽  
Shigeru Shimamoto
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
Bond Formation ◽  
Difficult Problem ◽  
Chemical Methods ◽  
Mutant Proteins ◽  
Folding Intermediates ◽  
Peptide Chemistry ◽  
Recent Developments

AbstractDisulfide-containing proteins are ideal models for studies of protein folding as the folding intermediates can be observed, trapped, and separated by HPLC during the folding reaction. However, regulating or analyzing the structures of folding intermediates of peptides and proteins continues to be a difficult problem. Recently, the development of several techniques in peptide chemistry and biotechnology has resulted in the availability of some powerful tools for studying protein folding in the context of the structural analysis of native, mutant proteins, and folding intermediates. In this review, recent developments in the field of disulfide-coupled peptide and protein folding are discussed, from the viewpoint of chemical and biotechnological methods, such as analytical methods for the detection of disulfide pairings, chemical methods for disulfide bond formation between the defined Cys residues, and applications of diselenide bonds for the regulation of disulfide-coupled peptide and protein folding.

scholarly journals In Vitro and In Vivo Assessment of the Potential of Escherichia coli Phages to Treat Infections and Survive Gastric Conditions

2021 ◽  
Vol 9 (9) ◽  
pp. 1869
Author(s):  
Joanna Kaczorowska ◽  
Eoghan Casey ◽  
Gabriele A. Lugli ◽  
Marco Ventura ◽  
David J. Clarke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Galleria Mellonella ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Composite Mixture ◽  
Ph Conditions ◽  
The Stability ◽  
Higher Sensitivity

Enterotoxigenic Escherichia coli (ETEC) and Shigella ssp. infections are associated with high rates of mortality, especially in infants in developing countries. Due to increasing levels of global antibiotic resistance exhibited by many pathogenic organisms, alternative strategies to combat such infections are urgently required. In this study, we evaluated the stability of five coliphages (four Myoviridae and one Siphoviridae phage) over a range of pH conditions and in simulated gastric conditions. The Myoviridae phages were stable across the range of pH 2 to 7, while the Siphoviridae phage, JK16, exhibited higher sensitivity to low pH. A composite mixture of these five phages was tested in vivo in a Galleria mellonella model. The obtained data clearly shows potential in treating E. coli infections prophylactically.

scholarly journals Phenotypic Divergence of P Proteins of Australian Bat Lyssavirus Lineages Circulating in Microbats and Flying Foxes

2021 ◽  
Author(s):  
Celine Deffrasnes ◽  
Meng-Xiao Luo ◽  
Linda Wiltzer ◽  
Cassandra T David ◽  
Kim G Lieu ◽  
...  
Keyword(s):  
Molecular Mapping ◽  
Protein A ◽  
Fine Tuning ◽  
Nuclear Trafficking ◽  
P Protein ◽  
Phenotypic Divergence ◽  
Reservoir Species ◽  
Pathogenic Viruses ◽  
P Proteins ◽  
Australian Bat Lyssavirus

Bats are reservoirs of many pathogenic viruses including the lyssaviruses rabies virus (RABV) and Australian bat lyssavirus (ABLV). Lyssavirus strains are closely associated with particular host reservoir species, with evidence of specific adaptation. Associated phenotypic changes remain poorly understood but are likely to involve P protein, a key mediator of the intracellular virus-host interface. Here, we examine the phenotype of P protein of ABLV, which circulates as two defined lineages associated with frugivorous and insectivorous bats, providing the opportunity compare proteins of viruses adapted to divergent bat species. We report that key functions of P protein in interferon/STAT1 antagonism and the capacity of P protein to undergo nuclear trafficking differ between lineages. Molecular mapping indicates that these differences are functionally distinct, and appear to involve modulatory effects on regulatory regions or structural impact, rather than changes to defined interaction sequences. This results in partial but significant phenotypic divergence, consistent with ‘fine-tuning’ to host biology, and with potentially distinct properties in the virus-host interface between bat families that represent key zoonotic reservoirs.

