Double Electron-electron Resonance Research Articles

Modeling of protein conformational changes with Rosetta guided by limited experimental data

Conformational changes are an essential component of functional cycles of many proteins, but their characterization often requires an integrative structural biology approach. Here, we introduce and benchmark ConfChangeMover (CCM), a new method built into the widely used macromolecular modeling suite Rosetta that is tailored to model conformational changes in proteins using sparse experimental data. CCM can rotate and translate secondary structural elements and modify their backbone dihedral angles in regions of interest. We benchmarked CCM on soluble and membrane proteins with simulated Cα-Cα distance restraints and sparse experimental double electron-electron resonance (DEER) restraints, respectively. In both benchmarks, CCM outperformed state-of-the-art Rosetta methods, showing that it can model a diverse array of conformational changes. In addition, the Rosetta framework allows a wide variety of experimental data to be integrated with CCM, thus extending its capability beyond DEER restraints. This method will contribute to the biophysical characterization of protein dynamics.

Functional Dynamics of Hsp70/CHIP/E2‐Ub Complex in Ubiquitination of Substrate Proteins

Heat shock protein (Hsp) 70 is the main mediator of protein quality control in the cytosol of eukaryotic cells. One of the major functions of Hsp70 is mediate the ubiquitination of client proteins which subsequently leads to proteasomal degradation. This process occurs via a multiprotein complex composed of Hsp70, carboxy terminus of Hsc70 interacting protein (CHIP), ubiquitin‐conjugating enzyme (E2), and ubiquitin (Ub). This complex is thought to be flexible, however, knowledge of the degree of dynamics are still lacking and detailed experimental evidence is needed. Here we present the models derived from Electron Paramagnetic Resonance (EPR) data and small‐angle X‐ray scattering (SAXS) ensembles for the complexes of Hsp70/CHIP/E2‐Ub with ATP or ADP. The model of ATP bound Hsp70/CHIP/E2‐Ub complex has an average distance of ~120 Å between the substrate‐binding domain (SBD) and the E2‐ubiquitin active site. The same distance of the multiprotein complex is found to be decreased to ~60 Å when Hsp70 is bound to ADP. Since the ADP bound conformation is considered as the active conformation that tightly binds client proteins, the alteration of the average distance between the Hsp70 SBD and the E2‐Ub active site implies that the Lys residue of the substrate might need to be positioned within appropriate distance to span the gap in order to efficiently promote ubiquitination. Here, we used molecular dynamic simulations, double electron‐electron resonance (DEER) EPR spectroscopy and in‐vitroubiquitination assays with designed model substrate proteins that place lysine residues at variable positions. We found the dynamics of the Hsp70/CHIP/E2‐Ubiquitin complex is playing a major role in the ubiquitination of various client proteins and these dynamics are necessary to overcome the distance barrier between E2‐Ub active site and the Hsp70 SBD. Our data also suggests that the Hsp70/CHIP complex is capable of ubiquitinating a client protein substrate when lysine residues are placed at several disparate positions. In sum, our data reveals the functional dynamics of the Hsp70/CHIP/E2‐Ub complex which enable ubiquitination of a client protein.

Compactness regularization in the analysis of dipolar EPR spectroscopy data

Dipolar electron paramagnetic resonance (EPR) experiments, such as double electron–electron resonance (DEER), measure distributions of nanometer-scale distances between paramagnetic centers, which are valuable for structural characterization of proteins and other macromolecular systems. One challenge in the least-squares fitting analysis of dipolar EPR data is the separation of the inter-molecular contribution (background) and the intra-molecular contribution. For noisy experimental traces of insufficient length, this separation is not unique, leading to identifiability problems for the background model parameters and the long-distance region of the intra-molecular distance distribution. Here, we introduce a regularization approach that mitigates this by including an additional penalty term in the objective function that is proportional to the variance of the distance distribution and thereby penalizes non-compact distributions. We examine the reliability of this approach statistically on a large set of synthetic data and illustrate it with an experimental example. The results show that the introduction of compactness can improve identifiability.

