Expression and Purification of Intrinsically Disordered Aβ Peptide and Setup of Reproducible Aggregation Kinetics Experiment

2020 ◽  
pp. 731-754 ◽  
Author(s):  
Sara Linse
Keyword(s):  
Aggregation Kinetics ◽  
Aβ Peptide ◽  
Expression And Purification ◽  
Intrinsically Disordered

scholarly journals An optimized Npro-based method for the expression and purification of intrinsically disordered proteins for an NMR study

2015 ◽  
Vol 3 (1) ◽  
pp. e1011004 ◽  
Author(s):  
Natsuko Goda ◽  
Naoki Matsuo ◽  
Takeshi Tenno ◽  
Sonoko Ishino ◽  
Yoshizumi Ishino ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Expression And Purification ◽  
Intrinsically Disordered ◽  
Nmr Study

Reading the phosphorylation code: binding of the 14-3-3 protein to multivalent client phosphoproteins

2020 ◽  
Vol 477 (7) ◽  
pp. 1219-1225 ◽  
Author(s):  
Nikolai N. Sluchanko
Keyword(s):  
Protein Function ◽  
Intrinsically Disordered Protein ◽  
Structural Basis ◽  
Major Protein ◽  
Post Translational Modifications ◽  
Protein Protein Interaction ◽  
Intrinsically Disordered ◽  
Biochemical Journal ◽  
Multiple Binding ◽  
And Control

Many major protein–protein interaction networks are maintained by ‘hub’ proteins with multiple binding partners, where interactions are often facilitated by intrinsically disordered protein regions that undergo post-translational modifications, such as phosphorylation. Phosphorylation can directly affect protein function and control recognition by proteins that ‘read’ the phosphorylation code, re-wiring the interactome. The eukaryotic 14-3-3 proteins recognizing multiple phosphoproteins nicely exemplify these concepts. Although recent studies established the biochemical and structural basis for the interaction of the 14-3-3 dimers with several phosphorylated clients, understanding their assembly with partners phosphorylated at multiple sites represents a challenge. Suboptimal sequence context around the phosphorylated residue may reduce binding affinity, resulting in quantitative differences for distinct phosphorylation sites, making hierarchy and priority in their binding rather uncertain. Recently, Stevers et al. [Biochemical Journal (2017) 474: 1273–1287] undertook a remarkable attempt to untangle the mechanism of 14-3-3 dimer binding to leucine-rich repeat kinase 2 (LRRK2) that contains multiple candidate 14-3-3-binding sites and is mutated in Parkinson's disease. By using the protein-peptide binding approach, the authors systematically analyzed affinities for a set of LRRK2 phosphopeptides, alone or in combination, to a 14-3-3 protein and determined crystal structures for 14-3-3 complexes with selected phosphopeptides. This study addresses a long-standing question in the 14-3-3 biology, unearthing a range of important details that are relevant for understanding binding mechanisms of other polyvalent proteins.

Functional Fractionation of Platelets: Aggregation Kinetics and Glycoprotein Labeling of Differing Platelet Populations

1982 ◽  
Vol 48 (02) ◽  
pp. 211-216 ◽  
Author(s):  
V M Haver ◽  
A R L Gear
Keyword(s):  
Galactose Oxidase ◽  
Aggregation Kinetics ◽  
Maximal Rate ◽  
Membrane Glycoproteins ◽  
Whole Cells ◽  
Reversible Aggregation ◽  
Significant Difference ◽  
Small Doses ◽  
Major Loss ◽  
Kinetics Of

SummaryPlatelet heterogeneity has been studied with a technique called functional fractionation which employs gentle centrifugation to yield subpopulations (“reactive” and “less-reactive” platelets) after exposure to small doses of aggregating agent. Aggregation kinetics of the different platelet populations were investigated by quenched-flow aggregometry. The large, “reactive” platelets were more sensitive to ADP (Ka = 1.74 μM) than the smaller “less-reactive” platelets (Ka = 4.08 μM). However, their maximal rate of aggregation (Vmax, % of platelets aggregating per sec) of 23.3 was significantly lower than the “less-reactive” platelets (Vmax = 34.7). The “reactive” platelets had a 2.2 fold higher level of cyclic AMP.Platelet glycoproteins were labeled using the neuraminidase-galactose oxidase – [H3]-NaBH4 technique. When platelets were labeled after reversible aggregation, the “reactive” platelets showed a two-fold decrease in labeling efficiency (versus control platelets). However, examination of whole cells or membrane preparations from reversibly aggregated platelets revealed no significant difference in Coomassie or PAS (Schiff) staining.These results suggest that the large, “reactive” platelets are more sensitive to ADP but are not hyperaggregable in a kinetic sense. Reversible aggregation may cause a re-orientation of membrane glycoproteins that is apparently not characterized by a major loss of glycoprotein material.