scholarly journals Study of the Assembly of Vesicular Stomatitis Virus N Protein: Role of the P Protein

2000 ◽  
Vol 74 (20) ◽  
pp. 9515-9524 ◽  
Author(s):  
Todd J. Green ◽  
Silvia Macpherson ◽  
Shihong Qiu ◽  
Jacob Lebowitz ◽  
Gail W. Wertz ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Analytical Ultracentrifugation ◽  
Structural Information ◽  
Protein Complexes ◽  
Outer Diameter ◽  
N Protein ◽  
Molar Ratios ◽  
P Protein ◽  
Vesicular Stomatitis ◽  
P Proteins

ABSTRACT To derive structural information about the vesicular stomatitis virus (VSV) nucleocapsid (N) protein, the N protein and the VSV phosphoprotein (P protein) were expressed together in Escherichia coli. The N and P proteins formed soluble protein complexes of various molar ratios when coexpressed. The major N/P protein complex was composed of 10 molecules of the N protein, 5 molecules of the P protein, and an RNA. A soluble N protein-RNA oligomer free of the P protein was isolated from the N/P protein-RNA complex using conditions of lowered pH. The molecular weight of the N protein-RNA oligomer, 513,879, as determined by analytical ultracentrifugation, showed that it was composed of 10 molecules of the N protein and an RNA of approximately 90 nucleotides. The N protein-RNA oligomer had the appearance of a disk with outer diameter, inner diameter, and thickness of 148 ± 10 Å, 78 ± 9 Å, and 83 ± 8 Å, respectively, as determined by electron microscopy. RNA in the complexes was protected from RNase digestion and was stable at pH 11. This verified that N/P protein complexes expressed in E. coli were competent for encapsidation. In addition to coexpression with the full-length P protein, the N protein was expressed with the C-terminal 72 amino acids of the P protein. This portion of the P protein was sufficient for binding to the N protein, maintaining it in a soluble state, and for assembly of N protein-RNA oligomers. With the results provided in this report, we propose a model for the assembly of an N/P protein-RNA oligomer.

Contribution of Disulfide Bonds and Calcium to Molluscan Hemocyanin Stability

2004 ◽  
Vol 59 (3-4) ◽  
pp. 281-287 ◽  
Author(s):  
Dessislava N. Georgieva ◽  
Nicolay Genov ◽  
Markus Perbandt ◽  
Wolfgang Voelter ◽  
Christian Betzel
Keyword(s):  
Free Energy ◽  
Calcium Ions ◽  
Disulfide Bonds ◽  
Functional Unit ◽  
Helical Structure ◽  
Practical Application ◽  
Destructive Effect ◽  
X Ray ◽  
Respiratory Proteins ◽  
The Stability

Disulfide bonds and calcium ions contribute significantly to the stability of the hemocyanin from the mollusc Rapana thomasiana grosse (gastropod). An extremely powerful protective effect of Ca2+ at a concentration of 100 mᴍ (100% protection) against the destructive effect of reductants like dithiothreitol was observed. This is important for the practical application of molluscan hemocyanins in experimental biochemistry, immunology and medicine. The reduction of the disulfide bonds in the Rapana hemocyanin leads to a 20% decrease of the α-helical structure. The S-S bonds contribute significantly to the free energy of stabilization in water increasing ⊿GD H2O by 6.9 kJ mol-1. The data are related to the X-ray model of the Rapana hemocyanin functional unit RtH2e. The results of this study can be of common validity for related respiratory proteins because the cysteine residues are conserved in all sequences of molluscan hemocyanins published so far.

scholarly journals The impact of O-glycan chemistry on the stability of intrinsically disordered proteins

2018 ◽  
Vol 9 (15) ◽  
pp. 3710-3715 ◽  
Author(s):  
Erica T. Prates ◽  
Xiaoyang Guan ◽  
Yaohao Li ◽  
Xinfeng Wang ◽  
Patrick K. Chaffey ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Protein Glycosylation ◽  
Disordered Proteins ◽  
Biological Functions ◽  
Post Translational Modification ◽  
Intrinsically Disordered ◽  
P Protein ◽  
The Stability ◽  
The Impact

Protein glycosylation is a diverse post-translational modification that serves myriad biological functions.