DEER experiments reveal fundamental differences between calmodulin complexes with IQ and MARCKS peptides in solution

Calmodulin (CaM) is a calcium-binding protein that regulates the function of many proteins by indirectly conferring Ca2+ sensitivity, and it undergoes a large conformational change on partners' binding. We compared the solution binding mode of the target peptides MARCKS and IQ by double electron-electron resonance (DEER) distance measurements and paramagnetic NMR. We combined nitroxide and Gd(III) spin labels, including specific substitution of one of the Ca2+ ions in the CaM mutant N60D by a Gd(III) ion. The binding of MARCKS to holo-CaM resulted neither in a closed conformation nor in a unique relative orientation between the two CaM domains, in contrast with the crystal structure. Binding of IQ to holo-CaM did generate a closed conformation. Using elastic network modeling and 12 distance restraints obtained from multiple holo-CaM/IQ DEER data, we derived a model of the solution structure, which is in reasonable agreement with the crystal structure.

Calculation of pulsed EPR DEER signal for two coupled Gd3+ ions by dipolar interaction using rotating frames

A method, based on the use of rotating frames, to either consider only the rotating frame of the observer spin, which is the main rotating frame, or to consider only the rotating frame of the pump spin, used only during the application of the pump pulse, has been exploited to calculate accurately the four-pulse EPR (Electron Paramagnetic Resonance) DEER (Double Electron-Electron Resonance) signal for two Gd3+ ions, coupled by the dipolar interaction in a biradical in a biological system, including the zero-field splitting of each Gd3+ ion. It has been illustrated here by calculating the four-pulse DEER signals at W-band for three values of the dipolar constant: ωdd=0.63MHz, ωdd=0.49MHz and ωdd=0.39MHz, corresponding to the range of typical Gd3+−Gd3+ distance (r) values, r=4.36 nm, 4.73 nm, 5.10 nm, respectively, in a sample of Gd-DOTA.

Enhancement of the creation yield of NV ensembles in a chemically vapour deposited diamond

In this work we investigate the properties of negatively charged nitrogen-vacancy (NV−) centres created during single crystal diamond growth by chemical vapour deposition (CVD) on [113]-oriented substrates and with N2O as a dopant gas. The use of spin echo and double electron-electron resonance (DEER) allows us to assess NV− ratio with respect to substitutional nitrogen impurities (Ns0) incorporated during growth, a critical figure of merit for quantum technologies. We demonstrate that, at moderate growth temperatures (800 °C), dense NV− ensembles of several hundreds of ppb (800 ppb for 50 ppm of added N2O) and with exceptionally high NV−/ Ns0 ratios of up to 25% can be achieved. This NV− creation yield is higher by at least an order of magnitude to that typically obtained on standard [100]-grown diamonds and comparable to the best values reported for electron-irradiated diamonds. The material obtained here thus advantageously combines a high NV− density, high creation yield, long coherence times of several tens of μs together with a partial preferential orientation. These are highly desirable requirements for diamond-based quantum sensors that may spur new developments with this crystalline orientation leading to improved performance and sensitivity.

Neural networks in pulsed dipolar spectroscopy: A practical guide

This is a methodological guide to the use of deep neural networks in the processing of pulsed dipolar spectroscopy (PDS) data encountered in structural biology, organic photovoltaics, photosynthesis research, and other domains featuring long-lived radical pairs and paramagnetic metal ions. PDS uses distance dependence of magnetic dipolar interactions; measuring a single well-defined distance is straightforward, but extracting distance distributions is a hard and mathematically ill-posed problem requiring careful regularisation and background fitting. Neural networks do this exceptionally well, but their “robust black box” reputation hides the complexity of their design and training – particularly when the training dataset is effectively infinite. The objective of this paper is to give insight into training against simulated databases, to discuss network architecture choices, to describe options for handling DEER (double electron-electron resonance) and RIDME (relaxation-induced dipolar modulation enhancement) experiments, and to provide a practical data processing flowchart.

