Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Probing the Binding Modes of a Multidomain Protein to Lipid-based Nanoparticles by Relaxation-based NMR.

ABSTRACT: The interactions of two model multidomain proteins-covalently linked diubiquitins, Ub2-with lipid-based nanoparticles have been quantitatively probed by the measurements of NMR lifetime line broadening, ?R2. By combined analysis of ?R2 profiles arising from interactions with liposomes of varying sizes, an approach recently developed for the characterization of interactions of monoubiquitin with liposomes, we determine how the parameters of exchange (liposome binding) and dynamics of each individual domain of Ub2 on the surface of liposomes change when the domains are covalently attached to one another by a flexible linker. Two different covalent linkages were used: K63-linked and K48-linked Ub2. The possibility of three distinct modes of binding of Ub2 to liposomes requires the introduction of simple but important modifications to the strategy of analysis originally developed for monoubiquitin.

SUBMITTER: Ceccon A

PROVIDER: S-EPMC5576874 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Probing the Binding Modes of a Multidomain Protein to Lipid-based Nanoparticles by Relaxation-based NMR.

Ceccon Alberto A Tugarinov Vitali V Boughton Andrew J AJ Fushman David D Clore G Marius GM

The journal of physical chemistry letters 20170524 11

The interactions of two model multidomain proteins-covalently linked diubiquitins, Ub2-with lipid-based nanoparticles have been quantitatively probed by the measurements of NMR lifetime line broadening, ΔR2. By combined analysis of ΔR2 profiles arising from interactions with liposomes of varying sizes, an approach recently developed for the characterization of interactions of monoubiquitin with liposomes, we determine how the parameters of exchange (liposome bind ...[more]

PMID: 28530812

Similar Datasets

Global Dynamics of a Protein on the Surface of Anisotropic Lipid Nanoparticles Derived from Relaxation-Based NMR Spectroscopy.

Project description:The global motions of ubiquitin, a model protein, on the surface of anisotropically tumbling 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG):1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles are described. The shapes of POPG:DHPC bicelles prepared with high molar ratios q of POPG to DHPC can be approximated by prolate ellipsoids, with the ratio of ellipsoid dimensions and dimensions themselves increasing with higher values of q. Adaptation of the nuclear magnetic resonance (NMR) relaxation-based approach that we previously developed for interactions of ubiquitin with spherical POPG liposomes (Ceccon, A. J. Am. Chem. Soc. 2016, 138, 5789-5792) allowed us to quantitatively analyze the variation in lifetime line broadening of NMR signals (ΔR2) measured for ubiquitin in the presence of q = 2 POPG:DHPC bicelles and the associated transverse spin relaxation rates (R2,B) of bicelle-bound ubiquitin. Ubiquitin, transiently bound to POPG:DHPC bicelles, undergoes internal rotation about an axis orthogonal to the surface of the bicelle and perpendicular to the principal axis of its rotational diffusion tensor on the low microsecond time scale (∼3 μs), while the rotation axis itself wobbles in a cone on a submicrosecond time scale (≤ 500 ns).

| S-EPMC9357214 | biostudies-literature

Probing the transient dark state of substrate binding to GroEL by relaxation-based solution NMR.

Project description:The mechanism whereby the prototypical chaperonin GroEL performs work on substrate proteins has not yet been fully elucidated, hindered by lack of detailed structural and dynamic information on the bound substrate. Previous investigations have produced conflicting reports on the state of GroEL-bound polypeptides, largely due to the transient and dynamic nature of these complexes. Here, we present a unique approach, based on combined analysis of four complementary relaxation-based NMR experiments, to probe directly the "dark" NMR-invisible state of the model, intrinsically disordered, polypeptide amyloid ? (A?40) bound to GroEL. The four NMR experiments, lifetime line-broadening, dark-state exchange saturation transfer, relaxation dispersion, and small exchange-induced chemical shifts, are dependent in different ways on the overall exchange rates and populations of the free and bound states of the substrate, as well as on residue-specific dynamics and structure within the bound state as reported by transverse magnetization relaxation rates and backbone chemical shifts, respectively. Global fitting of all the NMR data shows that the complex is transient with a lifetime of <1 ms, that binding involves two predominantly hydrophobic segments corresponding to predicted GroEL consensus binding sequences, and that the structure of the bound polypeptide remains intrinsically and dynamically disordered with minimal changes in secondary structure propensity relative to the free state. Our results establish a unique method to observe NMR-invisible dynamic states of GroEL-bound substrates and to describe at atomic resolution the events between substrate binding and encapsulation that are crucial for understanding the normal and stress-related metabolic function of chaperonins.

| S-EPMC3710837 | biostudies-other

Probing initial transient oligomerization events facilitating Huntingtin fibril nucleation at atomic resolution by relaxation-based NMR.

