Conformational Exchange Rates Research Articles

Liquid-Liquid Phase Separation of an Intrinsically Disordered Region of a Germ Cell-Specific Protein Modulates the Stability and Conformational Exchange Rate of SH3 Domain.

The phenomenon of liquid-liquid phase separation is found in numerous biological processes. The biomolecules enveloped in the phase-separated droplets experience an obviously different environment from those in cellular or aqueous solution. Herein, we quantitatively characterized the thermodynamics and exchange kinetics of a model protein SH3 domain in the condensed phase of an intrinsically disordered region of a germ cell-specific protein DDX4N1 by using 19F-NMR spectroscopy. The stability and exchange rate of the SH3 domain are different from those in buffer and macromolecular crowding conditions. Our finding indicates that the local transient ordered microstructure and heterogeneity in the condensates play significant roles in modulating the biophysical properties of the enveloped proteins, and this finding may be essential to further our understanding how phase separation regulates the function of proteins in cells.

Dynamic Exchange of the Metal Chelating Moiety: A Key Factor in Determining the Rigidity of Protein-Tag Conjugates in Paramagnetic NMR.

Site-specific labeling of proteins with a paramagnetic tag is an efficient way to provide atomic-resolution information about the dynamics, interactions, and structures of the proteins and protein-ligand complexes. The paramagnetic effects manifested in NMR spectroscopy generally contain paramagnetic relaxation enhancement, pseudocontact shifts (PCSs), and residual dipolar coupling (RDC), and these effects correlate closely with the flexibility of protein-tag conjugates. The rigidity of the paramagnetic tag is greatly important in decoding the structural details of macromolecular complexes, because paramagnetic averaging reduces the PCSs and RDCs. Here we show that the dynamic exchange of the metal chelating moiety is a key factor in determining the rigidity of the paramagnetic tag in the protein conjugates. Decreasing the conformational exchange rates in the metal chelating moiety greatly minimizes the paramagnetic averaging and thus increases PCSs and RDCs. This effect has been demonstrated in an open-chain tag, Py-l-Cys-DTPA, which generates large PCSs and RDCs that are comparable to those of the reported cyclic DOTA-like tags. The proposed route offers a unique way to design suitable paramagnetic tags for applications in biological systems.

Expanding the membrane-protein NMR toolkit.

An NMR method to monitor conformational states of challenging large protein targets is described. The method, which can be used to evaluate distances between two labels and to measure conformational exchange rates, revealed an unanticipated outward-facing state in a glutamate transporter.

Deuteron Solid-State NMR Relaxation Measurements Reveal Two Distinct Conformational Exchange Processes in the Disordered N-Terminal Domain of Amyloid-β Fibrils.

We employed deuterium solid-state NMR techniques under static conditions to discern the details of the μs-ms timescale motions in the flexible N-terminal subdomain of Aβ1-40 amyloid fibrils, which spans residues 1-16. In particular, we utilized a rotating frame (R1ρ ) and the newly developed time domain quadrupolar Carr-Purcell-Meiboom-Gill (QCPMG) relaxation measurements at the selectively deuterated side chains of A2, H6, and G9. The two experiments are complementary in terms of probing somewhat different timescales of motions, governed by the tensor parameters and the sampling window of the magnetization decay curves. The results indicated two mobile "free" states of the N-terminal domain undergoing global diffusive motions, with isotropic diffusion coefficients of 0.7-1 ⋅ 108 and 0.3-3 ⋅ 106 ad2 s-1 . The free states are also involved in the conformational exchange with a single bound state, in which the diffusive motions are quenched, likely due to transient interactions with the structured hydrophobic core. The conformational exchange rate constants are 2-3 ⋅ 105 s-1 and 2-3 ⋅ 104 s-1 for the fast and slow diffusion free states, respectively.

