Biophysical Characterization of Pro-apoptotic BimBH3 Peptides Reveals an Unexpected Capacity for Self-Association

Bcl-2 proteins orchestrate the mitochondrial pathway of apoptosis, pivotal for cell death. Yet, the structural details of the conformational changes of pro- and antiapoptotic proteins and their interactions remain unclear. Pulse dipolar spectroscopy (double electron-electron resonance [DEER], also known as PELDOR) in combination with spin-labeled apoptotic Bcl-2 proteins unveils conformational changes and interactions of each protein player via detection of intra- and inter-protein distances. Here, we present the synthesis and characterization of pro-apoptotic BimBH3 peptides of different lengths carrying cysteines for labeling with nitroxide or gadolinium spin probes. We show by DEER that the length of the peptides modulates their homo-interactions in the absence of other Bcl-2 proteins and solve by X-ray crystallography the structure of a BimBH3 tetramer, revealing the molecular details of the inter-peptide interactions. Finally, we prove that using orthogonal labels and three-channel DEER we can disentangle the Bim-Bim, Bcl-xL-Bcl-xL, and Bim-Bcl-xL interactions in a simplified interactome.This work was funded by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—EXC-2033—Projektnummer 390677874, the DFG Priority Program SPP1601 “New Frontiers in Sensitivity in EPR Spectroscopy” (to E.B.), DFG BO 3000/5-1 (to E.B.), SFB958 – Z04 (to E.B.), DFG grant INST 130/972-1 FUGG (to E.B.). P.E.C. is supported by an Australian NHMRC fellowship (1079700

Improved signal fidelity in 4-pulse DEER with Gaussian pulses

The introduction of arbitrary waveform generator (AWG) technology and the availability of high power microwave amplifiers mark a ‘‘new era” in pulse EPR due to significant sensitivity improvements and the possibility to perform novel types of experiments. We present an optimized 4-pulse DEER setup that uses Gaussian observer pulses (GaussDEER) in connection with a Gaussian/shaped pump pulse. Gaussian pulses allow to experimentally remove the ‘‘2+1” pulse train ESE signal which is intrinsically present in any DEER experiment performed with rectangular pulses. Further signal improvements are obtained with shaped pump pulses, which can significantly increase the modulation depth of the DEER experiment due to their tailored excitation bandwidth. Although sequences like CP (Carr-Purcell) DEER offer advan- tages such as a prolongation of the dipolar evolution time, they suffer from post-processing of the time- domain data to remove artifacts. Therefore, it is worth having a 4-pulse DEER experiment free of residual ‘‘2+1” signal since this is still the main dipolar spectroscopic technique used in structural biology. In this work we focus on nitroxides, which are the spin probes primarily used in site-directed spin labeling studies of biomolecules, however, the advantages introduced by Gaussian pulses can be extended to any spin type.</p

Method development and state of the art EPR techniques for new insights into apoptosis

Diese Arbeit strebt danach eine Brücke zwischen Elektronenspinresonanz (EPR) -Methodenentwicklung und der Anwendung modernster EPR-Techniken auf ein komplexes Proteinsystem zu bauen. Die Sensitivität und Signaltreue des Doppel-Elektron-Elektron-Resonanz (DEER) Experiments kann durch die Verwendung von gaußförmigen Pulsen erheblich verbessert werden. Außerdem wird experimentell gezeigt wie Signalübersprechen zwischen DEER Kanälen in Proben entsteht, die orthogonal mit Nitroxiden und Gadolinium spinmarkiert sind. Dazu werden Identifikations- und Unterdrückungsstrategien eingeführt und diskutiert. Die Anwendung von orthogonalen Spinmarkierungsstrategien zur Erforschung der Bcl-2-Familie, die der primäre Regulator des mitochondrialen Pfades der Apoptose ist, bietet neue Einblicke in das Bcl-2-Interaktom. Dem Bestreben folgend Proteine in physiologischeren Umgebungen zu messen, wird untersucht, wie geeignete Protein/Spinmarkierungs-Kombinationen für In-Zell-Studien gefunden werden können.This thesis strives to span a bridge between electron paramagnetic resonance (EPR) method development and the application of state of the art EPR techniques to a complex protein system. Double electron-electron resonance (DEER) sensitivity and signal fidelity can be considerably improved by the utilization of Gaussian pulses that allow to remove the "2+1" pulse train artifact. Moreover, it is shown experimentally how DEER channel cross-talk signals appear in samples that are orthogonally spin-labeled with nitroxide and gadolinium labels. Both identification and suppression strategies are introduced and discussed. The introduction of orthogonal spin labeling strategies to the Bcl-2 family, which is the primary regulator of the mitochondrial pathway of apoptosis, offers new insights into the Bcl-2 interactome. Following the aspiration to measure proteins in more physiological environments, it is addressed how suitable protein/spin label combinations can be found for in-cell studies