Project description:The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (httNT), a polyglutamine (Q n ) tract, and a proline-rich sequence. Polyglutamine expansion results in an aggregation-prone protein responsible for Huntington's disease. Here, we study the earliest events involved in oligomerization of a minimalistic construct, httNTQ7, which remains largely monomeric over a sufficiently long period of time to permit detailed quantitative NMR analysis of the kinetics and structure of sparsely populated [Formula: see text] oligomeric states, yet still eventually forms fibrils. Global fitting of concentration-dependent relaxation dispersion, transverse relaxation in the rotating frame, and exchange-induced chemical shift data reveals a bifurcated assembly mechanism in which the NMR observable monomeric species either self-associates to form a productive dimer (τex ∼ 30 μs, K diss ∼ 0.1 M) that goes on to form a tetramer ([Formula: see text] μs; K diss ∼ 22 μM), or exchanges with a "nonproductive" dimer that does not oligomerize further (τex ∼ 400 μs; K diss ∼ 0.3 M). The excited state backbone chemical shifts are indicative of a contiguous helix (residues 3-17) in the productive dimer/tetramer, with only partial helical character in the nonproductive dimer. A structural model of the productive dimer/tetramer was obtained by simulated annealing driven by intermolecular paramagnetic relaxation enhancement data. The tetramer comprises a D 2 symmetric dimer of dimers with largely hydrophobic packing between the helical subunits. The structural model, validated by EPR distance measurements, illuminates the role of the httNT domain in the earliest stages of prenucleation and oligomerization, before fibril formation.

| S-EPMC6397591 | biostudies-literature

Interplay between conformational selection and induced fit in multidomain protein-ligand binding probed by paramagnetic relaxation enhancement.

Project description:The binding of ligands and substrates to proteins has been extensively studied for many years and can be described, in its simplest form, by two limiting mechanisms: conformational selection and induced fit. Conformational selection involves the binding of ligand to a pre-existing sparsely-populated conformation of the free protein that is the same as that in the final protein-ligand complex. In the case of induced fit, the ligand binds to the major conformation of the free protein and only subsequent to binding undergoes a conformational change to the final protein-ligand complex. While these two mechanisms can be dissected and distinguished by transient kinetic measurements, direct direction, characterization and visualization of transient, sparsely-populated states of proteins are experimentally challenging. Unless trapped, sparsely-populated states are generally invisible to conventional structural and biophysical techniques, including crystallography and most NMR measurements. In this review we summarize some recent developments in the use of paramagnetic relaxation enhancement to directly study sparsely-populated states of proteins and illustrate the application of this approach to two proteins, maltose binding protein and calmodulin, both of which undergo large rigid body conformational rearrangements upon ligand binding from an open apo state to a closed ligand-bound holo state. We show that the apo state ensemble comprises a small population of partially-closed configurations that are similar but not identical to that of the holo state. These results highlight the complementarity and interplay of induced fit and conformational selection and suggest that the existence of partially-closed states in the absence of ligand facilitates the transition to the closed ligand-bound state.

| S-EPMC3944927 | biostudies-literature

Probing driving forces for binding between nanoparticles and amino acids by saturation-transfer difference NMR.

Project description:As nanotechnology becomes increasingly used in biomedicine, it is important to have techniques by which to examine the structure and dynamics of biologically-relevant molecules on the surface of engineered nanoparticles. Previous work has shown that Saturation-Transfer Difference (STD)-NMR can be used to explore the interaction between small molecules, including amino acids, and the surface of polystyrene nanoparticles. Here we use STD-NMR to further explore the different driving forces that are responsible for these interactions. Electrostatic effects are probed by using zwitterionic polystyrene beads and performing STD-NMR experiments at high, low, and neutral pH, as well as by varying the salt concentration and observing the effect on the STD buildup curve. The influence of dispersion interactions on ligand-nanoparticle binding is also explored, by establishing a structure-activity relationship for binding using a series of unnatural amino acids with different lengths of hydrophobic side chains. These results will be useful for predicting which residues in a peptide are responsible for binding and for understanding the driving forces for binding between peptides and nanoparticles in future studies.

| S-EPMC7378059 | biostudies-literature

Microscopic insights into the NMR relaxation-based protein conformational entropy meter.

Project description:Conformational entropy is a potentially important thermodynamic parameter contributing to protein function. Quantitative measures of conformational entropy are necessary for an understanding of its role but have been difficult to obtain. An empirical method that utilizes changes in conformational dynamics as a proxy for changes in conformational entropy has recently been introduced. Here we probe the microscopic origins of the link between conformational dynamics and conformational entropy using molecular dynamics simulations. Simulation of seven proteins gave an excellent correlation with measures of side-chain motion derived from NMR relaxation. The simulations show that the motion of methyl-bearing side chains are sufficiently coupled to that of other side chains to serve as excellent reporters of the overall side-chain conformational entropy. These results tend to validate the use of experimentally accessible measures of methyl motion--the NMR-derived generalized order parameters--as a proxy from which to derive changes in protein conformational entropy.

| S-EPMC3821934 | biostudies-literature

Global Dynamics and Exchange Kinetics of a Protein on the Surface of Nanoparticles Revealed by Relaxation-Based Solution NMR Spectroscopy.

