NMR Paramagnetic Relaxation Enhancement Research Articles

Integrating NMR, SAXS, and Atomistic Simulations: Structure and Dynamics of a Two-Domain Protein

Multidomain proteins with two or more independently folded functional domains are prevalent in nature. Whereas most multidomain proteins are linked linearly in sequence, roughly one-tenth possess domain insertions where a guest domain is implanted into a loop of a host domain, such that the two domains are connected by a pair of interdomain linkers. Here, we characterized the influence of the interdomain linkers on the structure and dynamics of a domain-insertion protein in which the guest LysM domain is inserted into a central loop of the host CVNH domain. Expanding upon our previous crystallographic and NMR studies, we applied SAXS in combination with NMR paramagnetic relaxation enhancement to construct a structural model of the overall two-domain system. Although the two domains have no fixed relative orientation, certain orientations were found to be preferred over others. We also assessed the accuracies of molecular mechanics force fields in modeling the structure and dynamics of tethered multidomain proteins by integrating our experimental results with microsecond-scale atomistic molecular dynamics simulations. In particular, our evaluation of two different combinations of the latest force fields and water models revealed that both combinations accurately reproduce certain structural and dynamical properties, but are inaccurate for others. Overall, our study illustrates the value of integrating experimental NMR and SAXS studies with long timescale atomistic simulations for characterizing structural ensembles of flexibly linked multidomain systems.

Application of Lysine-specific Labeling to Detect Transient Interactions Present During Human Lysozyme Amyloid Fibril Formation

Populating transient and partially unfolded species is a crucial step in the formation and accumulation of amyloid fibrils formed from pathogenic variants of human lysozyme linked with a rare but fatal hereditary systemic amyloidosis. The partially unfolded species possess an unstructured β-domain and C-helix with the rest of the α-domain remaining native-like. Here we use paramagnetic relaxation enhancement (PRE) measured by NMR spectroscopy to study the transient intermolecular interactions between such intermediate species. Nitroxide spin labels, introduced specifically at three individual lysine residues, generate distinct PRE profiles, indicating the presence of intermolecular interactions between residues within the unfolded β-domain. This study describes the applicability to PRE NMR measurements of selective lysine labeling, at different sites within a protein, as an alternative to the introduction of spin labels via engineered cysteine residues. These results reveal the importance of the β-sheet region of lysozyme for initiating self-assembly into amyloid fibrils.

Abstract 5238: Structural and biochemical characterization of membrane bound KRAS

Abstract KRAS4b functions as a molecular switch cycling between an active GTP bound state and inactive GDP state. Active KRAS4b binds tightly to RAF1 via its Ras binding domain (RBD), however activation of RAF1 competent for MAPK signal transduction requires membrane bound KRAS4b. Using recombinant farnesylated and methylated KRAS4b (KRAS4b-FMe) we have used a variety of biophysical methodologies including paramagnetic relaxation enhancement NMR, neutron reflectivity, small angle neutron and x-ray scattering and protein footprinting to gain insights into the orientation and structure of KRAS-FMe bound to a membrane. In these experiments we have used Nanodiscs or tethered lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline or -serine (70% POPC 30% POPS). Our data indicate that KRAS-FMe is oriented perpendicular to the membrane with minimal direct contacts to the membrane. The nucleotide bound state does not significantly change the orientation relative to the membrane. We will also present data on the effect of the RBD domain of RAF1 binding to KRAS-FMe at the membrane. Citation Format: Andrew G. Stephen, Que Van, Marco Tonelli, William Gillette, Dominic Esposito, Ben Niu, Michael Gross, Frank Heinrich, Arvind Ramanathan, Christopher Stanley. Structural and biochemical characterization of membrane bound KRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5238. doi:10.1158/1538-7445.AM2017-5238

Cu(II)-Based Paramagnetic Probe to Study RNA-Protein Interactions by NMR.