Orthogonal spin labeling and pulsed dipolar spectroscopy for protein studies

Different types of spin labels are currently available for structural studies of biomolecules both in vitro and in cells using Electron Paramagnetic Resonance (EPR) and pulse dipolar spectroscopy (PDS). Each type of label has its own advantages and disadvantages, that will be addressed in this chapter. The spectroscopically distinct properties of the labels have fostered new applications of PDS aimed to simultaneously extract multiple inter-label distances on the same sample. In fact, combining different labels and choosing the optimal strategy to address their inter-label distances can increase the information content per sample, and this is pivotal to better characterize complex multi-component biomolecular systems. In this review, we provide a brief background of the spectroscopic properties of the four most common orthogonal spin labels for PDS measurements and focus on the various methods at disposal to extract homo- and hetero-label distances in proteins. We also devote a section to possible artifacts arising from channel crosstalk and provide few examples of applications in structural biology.</p

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 very high incident microwave powers (>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 as low as 14±2 ns achieved using less than 1 W of power at X-band (nominally 9.5 GHz) on a molecular ruler and a T4-lysozyme sample. These low-power experiments were performed using an active volume 120 times smaller than that of a standard pulse EPR resonator, while only a sixfold 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 the 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

Orthogonally spin-labeled rulers help to identify crosstalk signals and improve DEER signal fidelity

DEER spectroscopy applied to orthogonally spin-labeled proteins is a versatile technique which allows simplifying the assignment of distances in complex spin systems and thereby increasing the information content that can be obtained per sample. In fact, orthogonal spin labels can be independently addressed in DEER experiments due to spectroscopically non-overlapping central transitions, distinct relaxation times and/or transition moments. Here we focus on molecular rulers orthogonally labeled with nitroxide (NO) and gadolinium (Gd) spins, which give access to three distinct DEER channels, probing NO-NO, NO-Gd and Gd-Gd distances. It has been previously suggested that crosstalk signals between individual DEER channels might occur, for example, between NO and Gd due to their inevitable spectral overlap. However, a systematic study to address these issues has not yet been carried out. Here, we perform a thorough three-channel DEER analysis on mixtures of NO-NO, NO-Gd and Gd-Gd molecular rulers characterized by distinct, non-overlapping distance distributions to study under which conditions crosstalk signals occur and how they can be identified or suppressed to improve signal fidelity. This study will help to improve the assignment of the correct distances in homo- and hetero-complexes of orthogonally spin-labeled proteins.</p

A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels

Bax is a Bcl-2 protein crucial for apoptosis initiation and execution, whose active conformation is only partially understood. Dipolar EPR spectroscopy has proven to be a valuable tool to determine coarse-grained models of membrane-embedded Bcl-2 proteins. Here we show how the combination of spectroscopically distinguishable nitroxide and gadolinium spin labels and Double Electron-Electron Resonance can help to gain new insights into the quaternary structure of active, membrane-embedded Bax oligomers. We show that attaching labels bulkier than the conventional MTSL may affect Bax fold and activity, depending on the protein/label combination. However, we identified a suitable pair of spectroscopically distinguishable labels, which allows to study complex distance networks in the oligomers that could not be disentangled before. Additionally, we compared the stability of the different spin-labeled protein variants in E . coli and HeLa cell extracts. We found that the gem -diethyl nitroxide-labeled Bax variants were reasonably stable in HeLa cell extracts. However, when transferred into human cells, Bax was found to be mislocalized, thus preventing its characterization in a physiological environment. The successful use of spectroscopically distinguishable labels on membrane-embedded Bax-oligomers opens an exciting new path towards structure determination of membrane-embedded homo- or hetero-oligomeric Bcl-2 proteins via EPR.</p

Strategies to identify and suppress crosstalk signals in double electron–electron resonance (DEER) experiments with gadoliniumIII^{III} and nitroxide spin-labeled compounds