Solid-state NMR reveals a comprehensive view of the dynamics of the flexible, disordered N-terminal domain of amyloid-β fibrils

Amyloid fibril deposits observed in Alzheimer's disease comprise amyloid-β (Aβ) protein possessing a structured hydrophobic core and a disordered N-terminal domain (residues 1-16). The internal flexibility of the disordered domain is likely essential for Aβ aggregation. Here, we used 2H static solid-state NMR methods to probe the dynamics of selected side chains of the N-terminal domain of Aβ1-40 fibrils. Line shape and relaxation data suggested a two-state model in which the domain's free state undergoes a diffusive motion that is quenched in the bound state, likely because of transient interactions with the structured C-terminal domain. At 37 °C, we observed freezing of the dynamics progressively along the Aβ sequence, with the fraction of the bound state increasing and the rate of diffusion decreasing. We also found that without solvation, the diffusive motion is quenched. The solvent acted as a plasticizer reminiscent of its role in the onset of global dynamics in globular proteins. As the temperature was lowered, the fraction of the bound state exhibited sigmoidal behavior. The midpoint of the freezing curve coincided with the bulk solvent freezing for the N-terminal residues and increased further along the sequence. Using 2H R1ρ measurements, we determined the conformational exchange rate constant between the free and bound states under physiological conditions. Zinc-induced aggregation leads to the enhancement of the dynamics, manifested by the faster conformational exchange, faster diffusion, and lower freezing-curve midpoints.

Deuterium Rotating Frame NMR Relaxation Measurements in the Solid State under Static Conditions for Quantification of Dynamics.

The feasibility of static deuterium rotating frame NMR relaxation measurements for characterization of slow timescale motions in powder systems is demonstrated. Using a model compound dimethyl sulfone-d6 , we show that these measurements yield conformational exchange rates and activation energy values in accordance with results obtained with other techniques. Furthermore, we demonstrate that the full Liouvillian approach as opposed to the Redfield approximation is necessary to analyze the experimental data.

Analysis of Saturation Recovery Amplitudes to Characterize Conformational Exchange in Spin-Labeled Proteins

Analysis of saturation recovery data from spin-labeled proteins is extended to include the amplitudes in addition to the recovery rates for two-site exchange. It is shown that the recovery amplitudes depend strongly on the exchange rate between states as well as their populations and this dependence provides a simple criterion to identify exchange rates in the 10–1000 kHz range. Analysis of experimental SR relaxation curves via the uniform penalty (UPEN) method allows for reliable identification of single, double, or other multiple-component traces, and global fitting of a set of relaxation curves using both relaxation rates and amplitudes determined from the UPEN fits allows for the estimation of exchange rate in the above domain. The theory is tested on simple model systems, and applied to the determination of conformational exchange rates in spin-labeled mutants of T4 Lysozyme and intestinal fatty acid binding protein. Finally, an example of T 1-weighted spectral editing is provided for systems in the slow exchange limit.

Elevated μs-ms timescale backbone dynamics in the transition state analog form of arginine kinase.

Arginine kinase catalyzes reversible phosphoryl transfer between arginine and ATP. Crystal structures of arginine kinase in an open, substrate-free form and closed, transition state analog (TSA) complex indicate that the enzyme undergoes substantial domain and loop rearrangements required for substrate binding, catalysis, and product release. Nuclear magnetic resonance (NMR) has shown that substrate-free arginine kinase is rigid on the ps-ns timescale (average S2=0.84±0.08) yet quite dynamic on the µs-ms timescale (35 residues with Rex, 12%), and that movements of the N-terminal domain and the loop comprising residues I182-G209 are rate-limiting on catalysis. Here, NMR of the TSA-bound enzyme shows similar rigidity on the ps-ns timescale (average S2=0.91±0.05) and substantially increased μs-ms timescale dynamics (77 residues; 22%). Many of the residues displaying μs-ms dynamics in NMR Carr-Purcell-Meiboom-Gill (CPMG) 15N backbone relaxation dispersion experiments of the TSA complex are also dynamic in substrate-free enzyme. However, the presence of additional dynamic residues in the TSA-bound form suggests that dynamics extend through much of the C-terminal domain, which indicates that in the closed form, a larger fraction of the protein takes part in conformational transitions to the excited state(s). Conformational exchange rate constants (kex) of the TSA complex are all approximately 2500s-1, higher than any observed in the substrate-free enzyme (800-1900s-1). Elevated μs-ms timescale protein dynamics in the TSA-bound enzyme is more consistent with recently postulated catalytic networks involving multiple interconnected states at each step of the reaction, rather than a classical single stabilized transition state.

Geometric Approximation: A New Computational Approach To Characterize Protein Dynamics from NMR Adiabatic Relaxation Dispersion Experiments.

