Search results for "intrinsically disordered protein"
showing 10 items of 33 documents
Secondary structure and dynamics study of the intrinsically disordered silica-mineralizing peptide P5S3during silicic acid condensation and silica de…
2017
The silica forming repeat R5 of sil1 from Cylindrotheca fusiformis was the blueprint for the design of P5 S3 , a 50-residue peptide which can be produced in large amounts by recombinant bacterial expression. It contains 5 protein kinase A target sites and is highly cationic due to 10 lysine and 10 arginine residues. In the presence of supersaturated orthosilicic acid P5 S3 enhances silica-formation whereas it retards the dissolution of amorphous silica (SiO2 ) at globally undersaturated concentrations. The secondary structure of P5 S3 during these 2 processes was studied by circular dichroism (CD) spectroscopy, complemented by nuclear magnetic resonance (NMR) spectroscopy of the peptide in …
Probing Differential Binding Mechanisms of Phenylalanine-Glycine-Rich Nucleoporins by Single-Molecule FRET
2018
Abstract Phenylalanine-glycine-rich nucleoporins (FG-Nups) are intrinsically disordered proteins, constituting the selective barrier of the nuclear pore complex. They are highly dynamic under physiological conditions and studying their interaction with nuclear transport receptors (NTRs) is key to understanding the molecular mechanism of nucleocytoplasmic transport. Distinct conformational features of FG-Nups interacting with diverse NTRs can be detected by multiparameter single-molecule fluorescence energy transfer (smFRET), which is a powerful technique for studying the dynamics and interactions of biomolecules in solution. Here we provide a detailed protocol utilizing smFRET to reveal dif…
Two differential binding mechanisms of FG-nucleoporins and nuclear transport receptors
2018
Summary Phenylalanine-glycine-rich nucleoporins (FG-Nups) are intrinsically disordered proteins, constituting the selective barrier of the nuclear pore complex (NPC). Previous studies showed that nuclear transport receptors (NTRs) were found to interact with FG-Nups by forming an “archetypal-fuzzy” complex through the rapid formation and breakage of interactions with many individual FG motifs. Here, we use single-molecule studies combined with atomistic simulations to show that, in sharp contrast, FG-Nup214 undergoes a coupled reconfiguration-binding mechanism when interacting with the export receptor CRM1. Association and dissociation rate constants are more than an order of magnitude lowe…
Evolving Notch polyQ tracts reveal possible solenoid interference elements.
2016
ABSTRACTPolyglutamine (polyQ) tracts in regulatory proteins are extremely polymorphic. As functional elements under selection for length, triplet repeats are prone to DNA replication slippage and indel mutations. Many polyQ tracts are also embedded within intrinsically disordered domains, which are less constrained, fast evolving, and difficult to characterize. To identify structural principles underlying polyQ tracts in disordered regulatory domains, here I analyze deep evolution of metazoan Notch polyQ tracts, which can generate alleles causing developmental and neurogenic defects. I show that Notch features polyQ tract turnover that is restricted to a discrete number of conserved “polyQ …
Comment on “Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water”
2018
Editors at Science requested our input on the above discussion (comment by Best et al . and response by Riback et al .) because both sets of authors use our data from Fuertes et al . (2017) to support their arguments. The topic of discussion pertains to the discrepant inferences drawn from SAXS versus FRET measurements regarding the dimensions of intrinsically disordered proteins (IDPs) in aqueous solvents. Using SAXS measurements on labeled and unlabeled proteins, we ruled out the labels used for FRET measurements as the cause of discrepant inferences between the two methods. Instead, we propose that FRET and SAXS provide complementary readouts because of a decoupling of size and shape fl…
Lunasin is a redox sensitive intrinsically disordered peptide with two transiently populated α-helical regions.
2016
Lunasin is a 43 amino acid peptide with anti-cancer, antioxidant, anti-inflammatory and cholesterol-lowering properties. Although the mechanism of action of lunasin has been characterized to some extent, its exact three-dimensional structure as well as the function of the N-terminal sequence remains unknown. We established a novel method for the production of recombinant lunasin that allows efficient isotope labeling for NMR studies. Initial studies showed that lunasin can exist in a reduced or oxidized state with an intramolecular disulfide bond depending on solution conditions. The structure of both forms of the peptide at pH 3.5 and 6.5 was characterized by CD spectroscopy and multidimen…
FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.
2021
International audience; Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever- increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among d…
Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2
2016
We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728–1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nM). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this …
Dispersion from Cα or NH: 4D experiments for backbone resonance assignment of intrinsically disordered proteins
2020
AbstractResonance assignment of intrinsically disordered proteins is remarkably challenging due to scant chemical shift dispersion arising from conformational heterogeneity. The challenge is even greater if repeating segments are present in the amino acid sequence. To forward unambiguous resonance assignment of intrinsically disordered proteins, we present iHACANCO, HACACON and (HACA)CONCAHA, three Hα-detected 4D experiments with Cα as an additional dimension. In addition, we present (HACA)CON(CA)NH and (HACA)N(CA)CONH, new 4D Hα-start, HN-detect experiments which have two NH dimensions to enhance peak dispersion in a sequential walk through C′, NH and HN, and provide more accurate NH/HN ch…
1H, 13C, and 15N NMR chemical shift assignment of the complex formed by the first EPEC EspF repeat and N-WASP GTPase binding domain
2021
AbstractLEE-encoded effector EspF (EspF) is an effector protein part of enteropathogenic Escherichia coli’s (EPEC’s) arsenal for intestinal infection. This intrinsically disordered protein contains three highly conserved repeats which together compose over half of the protein’s complete amino acid sequence. EPEC uses EspF to hijack host proteins in order to promote infection. In the attack EspF is translocated, together with other effector proteins, to host cell via type III secretion system. Inside host EspF stimulates actin polymerization by interacting with Neural Wiskott-Aldrich syndrome protein (N-WASP), a regulator in actin polymerization machinery. It is presumed that EspF acts by di…