Double electron-electron resonance (DEER) spectroscopy applied to orthogonally spin-labeled biomolecular complexes simplifies the assignment of intra- and intermolecular distances, thereby increasing the information content per sample. In fact, various spin labels can be addressed independently in DEER experiments due to spectroscopically nonoverlapping central transitions, distinct relaxation times, and/or transition moments; hence, they are referred to as spectroscopically orthogonal. Molecular complexes which are, for example, orthogonally spin-labeled with nitroxide (NO) and gadolinium (Gd) labels give access to three distinct DEER channels that are optimized to selectively probe NO–NO, NO–Gd, and Gd–Gd distances. Nevertheless, it has been previously recognized that crosstalk signals between individual DEER channels can occur, for example, when a Gd–Gd distance appears in a DEER channel optimized to detect NO–Gd distances. This is caused by residual spectral overlap between NO and Gd spins which, therefore, cannot be considered as perfectly orthogonal. Here, we present a systematic study on how to identify and suppress crosstalk signals that can appear in DEER experiments using mixtures of NO–NO, NO–Gd, and Gd–Gd molecular rulers characterized by distinct, nonoverlapping distance distributions. This study will help to correctly assign the distance peaks in homo- and heterocomplexes of biomolecules carrying not perfectly orthogonal spin labels

Dissecting the Molecular Origin of <i>g</i> -Tensor Heterogeneity and Strain in Nitroxide Radicals in Water: Electron Paramagnetic Resonance Experiment versus Theory

Nitroxides are common EPR sensors of microenvironmental properties such as polarity, numbers of H-bonds, pH, and so forth. Their solvation in an aqueous environment is facilitated by their high propensity to form H-bonds with the surrounding water molecules. Their g- and A-tensor elements are key parameters to extracting the properties of their microenvironment. In particular, the gxx value of nitroxides is rich in information. It is known to be characterized by discrete values representing nitroxide populations previously assigned to have different H-bonds with the surrounding waters. Additionally, there is a large g-strain, that is, a broadening of g-values associated with it, which is generally correlated with environmental and structural micro-heterogeneities. The g-strain is responsible for the frequency dependence of the apparent line width of the EPR spectra, which becomes evident at high field/frequency. Here, we address the molecular origin of the gxx heterogeneity and of the g-strain of a nitroxide moiety (HMI: 2,2,3,4,5,5-hexamethylimidazolidin-1-oxyl, C9H19N2O) in water. To treat the solvation effect on the g-strain, we combined a multi-frequency experimental approach with ab initio molecular dynamics simulations for structural sampling and quantum chemical EPR property calculations at the highest realistically affordable level, including an explicitly micro-solvated HMI ensemble and the embedded cluster reference interaction site model. We could clearly identify the distinct populations of the H-bonded nitroxides responsible for the gxx heterogeneity experimentally observed, and we dissected the role of the solvation shell, H-bond formation, and structural deformation of the nitroxide in the creation of the g-strain associated with each nitroxide subensemble. Two contributions to the g-strain were identified in this study. The first contribution depends on the number of hydrogen bonds formed between the nitroxide and the solvent because this has a large and well-understood effect on the gxx-shift. This contribution can only be resolved at high resonance frequencies, where it leads to distinct peaks in the gxx region. The second contribution arises from configurational fluctuations of the nitroxide that necessarily lead to g-shift heterogeneity. These contributions cannot be resolved experimentally as distinct resonances but add to the line broadening. They can be quantitatively analyzed by studying the apparent line width as a function of microwave frequency. Interestingly, both theory and experiment confirm that this contribution is independent of the number of H-bonds. Perhaps even more surprisingly, the theoretical analysis suggests that the configurational fluctuation broadening is not induced by the solvent but is inherently present even in the gas phase. Moreover, the calculations predict that this broadening decreases upon solvation of the nitroxide.</p

Spectroscopically Orthogonal Spin Labels in Structural Biology at Physiological Temperatures

Electron paramagnetic resonance spectroscopy (EPR) is mostly used in structural biology in conjunction with pulsed dipolar spectroscopy (PDS) methods to monitor interspin distances in biomacromolecules at cryogenic temperatures both in vitro and in cells. In this context, spectroscopically orthogonal spin labels were shown to increase the information content that can be gained per sample. Here, we exploit the characteristic properties of gadolinium and nitroxide spin labels at physiological temperatures to study side chain dynamics via continuous wave (cw) EPR at X band, surface water dynamics via Overhauser dynamic nuclear polarization at X band and short-range distances via cw EPR at high fields. The presented approaches further increase the accessible information content on biomolecules tagged with orthogonal labels providing insights into molecular interactions and dynamic equilibria that are only revealed under physiological conditions