A new computational strategy is reported that provides a fast approximation of numerical solutions of differential equations in general. The method is demonstrated with the analysis of NMR adiabatic relaxation dispersion experiments to reveal biomolecular dynamics. When an analytical solution to the theoretical equations describing a physical process is not available, the new approach can significantly accelerate the computational speed of the conventional numerical integration up to 10(5) times. NMR adiabatic relaxation dispersion experiments enhanced with optimized proton-decoupled pulse sequences, although extremely powerful, have previously been refractory to quantitative analysis. Both simulations and experimental validation demonstrate detectable "slow" (microsecond to millisecond) conformational exchange rates from 10(2) to 10(5) s(-1). This greatly expanded time-scale range enables the characterization of a wide array of conformational fluctuations for individual residues, which correlate with biomolecular function and were previously inaccessible. Moreover, the new computational method can be potentially generalized for analysis of new types of relaxation dispersion experiments to characterize the various dynamics of biomolecular systems.

α-synuclein-lanthanide metal ions interaction: binding sites, conformation and fibrillation.

BackgroundThe pathological hallmark of Parkinson’s disease is the deposition of cytoplasmic neuronal inclusions termed Lewy bodies. The major component of Lewy bodies is amyloid fibrils of α-synuclein. To investigate what causes α-synuclein aggregation is essential to understand its pathological roles in Parkinson’s disease. Various metal ions, including iron and copper, have been implicated in the pathogenesis of Parkinson’s disease. Divalent metal ions can regulate α-synuclein fibrillation rate, however, few studies have been performed to investigate how trivalent metal ions interact with α-synuclein and their effect on α-synuclein fibrillation. The study of the interaction between divalent and trivalent metal ions with α-synuclein is of vital importance to realize the mechanism of α-synuclein fibrillation.ResultsHere we used nuclear magnetic resonance spectroscopy to determine the trivalent metal ions (lanthanides) binding sites in α-synuclein. We found that lanthanide metal ions not only bind non-specifically to the C-terminal domain of α-synuclein, but also transiently interact with residues contain carboxyl groups in the N-terminal and NAC regions, the latter binding sites were not found for divalent cations. In addition, lanthanide ions bound α-synuclein exhibits slower conformational exchange rate. Compare to divalent cations, lanthanide ions accelerate α-synuclein fibrillation much faster.ConclusionsWe identified the lanthanide metal ions binding sites in α-synuclein and found a hierarchal effect for lanthanide ions binding to α-synuclein, driven by the interaction with aspartic acids and glutamic acids residues. Lanthanide ions binding also induced conformational dynamics change of α-synuclein. Compared to divalent cations, lanthanide metal ions significantly accelerated α-synuclein fibrillation, possibly due to the different inherent properties such as charge, binding sites and coordination modes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13628-016-0026-1) contains supplementary material, which is available to authorized users.

Asymmetric protonation of EmrE.

The small multidrug resistance transporter EmrE is a homodimer that uses energy provided by the proton motive force to drive the efflux of drug substrates. The pKa values of its "active-site" residues--glutamate 14 (Glu14) from each subunit--must be poised around physiological pH values to efficiently couple proton import to drug export in vivo. To assess the protonation of EmrE, pH titrations were conducted with (1)H-(15)N TROSY-HSQC nuclear magnetic resonance (NMR) spectra. Analysis of these spectra indicates that the Glu14 residues have asymmetric pKa values of 7.0 ± 0.1 and 8.2 ± 0.3 at 45°C and 6.8 ± 0.1 and 8.5 ± 0.2 at 25°C. These pKa values are substantially increased compared with typical pKa values for solvent-exposed glutamates but are within the range of published Glu14 pKa values inferred from the pH dependence of substrate binding and transport assays. The active-site mutant, E14D-EmrE, has pKa values below the physiological pH range, consistent with its impaired transport activity. The NMR spectra demonstrate that the protonation states of the active-site Glu14 residues determine both the global structure and the rate of conformational exchange between inward- and outward-facing EmrE. Thus, the pKa values of the asymmetric active-site Glu14 residues are key for proper coupling of proton import to multidrug efflux. However, the results raise new questions regarding the coupling mechanism because they show that EmrE exists in a mixture of protonation states near neutral pH and can interconvert between inward- and outward-facing forms in multiple different protonation states.

Substrate-modulated ADP/ATP-transporter dynamics revealed by NMR relaxation dispersion.