Human CEACAM1 N-domain dimerization is independent from glycan modifications

Carcinoembryonic cellular adhesion molecules (CEACAMs) serve diverse roles in cell signaling, proliferation, and survival and are made up of one or several immunoglobulin (Ig)-like ectodomains glycosylated invivo. The physiological oligomeric state and how it contributes to protein function are central to understanding CEACAMs. Two putative dimer conformations involving different CEACAM1 N-terminal Ig-like domain (CCM1) protein faces (ABED and GFCC'C″) were identified from crystal structures. GFCC'C″ was identified as the dominant CCM1 solution dimer, but ambiguity regarding the effect of glycosylation on dimer formation calls its physiological relevance into question. We present the first crystal structure of minimally glycosylated CCM1 in the GFCC'C″ dimer conformation and characterization in solution by continuous-wave and double electron-electron resonance electron paramagnetic resonance spectroscopy. Our results suggest the GFCC'C″ dimer is dominant in solution with different levels of glycosylation, and structural conservation and co-evolved residues support that the GFCC'C″ dimer is conserved across CEACAMs.

Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer

Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterize Ab1303 and Ab1573, heterologously-neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding is observed only when Env trimers are not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures show that both antibodies recognize the CD4bs on Env trimer with an ‘occluded-open’ conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation includes outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, does not exhibit V1V2 displacement, 4-stranded gp120 bridging sheet, or co-receptor binding site exposure. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggest an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.

In situ distance measurements in a membrane transporter using maleimide functionalized orthogonal spin labels and 5-pulse electron-electron double resonance spectroscopy

Spectroscopic investigation of membrane proteins in their native environment is a challenging task. Earlier we demonstrated the feasibility to measure precise distances within outer membrane proteins in E. coli and native membranes using methanethiosulfonate (MTS) functionalized labels combined with pulsed electron-electron double resonance spectroscopy. Here we show the application of maleimide functionalized Gd(III), nitroxide, and trityl labels for in situ distance measurement using the cobalamin transporter BtuB. These labels enabled distance measurements for BtuB in E. coli and native outer membranes and in the membranes maleimide-Gd-DOTA also is effective. Further, we show that the observable dipolar evolution time can be significantly prolonged in the native environments using the Carr-Purcell 5-pulse electron double resonance sequence. For a nitroxide-nitroxide pair, application of sech/tanh inversion pulses substantially suppressed the 4-pulse artifact at the Q-band frequency. In the case of a nitroxide-trityl pair, Gaussian pump pulses of varying amplitude are sufficient to suppress the artifact to the typical noise level. The feasibility of a range of bioresistant spin labels and the 5-pulse electron double resonance offers promising tools for investigating heterooligomeric membrane protein complexes in their native environment.

Accessing protein conformational distributions in mammalian cells with fast, bio-orthogonal spin-labeling and DEER spectroscopy

The ability to measure protein structure and dynamics directly within the cellular environment is fundamental to understanding the molecular mechanisms of protein function and dysfunction. Site-directed spin-labeling, in combination with double electron-electron resonance (DEER) spectroscopy, is a powerful method for determining both the structural states and the conformational equilibria of biomacromolecules. The application of DEER to expressed proteins within cells, however, has been hindered by issues of sensitivity, toxicity, specificity and extent of labeling, and spin-label stability.

Time-resolved DEER EPR and solid-state NMR afford kinetic and structural elucidation of substrate binding to Ca2+-ligated calmodulin

Recent advances in rapid mixing and freeze quenching have opened the path for time-resolved electron paramagnetic resonance (EPR)-based double electron-electron resonance (DEER) and solid-state NMR of protein-substrate interactions. DEER, in conjunction with phase memory time filtering to quantitatively extract species populations, permits monitoring time-dependent probability distance distributions between pairs of spin labels, while solid-state NMR provides quantitative residue-specific information on the appearance of structural order and the development of intermolecular contacts between substrate and protein. Here, we demonstrate the power of these combined approaches to unravel the kinetic and structural pathways in the binding of the intrinsically disordered peptide substrate (M13) derived from myosin light-chain kinase to the universal eukaryotic calcium regulator, calmodulin. Global kinetic analysis of the data reveals coupled folding and binding of the peptide associated with large spatial rearrangements of the two domains of calmodulin. The initial binding events involve a bifurcating pathway in which the M13 peptide associates via either its N- or C-terminal regions with the C- or N-terminal domains, respectively, of calmodulin/4Ca2+ to yield two extended "encounter" complexes, states A and A*, without conformational ordering of M13. State A is immediately converted to the final compact complex, state C, on a timescale τ ≤ 600 μs. State A*, however, only reaches the final complex via a collapsed intermediate B (τ ∼ 1.5 to 2.5 ms), in which the peptide is only partially ordered and not all intermolecular contacts are formed. State B then undergoes a relatively slow (τ ∼ 7 to 18 ms) conformational rearrangement to state C.