Recombinant Expression and Purification of Mouse Nectin-like 4 Glycoprotein in 293ET Cell Line

2018 ◽  
Vol 00 (00) ◽  
pp. 0-0
Author(s):  
Dongdong Li ◽  
Dongdong Li ◽  
Tai An ◽  
Xiao Liu ◽  
Bin Yin ◽  
...  
Keyword(s):  
Cell Line ◽  
Recombinant Expression ◽  
Expression And Purification

Self-Assembling Micelles Based on an Intrinsically Disordered Protein Domain

2018 ◽  
Author(s):  
Sarah Klass ◽  
Matthew J. Smith ◽  
Tahoe Fiala ◽  
Jessica Lee ◽  
Anthony Omole ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Fusion Proteins ◽  
Organic Molecules ◽  
Intrinsically Disordered Protein ◽  
Protein Domain ◽  
Enzymatic Cleavage ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
Self Assembling ◽  
Protein Tag

Herein, we describe a new series of fusion proteins that have been developed to self-assemble spontaneously into stable micelles that are 27 nm in diameter after enzymatic cleavage of a solubilizing protein tag. The sequences of the proteins are based on a human intrinsically disordered protein, which has been appended with a hydrophobic segment. The micelles were found to form across a broad range of pH, ionic strength, and temperature conditions, with critical micelle concentration (CMC) values below 1 µM being observed in some cases. The reported micelles were found to solubilize hydrophobic metal complexes and organic molecules, suggesting their potential suitability for catalysis and drug delivery applications.

Sequence Specificity Despite Intrinsic Disorder: How a Disease-Associated Val/Met Polymorphism Shifts Tertiary Interactions in a Long Disordered Protein

2019 ◽  
Author(s):  
Ruchi Lohia ◽  
Reza Salari ◽  
Grace Brannigan
Keyword(s):  
Secondary Structure ◽  
Electrostatic Interactions ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Sequence Specificity ◽  
Intrinsically Disordered ◽  
Specific Effects ◽  
Heterogeneous Polymer ◽  
Non Local ◽  
Dynamics Simulations

<div>The role of electrostatic interactions and mutations that change charge states in intrinsically disordered proteins (IDPs) is well-established, but many disease-associated mutations in IDPs are charge-neutral. The Val66Met single nucleotide polymorphism (SNP) encodes a hydrophobic-to-hydrophobic mutation at the midpoint of the prodomain of precursor brain-derived neurotrophic factor (BDNF), one of the earliest SNPs to be associated with neuropsychiatric disorders, for which the underlying molecular mechanism is unknown. Here we report on over 250 μs of fully-atomistic, explicit solvent, temperature replica exchange molecular dynamics simulations of the 91 residue BDNF prodomain, for both the V66 and M66 sequence.</div><div>The simulations were able to correctly reproduce the location of both local and non-local secondary changes due to the Val66Met mutation when compared with NMR spectroscopy. We find that the local structure change is mediated via entropic and sequence specific effects. We show that the highly disordered prodomain can be meaningfully divided into domains based on sequence alone. Monte Carlo simulations of a self-excluding heterogeneous polymer, with monomers representing each domain, suggest the sequence would be effectively segmented by the long, highly disordered polyampholyte near the sequence midpoint. This is qualitatively consistent with observed interdomain contacts within the BDNF prodomain, although contacts between the two segments are enriched relative to the self-excluding polymer. The Val66Met mutation increases interactions across the boundary between the two segments, due in part to a specific Met-Met interaction with a Methionine in the other segment. This effect propagates to cause the non-local change in secondary structure around the second methionine, previously observed in NMR. The effect is not mediated simply via changes in inter-domain contacts but is also dependent on secondary structure formation around residue 66, indicating a mechanism for secondary structure coupling in disordered proteins. </div>

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