Uptake, partitioning and utilization of carbon and nitrogen in the phloem bleeding tree, Tasmanian blue gum (Eucalyptus globulus)

2000 ◽  
Vol 27 (9) ◽  
pp. 869
Author(s):  
John S. Pate ◽  
David J. Arthur
Keyword(s):  
Eucalyptus Globulus ◽  
Assimilate Transport ◽  
Concentration Gradients ◽  
Phloem Sap ◽  
Empirical Modelling ◽  
Carbon And Nitrogen ◽  
C And N ◽  
Four Levels ◽  
Gains And Losses ◽  
Growing Shoot

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 An empirical modelling procedure was employed to follow uptake, transport and utilization of photo-assimilated carbon (C) and soil-derived nitrogen (N) over a 19-d period (November 1998) in 2-year-old plantation-grown trees of Eucalyptus globulus Labill. Models utilized data for gains and losses of C and N in dry matter (DM) of tree parts, CO2 exchanges and transpiration of foliage, respiratory losses of stems and roots, C:N weight ratios of xylem and phloem sap collected at different sites within the system, and phloem sap sugar concentration gradients along trunks and branches to indicate directions of assimilate flow. The model for C depicted the fate of exported fixed C from four levels of branches on the shoot system, cycling of 16% of the C supplied from shoot to root back to the shoot in xylem, major involvement of xylem-derived C in nourishment of rapidly growing branches, and a net daily respiratory output per tree equivalent to 39% of its net daytime photosynthetic gain in C by foliage. The model for N showed that upper growing shoot parts gained more N mobilized from lower branches than was being acquired from soil. It also indicated high rates of cycling of N through mature foliage, effective retention of xylem-derived N by growing branches and apices, and feedback of substantial amounts of phloem-exported N from lower branches into xylem moving further up the trunk. Transpiration loss per tree was equivalent to 272 mL g–1 DM accumulated. Data are discussed in relation to similarly executed C:N partitioning studies on herbaceous annual species.

Chemical chaperone-mediated protein folding: stabilization of P22 tailspike folding intermediates by glycerol

2007 ◽  
Vol 388 (8) ◽  
pp. 797-804 ◽  
Author(s):  
Rajesh Mishra ◽  
Rajiv Bhat ◽  
Robert Seckler
Keyword(s):  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Chemical Chaperone ◽  
Preferential Hydration ◽  
Denatured State ◽  
Folding Intermediates ◽  
Fluorescence Measurements ◽  
Phage P22 ◽  
The Stability ◽  
P22 Tailspike

Abstract Polyol co-solvents such as glycerol increase the thermal stability of proteins. This has been explained by preferential hydration favoring the more compact native over the denatured state. Although polyols are also expected to favor aggregation by the same mechanism, they have been found to increase the folding yields of some large, aggregation-prone proteins. We have used the homotrimeric phage P22 tailspike protein to investigate the origin of this effect. The folding of this protein is temperature-sensitive and limited by the stability of monomeric folding intermediates. At non-permissive temperature (≥35°C), tailspike refolding yields were increased significantly in the presence of 1–4 m glycerol. At low temperature, tailspike refolding is prevented when folding intermediates are destabilized by the addition of urea. Glycerol could offset the urea effect, suggesting that the polyol acts by stabilizing crucial folding intermediates and not by increasing solvent viscosity. The stabilization effect of glycerol on tailspike folding intermediates was confirmed in experiments using a temperature-sensitive folding mutant protein, by fluorescence measurements of subunit folding kinetics, and by temperature up-shift experiments. Our results suggest that the chemical chaperone effect of polyols observed in the folding of large proteins is due to preferential hydration favoring structure formation in folding intermediates.

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