Paramagnetic NMR techniques allow for studying three-dimensional structures of RNA-protein complexes. In particular, paramagnetic relaxation enhancement (PRE) data can provide valuable information about long-range distances between different structural components. For PRE NMR experiments, oligonucleotides are typically spin-labeled using nitroxide reagents. The current work describes an alternative approach involving a Cu(II) cyclen-based probe that can be covalently attached to an RNA strand in the vicinity of the protein's binding site using "click" chemistry. The approach has been applied to study binding of HIV-1 nucleocapsid protein 7 (NCp7) to a model RNA pentanucleotide, 5'-ACGCU-3'. Coordination of the paramagnetic metal to glutamic acid residue of NCp7 reduced flexibility of the probe, thus simplifying interpretation of the PRE data. NMR experiments showed attenuation of signal intensities from protein residues localized in proximity to the paramagnetic probe as the result of RNA-protein interactions. The extent of the attenuation was related to the probe's proximity allowing us to construct the protein's contact surface map.

NMR Studies of Conformational Selection of hnRNP H on RNA Splicing

Modularity provides proteins versatility in regulating many cellular processes. The RNA Recognition Motif (RRM) is a common protein family that adopts modularity to facilitate its biological activity. hnRNP H contains three RRMs that play important roles in RNA splicing by binding G-rich enhancer element2. However, the mechanism three RRMs use to recognize RNA is still unknown. Here, based on the analysis of paramagnetic relaxation enhancement (PRE), the data indicates that hnRNP H RRM12 adopts a closed conformation in the absence of RNA. Second, dynamics experiments, T1, T2 and NOE also reveals that RRM1 and RRM2 domain share similar motion, which suggests that compact (closed) form is major population in solution. Moreover, in closed form, one of two bindings site was buried in compact structure. Based on above observation, we propose a hypothesis that there is chemical equilibrium between open form and close form. Once hnRNP H recognizes its G-rich binding sequencing, it will release the blocking RRM from closed form and reestablish chemical equilibrium toward to open form through conformational selection in presence of RNA3. Integrated methods like NMR paramagnetic relaxation enhancement, dynamics experiments and SAXS would be utilized to address this hypothesis.

Prediction of Protein Structure Using Surface Accessibility Data

AbstractAn approach to the de novo structure prediction of proteins is described that relies on surface accessibility data from NMR paramagnetic relaxation enhancements by a soluble paramagnetic compound (sPRE). This method exploits the distance‐to‐surface information encoded in the sPRE data in the chemical shift‐based CS‐Rosetta de novo structure prediction framework to generate reliable structural models. For several proteins, it is demonstrated that surface accessibility data is an excellent measure of the correct protein fold in the early stages of the computational folding algorithm and significantly improves accuracy and convergence of the standard Rosetta structure prediction approach.

Prediction of Protein Structure Using Surface Accessibility Data.

An approach to the de novo structure prediction of proteins is described that relies on surface accessibility data from NMR paramagnetic relaxation enhancements by a soluble paramagnetic compound (sPRE). This method exploits the distance‐to‐surface information encoded in the sPRE data in the chemical shift‐based CS‐Rosetta de novo structure prediction framework to generate reliable structural models. For several proteins, it is demonstrated that surface accessibility data is an excellent measure of the correct protein fold in the early stages of the computational folding algorithm and significantly improves accuracy and convergence of the standard Rosetta structure prediction approach.

Hsp70 biases the folding pathways of client proteins

The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a (1)H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.

Weak self-association of cytochrome c peroxidase molecules observed by paramagnetic NMR.

There is growing experimental evidence that many proteins exhibit a tendency for (ultra)weak homo- or hetero- oligomerization interactions. With the development of paramagnetic relaxation enhancement NMR spectroscopy it has become possible to characterize weak complexes experimentally and even detect complexes with affinities in the 1–25 mM range. We present evidence for a weak complex between cytochrome c peroxidase (CcP) molecules. In a previous study, we attached nitroxide based spin labels at three positions on CcP with the intent of observing intramolecular PRE effects. However, several intermolecular PRE effects were also observed suggesting a weak self-association between CcP molecules. The CcP–CcP complex was characterized using paramagnetic NMR and protein docking. The interaction occurs between the surface that is also part of the stereo-specific binding site for its physiological partner, cytochrome c (Cc), and several small, positively charged patches on the “back” of CcP. The CcP–CcP complex is not a stereo-specific complex. It is a dynamic ensemble of orientations, characteristic of an encounter state. The contact areas resemble those observed for CcP molecules in crystals. The CcP–CcP complex formation competes with that of the CcP-Cc complex. However, the affinity for Cc is much larger and thus it is expected that, under physiological conditions, auto-inhibition will be limited.Graphical A weak self-association between cytochrome c peroxidase molecules was characterized using paramagnetic NMR.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-016-0035-z) contains supplementary material, which is available to authorized users.