The ADP/ATP carrier (AAC) transports ADP and ATP across the inner mitochondrial membrane. Unlike most transporters that have 2-fold direct or inverted quasi-symmetry, AAC has the apparent 3-fold rotational symmetry. Further, its transport rate is fast for transporters that carry large solutes. Here, we perform comprehensive NMR relaxation dispersion measurements for the yeast AAC carrier 3, which provide residue-specific information on the protein conformational exchange. Our data indicate that AAC is predominantly in the cytosol-facing open state and converts to a lowly populated state in an asymmetric manner despite its three-fold structural symmetry. Binding of the substrate ADP significantly increases the rate of conformational exchange, whereas the inhibitor CATR slows the exchange. These results suggest that while the transporter catalyzes the translocation of substrate, the substrate also facilitates interconversion between alternating states that may be relevant to the transport function.

Conformational selection in protein binding and function.

Protein binding and function often involves conformational changes. Advanced nuclear magnetic resonance (NMR) experiments indicate that these conformational changes can occur in the absence of ligand molecules (or with bound ligands), and that the ligands may "select" protein conformations for binding (or unbinding). In this review, we argue that this conformational selection requires transition times for ligand binding and unbinding that are small compared to the dwell times of proteins in different conformations, which is plausible for small ligand molecules. Such a separation of timescales leads to a decoupling and temporal ordering of binding/unbinding events and conformational changes. We propose that conformational-selection and induced-change processes (such as induced fit) are two sides of the same coin, because the temporal ordering is reversed in binding and unbinding direction. Conformational-selection processes can be characterized by a conformational excitation that occurs prior to a binding or unbinding event, while induced-change processes exhibit a characteristic conformational relaxation that occurs after a binding or unbinding event. We discuss how the ordering of events can be determined from relaxation rates and effective on- and off-rates determined in mixing experiments, and from the conformational exchange rates measured in advanced NMR or single-molecule fluorescence resonance energy transfer experiments. For larger ligand molecules such as peptides, conformational changes and binding events can be intricately coupled and exhibit aspects of conformational-selection and induced-change processes in both binding and unbinding direction.

Structure and flexibility of the thermophilic cold-shock protein of Thermus aquaticus

The thermophilic bacterium Thermus aquaticus is a well-known source of Taq polymerase. Here, we studied the structure and dynamics of the T. aquaticus cold-shock protein (Ta-Csp) to better understand its thermostability using NMR spectroscopy. We found that Ta-Csp has a five-stranded β-barrel structure with five salt bridges which are important for more rigid structure and a higher melting temperature (76°C) of Ta-Csp compared to mesophilic and psychrophilic Csps. Microsecond to millisecond time scale exchange processes occur only at the β1–β2 surface region of the nucleic acid binding site with an average conformational exchange rate constant of 674s−1. The results imply that thermophilic Ta-Csp has a more rigid structure and may not need high structural flexibility to accommodate nucleic acids upon cold shock compared to its mesophile and psychrophile counterparts.

Shaping up for structural glycomics: a predictive protocol for oligosaccharide conformational analysis applied to N-linked glycans

The human glycome comprises a vast untapped repository of 3D-structural information that holds the key to glycan recognition and a new era of rationally designed mimetic chemical probes, drugs, and biomaterials. Toward routine prediction of oligosaccharide conformational populations and exchange rates at thermodynamic equilibrium, we apply hardware-accelerated aqueous molecular dynamics to model μs motions in N-glycans that underpin inflammation and immunity. In 10μs simulations, conformational equilibria of mannosyl cores, sialyl Lewis (sLe) antennae, and constituent sub-sequences agreed with prior refinements (X-ray and NMR). Glycosidic linkage and pyranose ring flexing were affected by branching, linkage position, and secondary structure, implicating sequence dependent motions in glycomic functional diversity. Linkage and ring conformational transitions that have eluded precise quantification by experiment and conventional (ns) simulations were predicted to occur on μs timescales. All rings populated non-chair shapes and the stacked galactose and fucose pyranoses of sLea and sLex were rigidified, suggesting an exploitable 3D-signature of cell adhesion protein binding. Analyses of sLex dynamics over 25μs revealed that only 10μs were sufficient to explore all aqueous conformers. This simulation protocol, which yields conformational ensembles that are independent of initial 3D-structure, is proposed as a route to understanding oligosaccharide recognition and structure–activity relationships, toward development of carbohydrate-based novel chemical entities.