EPR deer distance measurements on KCNE3 in proteoliposomes

KCNE3 is a single pass membrane protein involved in modulating the function and trafficking of variety of voltage gated potassium ion channels. A three dimensional structure of KCNE3 was recently determined using a solution NMR spectroscopy in isotropic DHPC/DMPG bicelles by the Sanders group. However, the structural topology and dynamic properties of KCNE3 are not fully understood in its native like environment. Double electron electron resonance (DEER), an advanced electron paramagnetic resonance (EPR) spectroscopic technique, is a very powerful and rapidly growing structure biology tool to study structural dynamics of a membrane protein by measuring distances within the range of 20-80 Å between two spin-labeled sites.

Is Cys(MTSL) the Best α-Amino Acid Residue to Electron Spin Labeling of Synthetically Accessible Peptide Molecules with Nitroxides?

Electron paramagnetic resonance spectroscopy, particularly its pulse technique double electron–electron resonance (DEER) (also termed PELDOR), is rapidly becoming an extremely useful tool for the experimental determination of side chain-to-side chain distances between free radicals in molecules fundamental for life, such as polypeptides. Among appropriate probes, the most popular are undoubtedly nitroxide electron spin labels. In this context, suitable biosynthetically derived, helical regions of proteins, along with synthetic peptides with amphiphilic properties and antibacterial activities, are the most extensively investigated compounds. A strict requirement for a precise distance measurement has been identified in a minimal dynamic flexibility of the two nitroxide-bearing α-amino acid side chains. To this end, in this study, we have experimentally compared in detail the side-chain mobility properties of the two currently most widely utilized residues, namely, Cys(MTSL) and 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). In particular, two double-labeled, chemically synthesized 20-mer peptide molecules have been adopted as appropriate templates for our investigation on the determination of the model intramolecular separations. These double-Cys(MTSL) and double-TOAC compounds are both analogues of the almost completely rigid backbone peptide ruler which we have envisaged and 3D structurally analyzed as our original, unlabeled compound. Here, we have clearly found that the TOAC side-chain labels are largely more 3D structurally restricted than the MTSL labels. From this result, we conclude that the TOAC residue offers more precise information than the Cys(MTSL) residue on the side chain-to-side chain distance distribution in synthetically accessible peptide molecules.

Simple Cryoprotectant-Free Method to Advance Pulsed Dipolar ESR Spectroscopy for Capturing Protein Conformational Ensembles.

Double electron-electron resonance (DEER) is a powerful technique for studying protein conformations. To preserve the room-temperature ensemble, proteins are usually shock-frozen in liquid nitrogen prior to DEER measurements. The use of cryoprotectant additives is, therefore, necessary to ensure the formation of a vitrified state. Here, we present a simple modification of the freezing process using a flexible fused silica microcapillary, which increases the freezing rates and thus enables DEER measurement without the use of cryoprotectants. The Bid protein, which is highly sensitive to cryoprotectant additives, is used as a model. We show that DEER with the simple modification can successfully reveal the cold denaturation of Bid, which was not possible with the conventional DEER preparations. The DEER result reveals the nature of Bid folding. Our method advances DEER for capturing the chemically and thermally induced conformational changes of a protein in a cryoprotectant-free medium.

Fullerene-based triplet spin labels: methodology aspects for pulsed dipolar EPR spectroscopy.