Valence state alternation of copper species doped in HY zeolite as revealed by paramagnetic relaxation enhancement NMR spectroscopy

Paramagnetic relaxation enhancement (PRE) solid-state NMR (ssNMR) was used to monitor the valence state alternation of copper species doped in HY zeolite during catalytic reaction processes. The combination of PRE ssNMR and in-situ NMR spectroscopy facilitates the detection of copper species as well as the monitoring of evolution from reactants, intermediates to products in heterogeneously catalyzed processes, which is of great importance for elucidating the detailed catalytic reaction mechanism.

Chemoenzymatic Synthesis of Nonasulfated Tetrahyaluronan with a Paramagnetic Tag for Studying Its Complex with Interleukin-10.

Implants and artificial biomaterials containing sulfated hyaluronans have been shown to improve the healing of injured skin and bones. It is hypothesized that these effects are mediated by the binding of sulfated glycosaminoglycans (GAGs) to growth factors and cytokines, resulting in the sequestering of proteins to the wound healing site and in modulated protein activity. Given that no direct synthetic access to sulfated oligohyaluronans has been available, little is known about their protein binding and the structure of the resulting protein complexes. Here, the chemoenzymatic preparation of oligohyaluronans on the gram scale is described. Oligohyaluronans are converted into anomeric azides at the reducing end, enabling the attachment of analytical labels through an anomeric ligation reaction. A nonasulfated tetrahyaluronan-ethylenediaminetetraacetic acid derivative has been produced and used as a paramagnetic tag for the elucidation of the complex of this ligand with interleukin-10 using paramagnetic relaxation enhancement NMR analysis. The metal ion position is resolved with 1.0 Å, enabling a refined structural model of the complex.

Characterization of the Shigella and Salmonella Type III Secretion System Tip-Translocon Protein-Protein Interaction by Paramagnetic Relaxation Enhancement.

Many Gram-negative pathogens, such as Shigella and Salmonella, assemble the type III secretion system (T3SS) to inject virulence proteins directly into eukaryotic cells to initiate infectious diseases. The needle apparatus of the T3SS consists of a base, an extracellular needle, a tip protein complex, and a translocon. The atomic structure of the assembled tip complex and the translocon is unknown. Here, we show by NMR paramagnetic relaxation enhancement (PRE) that the mixed α-β domain at the distal region of the Shigella and Salmonella tip proteins interacts with the N-terminal ectodomain of their major translocon proteins. Our results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and provide insights about the assembly of the needle apparatus of the T3SS.

Paramagnetic Nanoparticles Leave Their Mark on Nuclear Spins of Transiently Adsorbed Proteins.

The successful application of nanomaterials in biosciences necessitates an in-depth understanding of how they interface with biomolecules. Transient associations of proteins with nanoparticles (NPs) are accessible by solution NMR spectroscopy, albeit with some limitations. The incorporation of paramagnetic centers into NPs offers new opportunities to explore bio-nano interfaces. We propose NMR paramagnetic relaxation enhancement as a new tool to detect NP-binding surfaces on proteins with increased sensitivity, also extending the applicability of NMR investigations to heterogeneous biomolecular mixtures. The adsorption of ubiquitin on gadolinium-doped fluoride-based NPs produced residue-specific NMR line-broadening effects mapping to a contiguous area on the surface of the protein. Importantly, an identical paramagnetic fingerprint was observed in the presence of a competing protein-protein association equilibrium, exemplifying possible interactions taking place in crowded biological media. The interaction was further characterized using isothermal titration calorimetry and upconversion emission measurements. The data indicate that the used fluoride-based NPs are not biologically inert but rather are capable of biomolecular recognition.

Structure and Function of Transient Encounters of Redox Proteins.