Project description:The global motions and exchange kinetics of a model protein, ubiquitin, bound to the surface of negatively charged lipid-based nanoparticles (liposomes) are derived from combined analysis of exchange lifetime broadening arising from binding to nanoparticles of differing size. The relative contributions of residence time and rotational tumbling to the total effective correlation time of the bound protein are modulated by nanoparticle size, thereby permitting the various motional and exchange parameters to be determined. The residence time of ubiquitin bound to the surface of both large and small unilamellar liposomes is ∼20 μs. Bound ubiquitin undergoes internal rotation about an axis approximately perpendicular to the lipid surface on a low microsecond time scale (∼2 μs), while simultaneously wobbling in a cone of semiangle 30-55° centered about the internal rotation axis on the nanosecond time scale. The binding interface of ubiquitin with liposomes is mapped by intermolecular paramagnetic relaxation enhancement using Gd(3+)-tagged vesicles, to a predominantly positively charged surface orthogonal to the internal rotation axis.

| S-EPMC5590658 | biostudies-literature

Probing excited states and activation energy for the integral membrane protein phospholamban by NMR CPMG relaxation dispersion experiments.

Project description:Phospholamban (PLN) is a dynamic single-pass membrane protein that inhibits the flow of Ca(2+) ions into the sarcoplasmic reticulum (SR) of heart muscle by directly binding to and inhibiting the SR Ca(2+)ATPase (SERCA). The PLN monomer is the functionally active form that exists in equilibrium between ordered (T state) and disordered (R state) states. While the T state has been fully characterized using a hybrid solution/solid-state NMR approach, the R state structure has not been fully portrayed. It has, however, been detected by both NMR and EPR experiments in detergent micelles and lipid bilayers. In this work, we quantitatively probed the mus to ms dynamics of the PLN excited states by observing the T state in DPC micelles using CPMG relaxation dispersion NMR spectroscopy under functional conditions for SERCA. The (15)N backbone and (13)C(delta1) Ile-methyl dispersion curves were fit using a two-state equilibrium model, and indicate that residues within domain Ia (residues 1-16), the loop (17-22), and domain Ib (23-30) of PLN undergo mus-ms dynamics (k(ex)=6100+/-800 s(-1) at 17 degrees C). We measured k(ex) at additional temperatures, which allowed for a calculation of activation energy equal to approximately 5 kcal/mol. This energy barrier probably does not correspond to the detachment of the amphipathic domain Ia, but rather the energy needed to unwind domain Ib on the membrane surface, likely an important mechanism by which PLN converts between high and low affinity states for its binding partners.

| S-EPMC3328355 | biostudies-literature

Determination of protein-ligand binding modes using fast multi-dimensional NMR with hyperpolarization.

Project description:Elucidation of small molecule-protein interactions provides essential information for understanding biological processes such as cellular signaling, as well as for rational drug development. Here, multi-dimensional NMR with sensitivity enhancement by dissolution dynamic nuclear polarization (D-DNP) is shown to allow the determination of the binding epitope of folic acid when complexed with the target dihydrofolate reductase. Protein signals are selectively enhanced by polarization transfer from the hyperpolarized ligand. A pseudo three-dimensional data acquisition with ligand-side Hadamard encoding results in protein-side [13C, 1H] chemical shift correlations that contain intermolecular nuclear Overhauser effect (NOE) information. A scoring function based on this data is used to select pre-docked ligand poses. The top five poses are within 0.76 Å root-mean-square deviation from a reference structure for the encoded five protons, showing improvements compared with the poses selected by an energy-based scoring function without experimental inputs. The sensitivity enhancement provided by the D-DNP combined with multi-dimensional NMR increases the speed and potentially the selectivity of structure elucidation of ligand binding epitopes.

| S-EPMC7441707 | biostudies-literature

Probing local backbone geometries in intrinsically disordered proteins by cross-correlated NMR relaxation.

Project description:An ultra-high-resolution NMR experiment for the measurement of intraresidue (1)H(i)-(15)N(i)-(13)C'(i) dipolar-chemical shift anisotropy relaxation interference is employed to extract information about local backbone geometries in intrinsically disordered proteins. The study of tumor suppressor BASP1 revealed a population shift of β-turn geometries at low pH conditions and a compaction of the BASP1 structural ensemble.

| S-EPMC3659411 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data