Triplet states of photoexcited organic molecules are promising spin labels with advanced spectroscopic properties for pulsed dipolar electron paramagnetic resonance (PD EPR) spectroscopy. Recently proposed triplet fullerene labels have shown great potential for double electron-electron resonance (DEER) distance measurements as "observer spins" due to a high quantum yield of the triplet state, hyperpolarization and relatively narrow EPR spectra. Here, we demonstrate the applicability of fullerene labels to other PD EPR techniques, such as relaxation induced dipolar modulation enhancement (RIDME) and laser induced magnetic dipolar spectroscopy (LaserIMD). In particular, a specific contaminating signal in LaserIMD experiments was observed, explained and mitigated. Comparative analyses of the signal-to-noise (SNR) ratios were performed for all employed methods. DEER on the fullerene-triarylmethyl pair shows the best performance, which allows state-of-the-art DEER acquisition at 100 nM with a SNR of ∼35 within reasonable 42 hours.

Dipolar pathways in dipolar EPR spectroscopy.

Dipolar electron paramagnetic resonance (EPR) experiments such as double electron-electron resonance (DEER) measure distributions of nanometer-scale distances between unpaired electrons, which provide valuable information for structural characterization of proteins and other macromolecular systems. To determine these distributions from the experimental signal, it is critical to employ an accurate model of the signal. For dilute samples of doubly spin-labeled molecules, the signal is a product of an intramolecular and an intermolecular contribution. We present a general model based on dipolar pathways valid for dipolar EPR experiments with spin-1/2 labels. Our results show that the intramolecular contribution consists of a sum and the intermolecular contribution consists of a product over individual dipolar pathway contributions. We examine several commonly used dipolar EPR experiments in terms of dipolar pathways and show experimental results confirming the theoretical predictions. This multi-pathway model makes it possible to analyze a wide range of dipolar EPR experiments within a single theoretical framework.

Tracking protein domain movements by EPR distance determination and multilateration.

Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful approach to supplement the large toolbox of methods for protein structure determination. The strength of EPR spectroscopy is the ability to map flexibility of protein domains and conformational ensembles. EPR distance determination in the nanometer range and subsequent multilateration enables the three-dimensional visualization of the localization of a spin label in a protein domain. Therefore, multilateration can not only be used to represent the degree of flexibility of protein structural elements, but also track movements of whole domains. We report a detailed protocol for all needed steps, beginning with the choice of the labeling sites, the spin labeling reaction, the EPR distance measurement by double electron-electron resonance (DEER) and finally the multilateration exploiting the EPR distance restraints.

Dipolar pathways in multi-spin and multi-dimensional dipolar EPR spectroscopy.

Dipolar electron paramagnetic resonance (EPR) experiments, such as double electron-electron resonance (DEER), measure distributions of nanometer-scale distances between unpaired electrons, which provide valuable information for structural characterization of proteins and other macromolecular systems. We present an extension to our previously published general model based on dipolar pathways valid for multi-dimensional dipolar EPR experiments with more than two spin-1/2 labels. We examine the 4-pulse DEER and TRIER experiments in terms of dipolar pathways and show experimental results confirming the theoretical predictions. This extension to the dipolar pathways model allows the analysis of previously challenging datasets and the extraction of multivariate distance distributions.

Milliwatt three- and four-pulse double electron electron resonance for protein structure determination.

Electron paramagnetic resonance (EPR) experiments for protein structure determination using double electron-electron resonance (DEER) spectroscopy rely on high-power microwave amplifiers (>300 W) to create the short pulse lengths needed to excite a sizable portion of the spectrum. The recently introduced self-resonant microhelix combines a high B1 conversion efficiency with an intrinsically large bandwidth (low Q-value) and a high absolute sensitivity. We report dead times in 3-pulse DEER experiments as low as 14 ± 2 ns achieved using less than 1 W of power at X-band (nominally 9.5 GHz) for experiments on a molecular ruler and a T4 lysozyme sample for concentrations down to 100 μM. These low-power experiments were performed using an active volume 120 times smaller than that of a standard pulse EPR resonator, while only a 11-fold decrease in the signal-to-noise ratio was observed. Small build sizes, as realized with the microhelix, give access to volume-limited samples, while shorter dead times allow the investigation of fast relaxing spin species. With the significantly reduced dead times, the 3-pulse DEER experiment can be revisited. Here, we show experimentally that 3-pulse DEER offers superior sensitivity over 4-pulse DEER. We assert that the microhelix paves the road for low-cost benchtop X-band pulse EPR spectrometers by eliminating the need for high-power amplifiers, accelerating the adoption of pulse EPR to a broader community.