Many biomolecular interactions proceed via lowly populated, transient intermediates. Believed to facilitate formation of a productive complex, these short-lived species are inaccessible to conventional biophysical and structural techniques and, until recently, could only be studied by theoretical simulations. Recent development of experimental approaches sensitive to the presence of minor species--in particular paramagnetic relaxation enhancement (PRE) NMR spectroscopy--has enabled direct visualization and detailed characterization of such lowly populated states. Collectively referred to as an encounter complex, the binding intermediates are particularly important in transient protein interactions, such as those orchestrating signaling cascades or energy-generating electron transfer (ET) chains. Here I discuss encounter complexes of redox proteins mediating biological ET reactions, which are essential for many vital cellular activities including oxidative phosphorylation and photosynthesis. In particular, this Account focuses on the complex of cytochrome c (Cc) and cytochrome c peroxidase (CcP), which is a paradigm of biomolecular ET and an attractive system for studying protein binding and enzymatic catalysis. The Cc-CcP complex formation proceeds via an encounter state, consisting of multiple protein-protein orientations sampled in the search of the dominant, functionally active bound form and exhibiting a broad spatial distribution, in striking agreement with earlier theoretical simulations. At low ionic strength, CcP binds another Cc molecule to form a weak ternary complex, initially inferred from kinetics experiments and postulated to account for the measured ET activity. Despite strenuous efforts, the ternary complex could not be observed directly and remained eagerly sought for the past two decades. Very recently, we have solved its structure in solution and shown that it consists of two binding forms: the dominant, ET-inactive geometry and an ensemble of lowly populated species with short separations between Cc and CcP cofactors, which summarily account for the measured ET rate. Unlike most protein complexes, which require accurate alignment of the binding surfaces in a single, well-defined orientation to carry out their function, redox proteins can form multiple productive complexes. As fast ET will occur any time the redox centers of the binding partners are close enough to ensure efficient electron tunneling across the interface, many protein-protein orientations are expected to be ET active. The present analysis confirms that the low-occupancy states can support the functional ET activity and contribute to the stability of redox protein complexes. As illustrated here, boundaries between the dominant and the encounter forms become blurred for many dynamic ET systems, which are more aptly described by ensembles of functionally and structurally heterogeneous bound forms.

(19)F Paramagnetic Relaxation Enhancement: A Valuable Tool for Distance Measurements in Proteins.

Fluorine NMR paramagnetic relaxation enhancement was evaluated as a versatile approach for extracting distance information in selectively F-labeled proteins. Proof of concept and initial applications are presented for the HIV-inactivating lectin cyanovirin-N. Single F atoms were introduced at the 4-, 5-, 6- or 7 positions of Trp49 and the 4-position of Phe4, Phe54, and Phe80. The paramagnetic nitroxide spin label was attached to Cys residues that were placed into the protein at positions 50 or 52. (19)F-T2 NMR spectra with different relaxation delays were recorded and the transverse (19)F-PRE rate, (19)F-Γ2 , was used to determine the average distance between the F nucleus and the paramagnetic center. Our data show that experimental (19)F PRE-based distances correspond to 0.93 of the (1)HN-PRE distances, in perfect agreement with the gyromagnetic γ(19)F/γ(1)H ratio, thereby demonstrating that (19)F PREs are excellent alternative parameters for quantitative distance measurements in selectively F-labeled proteins.

19F Paramagnetic Relaxation Enhancement: A Valuable Tool for Distance Measurements in Proteins

AbstractFluorine NMR paramagnetic relaxation enhancement was evaluated as a versatile approach for extracting distance information in selectively F‐labeled proteins. Proof of concept and initial applications are presented for the HIV‐inactivating lectin cyanovirin‐N. Single F atoms were introduced at the 4‐, 5‐, 6‐ or 7 positions of Trp49 and the 4‐position of Phe4, Phe54, and Phe80. The paramagnetic nitroxide spin label was attached to Cys residues that were placed into the protein at positions 50 or 52. 19F‐T2 NMR spectra with different relaxation delays were recorded and the transverse 19F‐PRE rate, 19F‐Γ2, was used to determine the average distance between the F nucleus and the paramagnetic center. Our data show that experimental 19F PRE‐based distances correspond to 0.93 of the 1HN‐PRE distances, in perfect agreement with the gyromagnetic γ19F/γ1H ratio, thereby demonstrating that 19F PREs are excellent alternative parameters for quantitative distance measurements in selectively F‐labeled proteins.

