The Effect of Counter Ions on the Conformation of Intrinsically Disordered Proteins Studied by Size-Exclusion Chromatography

2012 ◽  
pp. 319-330 ◽  
Author(s):  
Magdalena Wojtas ◽  
Tomasz M. Kapłon ◽  
Piotr Dobryszycki ◽  
Andrzej Ożyhar
Keyword(s):  
Size Exclusion Chromatography ◽  
Intrinsically Disordered Proteins ◽  
Size Exclusion ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Counter Ions ◽  
Exclusion Chromatography

scholarly journals Hsp70 inhibits aggregation of Islet amyloid polypeptide by binding to the heterogeneous prenucleation oligomers

2020 ◽  
Author(s):  
Neeraja Chilukoti ◽  
Bankanidhi Sahoo ◽  
S Deepa ◽  
Sreelakshmi Cherakara ◽  
Mithun Maddheshiya ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Islet Amyloid Polypeptide ◽  
Correlation Spectroscopy ◽  
Size Exclusion ◽  
Disordered Proteins ◽  
Strong Interactions ◽  
Islet Amyloid ◽  
Intrinsically Disordered ◽  
Exclusion Chromatography ◽  
In The Beginning

AbstractMolecular chaperone Hsp70 plays important roles in the pathology of amyloid diseases by inhibiting aberrant aggregation of proteins. However, mechanism of the interactions of Hsp70 with the amyloidogenic intrinsically disordered proteins (IDPs) is not clear. Here, we use Hsp70 from different organisms to show that it inhibits aggregation of Islet amyloid polypeptide (IAPP) at substoichiometric concentrations even in absence of ATP. The effect is found to be the strongest if Hsp70 is added in the beginning of aggregation but progressively less if added later, indicating role of Hsp70 in preventing primary nucleation possibly via interactions with the prefibrillar oligomers of IAPP. Fluorescence Correlation Spectroscopy (FCS) measurements of the solutions containing fluorescently labelled Hsp70 and IAPP exhibit fluorescence bursts suggesting formation of heterogeneous complexes of oligomeric IAPP binding to multiple molecules of Hsp70. Size exclusion chromatography and field flow fractionation are then used to fractionate the smaller complexes. Multiangle light scattering and FCS measurements suggest that these complexes comprise of monomers of Hsp70 and small oligomers of IAPP. However, concentration of the complexes is measured to be a few nanomolar amidst several μmolar of free Hsp70 and IAPP. Hence, our results indicate that Hsp70 interacts poorly with the monomers but strongly with oligomers of IAPP. This is likely a common feature of the interactions between the chaperones and the amyloidogenic IDPs. While strong interactions with the oligomers prevent aberrant aggregation, poor interaction with the monomers avert interference with the functions of the IDPs.

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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