Technological advances in site-directed spin labeling of proteins

Molecular flexibility over a wide time range is of central importance to the function of many proteins, both soluble and membrane. Revealing the modes of flexibility, their amplitudes, and time scales under physiological conditions is the challenge for spectroscopic methods, one of which is site-directed spin labeling EPR (SDSL-EPR). Here we provide an overview of some recent technological advances in SDSL-EPR related to investigation of structure, structural heterogeneity, and dynamics of proteins. These include new classes of spin labels, advances in measurement of long range distances and distance distributions, methods for identifying backbone and conformational fluctuations, and new strategies for determining the kinetics of protein motion.

Glassy Dynamics of Protein Methyl Groups Revealed by Deuteron NMR

We investigated site-specific dynamics of key methyl groups in the hydrophobic core of chicken villin headpiece subdomain (HP36) over the temperature range between 298 and 140 K using deuteron solid-state NMR longitudinal relaxation measurements. The relaxation of the longitudinal magnetization is weakly nonexponential (glassy) at high temperatures and exhibits a stronger degree of nonexponentiality below about 175 K. In addition, the characteristic relaxation times deviate from the simple Arrhenius law. We interpret this behavior via the existence of distribution of activation energy barriers for the three-site methyl jumps, which originates from somewhat different methyl environments within the local energy landscape. The width of the distribution of the activation barriers for methyl jumps is rather significant, about 1.4 kJ/mol. Our experimental results and modeling allow for the description of the apparent change at about 175 K without invoking a specific transition temperature. For most residues in the core, the relaxation behavior at high temperatures points to the existence of conformational exchange between the substates of the landscape, and our model takes into account the kinetics of this process. The observed dynamics are the same for dry and hydrated protein. We also looked at the effect of F58L mutation inside the hydrophobic core on the dynamics of one of the residues and observed a significant increase in its conformational exchange rate constant at high temperatures.

Origin of the Conformational Heterogeneity of Cardiolipin-Bound Cytochrome c

Interactions of cytochrome c (cyt c) with cardiolipin (CL) partially unfold the protein, activating its peroxidase function, a critical event in the execution of apoptosis. However, structural features of the altered protein species in the heterogeneous ensemble are difficult to probe with ensemble averaging. Analyses of the dye-to-heme distance distributions P(r) from time-resolved FRET (TR-FRET) have uncovered two distinct types of CL-bound cyt c conformations, extended and compact. We have combined TR-FRET, fluorescence correlation spectroscopy (FCS), and biolayer interferometry to develop a systematic understanding of the functional partitioning between the two conformations. The two subpopulations are in equilibrium with each other, with a submillisecond rate of conformational exchange reflecting the protein folding into a compact non-native state, as well as protein interactions with the lipid surface. Electrostatic interactions with the negatively charged lipid surface that correlate with physiologically relevant changes in CL concentrations strongly affect the kinetics of cyt c binding and conformational exchange. A predominantly peripheral binding mechanism, rather than deep protein insertion into the membrane, provides a rationale for the general denaturing effect of the CL surface and the large-scale protein unfolding. These findings closely relate to cyt c folding dynamics and suggest a general strategy for extending the time window in monitoring the kinetics of folding.

Specialized Hsp70 Chaperone (HscA) Binds Preferentially to the Disordered Form, whereas J-protein (HscB) Binds Preferentially to the Structured Form of the Iron-Sulfur Cluster Scaffold Protein (IscU)

The Escherichia coli protein IscU serves as the scaffold for Fe-S cluster assembly and the vehicle for Fe-S cluster transfer to acceptor proteins, such as apoferredoxin. IscU populates two conformational states in solution, a structured conformation (S) that resembles the conformation of the holoprotein IscU-[2Fe-2S] and a dynamically disordered conformation (D) that does not bind metal ions. NMR spectroscopic results presented here show that the specialized Hsp70 chaperone (HscA), alone or as the HscA-ADP complex, preferentially binds to and stabilizes the D-state of IscU. IscU is released when HscA binds ATP. By contrast, the J-protein HscB binds preferentially to the S-state of IscU. Consistent with these findings, we propose a mechanism in which cluster transfer is coupled to hydrolysis of ATP bound to HscA, conversion of IscU to the D-state, and release of HscB.

Evidence for dynamics in proteins as a mechanism for ligand dissociation

Signal transduction, regulatory processes, and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times, or koff, should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into koff for a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase that vary by six orders of magnitude in binding affinity. NMR relaxation dispersion experiments revealed a common set of residues near the binding site that undergo a concerted, millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with Ki and koff, suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although kconf,forward is faster than koff, use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.