Unveiling transient protein-protein interactions that modulate inhibition of alpha-synuclein aggregation by beta-synuclein, a pre-synaptic protein that co-localizes with alpha-synuclein.

Pathology in Parkinson’s disease is linked to self-association of α-Synuclein (αS) into pathogenic oligomeric species and highly ordered amyloid fibrils. Developing effective therapeutic strategies against this debilitating disease is critical and βS, a pre-synaptic protein that co-localizes with αS, can act as an inhibitor of αS assembly. Despite the potential importance of βS as an inhibitor of αS, the nature, location and specificity of the molecular interactions between these two proteins is unknown. Here we use NMR paramagnetic relaxation enhancement experiments, to demonstrate that βS interacts directly with αS in a transient dimer complex with high specificity and weak affinity. Inhibition of αS by βS arises from transient αS/βS heterodimer species that exist primarily in head- to- tail configurations while αS aggregation arises from a more heterogeneous and weaker range of transient interactions that include both head-to-head and head-to-tail configurations. Our results highlight that intrinsically disordered proteins can interact directly with one another at low affinity and that the transient interactions that drive inhibition versus aggregation are distinct by virtue of their plasticity and specificity.

Determining Oligomeric Order of a Membrane Protein by Double Electron-Electron Resonance Spectroscopy

Many different classes of membrane proteins are known to form oligomers in cellular membranes in order to carry out specific cellular functions. Detection and detailed structural characterization of protein oligomers in lipid milieu is by no means a trivial task. Here we demonstrate the use of spin-labeling and Double Electron-Electron Resonance (DEER) spectroscopy to determine the oligomeric order of a membrane protein. Specifically, we investigate oligomerization of a seven-helical membrane photoreceptor Anabaena Sensory Rhodopsin (ASR) from Anabaena sp. PCC7120. Recently, ASR structure has been solved by both x-ray protein crystallography (Science 2004, 306, 1390) and solid-state NMR (Nat Methods 2013, 10, 1007). Here we show that the same spin-labeling sites we employed for paramagnetic relaxation enhancement (PRE) NMR can also be used for DEER experiments. The results demonstrate that DEER restraints can not only differentiate between the dimer (x-ray) and trimer (ssNMR) models that have very different interfaces, but further rule out hypothetical tetramer and other higher order polygon models. The crux of our DEER-based approach relies on taking advantage of the multi-spin effects and analyzing experimental DEER traces by direct fitting to the multispin models. Overall, the observed profound effect of higher order spin correlations on the DEER trace allows for a reliable differentiation between oligomer models. In the specific case of ASR, the DEER trace modeling allowed us to unambiguously discard all but the trimer model. Furthermore, the addition of DEER electron-electron distances to the NMR restraints in the structure calculation protocol improves local RMSD, and allows for refinement of the orientation of helices. Supported by U.S. DOE Contract DE-FG02-02ER15354 to AIS and NSERC Discovery Grants RGPIN-2014-04547 to VL and RGPIN-2013-250202 to LSB.

Practical Aspects of Paramagnetic Relaxation Enhancement in Biological Macromolecules.

In this brief review, we summarize various aspects of NMR paramagnetic relaxation enhancement (PRE). We discuss the types of spin labels used in NMR studies, describe the relevant theory used to accurately calculate PREs from coordinates, including how to take into account the fact that paramagnetic labels tend to be highly mobile and sample a wide range of conformational space, and outline methods to refine structures or ensembles of structures directly against PRE data using simulated annealing. Finally, we show how the PRE can be used to detect, characterize, and visualize sparsely populated states of proteins and their complexes that are invisible to all other biophysical techniques.

PREdator: a python based GUI for data analysis, evaluation and fitting

The analysis of a series of experimental data is an essential procedure in virtually every field of research. The information contained in the data is extracted by fitting the experimental data to a mathematical model. The type of the mathematical model (linear, exponential, logarithmic, etc.) reflects the physical laws that underlie the experimental data. Here, we aim to provide a readily accessible, user-friendly python script for data analysis, evaluation and fitting. PREdator is presented at the example of NMR paramagnetic relaxation enhancement analysis.