Four-pulse Double Electron-electron Resonance Research Articles

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.

Conformational Differences Are Observed for the Active and Inactive Forms of Pinholin S21 Using DEER Spectroscopy.

Bacteriophages have evolved with an efficient host cell lysis mechanism to terminate the infection cycle and release the new progeny virions at the optimum time, allowing adaptation with the changing host and environment. Among the lytic proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time known as "holin triggering". Pinholin S21 is a prototype holin of phage Φ21 which makes many nanoscale holes and destroys the proton motive force, which in turn activates the signal anchor release (SAR) endolysin system to degrade the peptidoglycan layer of the host cell and destruction of the outer membrane by the spanin complex. Like many others, phage Φ21 has two holin proteins: active pinholin and antipinholin. The antipinholin form differs only by three extra amino acids at the N-terminus; however, it has a different structural topology and conformation with respect to the membrane. Predefined combinations of active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously, the dynamics and topology of active pinholin and antipinholin were investigated (Ahammad et al. JPCB 2019, 2020) using continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. However, detailed structural studies and direct comparison of these two forms of pinholin S21 are absent in the literature. In this study, the structural topology and conformations of active pinholin (S2168) and inactive antipinholin (S2168IRS) in DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) proteoliposomes were investigated using the four-pulse double electron-electron resonance (DEER) EPR spectroscopic technique to measure distances between transmembrane domains 1 and 2 (TMD1 and TMD2). Five sets of interlabel distances were measured via DEER spectroscopy for both the active and inactive forms of pinholin S21. Structural models of the active pinholin and inactive antipinholin forms in DMPC proteoliposomes were obtained using the experimental DEER distances coupled with the simulated annealing software package Xplor-NIH. TMD2 of S2168 remains in the lipid bilayer, and TMD1 is partially externalized from the bilayer with some residues located on the surface. However, both TMDs remain incorporated in the lipid bilayer for the inactive S2168IRS form. This study demonstrates, for the first time, clear structural topology and conformational differences between the two forms of pinholin S21. This work will pave the way for further studies of other holin systems using the DEER spectroscopic technique and will give structural insight into these biological clocks in molecular detail.

CW EPR and DEER Methods to Determine BCL-2 Family Protein Structure and Interactions: Application of Site-Directed Spin Labeling to BAK Apoptotic Pores.

The continuous wave (CW) and pulse electron paramagnetic resonance (EPR) methods enable the measurement of distances between spin-labeled residues in biopolymers including proteins, providing structural information. Here we describe the CW EPR deconvolution/convolution method and the four-pulse double electron-electron resonance (DEER) approach for distance determination, which were applied to elucidate the organization of the BAK apoptotic pores formed in the lipid bilayers.

Exploring the pH-Induced Functional Phase Space of Human Serum Albumin by EPR Spectroscopy

A systematic study on the self-assembled solution system of human serum albumin (HSA) and paramagnetic doxyl stearic acid (5-DSA and 16-DSA) ligands is reported covering the broad pH range 0.7–12.9, mainly using electron paramagnetic resonance (EPR) methods. It is tested to which extent the pH-induced conformational isomers of HSA reveal themselves in continuous wave (CW) EPR spectra from this spin probing approach in comparison to an established spin-labeling strategy utilizing 3-maleimido proxyl (5-MSL). Most analyses are conducted on empirical levels with robust strategies that allow for the detection of dynamic changes of ligand, as well as protein. Special emphasis has been placed on the EPR spectroscopic detection of a molten globule (MG) state of HSA that is typically found by the fluorescent probe 8-Anilino- naphthalene-1-sulfonic acid (ANS). Moreover, four-pulse double electron-electron resonance (DEER) experiments are conducted and substantiated with dynamic light scattering (DLS) data to determine changes in the solution shape of HSA with pH. All results are ultimately combined in a detailed scheme that describes the pH-induced functional phase space of HSA.

Double Electron–Electron Resonance Between Trapped Electron and Hole in a Semiconductor

We demonstrate the spin interactions between dispersedly trapped electrons and holes in a semiconductor using the double electron–electron resonance (DEER) method of the pulsed electron paramagnetic resonance (EPR) techniques. An aluminum-doped titanium dioxide crystal is adopted as a spin system, in which optically generated electrons and holes are trapped, to reveal EPR signals that appear close to each other at a selected crystal orientation under an external magnetic field. We used the four-pulse DEER method by applying two microwave frequencies to a microwave cavity for pumping electrons and probing holes at the optimum temperature of 32 K. The dipolar modulation in the probed signal by pumping interacting spins was successfully detected. The observed non-oscillating decay shape indicates that the detected interaction is caused by widely distributed trapped electron and hole spins over long distances. We were able to extract a spin-pair distribution function by the first derivative of a background-corrected curve, referring to a previously reported method.

Combining NMR and EPR to Determine Structures of Large RNAs and Protein-RNA Complexes in Solution.

Although functional significance of large noncoding RNAs and their complexes with proteins is well recognized, structural information for this class of systems is very scarce. Their inherent flexibility causes problems in crystallographic approaches, while their typical size is beyond the limits of state-of-the-art purely NMR-based approaches. Here, we review an approach that combines high-resolution NMR restraints with lower resolution long-range constraints based on site-directed spin labeling and measurements of distance distribution restraints in the range between 15 and 80Å by the four-pulse double electron-electron resonance (DEER) EPR technique. We discuss sample preparation, the basic assumptions behind data analysis in the EPR-based distance measurements, treatment of the label-based constraints in generation of the structure, and the back-calculation of distance distributions for structure validation. Step-by-step protocols are provided for DEER distance distribution measurements including data analysis and for CYANA based structure calculation using combined NMR and EPR data.

DEER-Stitch: Combining three- and four-pulse DEER measurements for high sensitivity, deadtime free data

Over approximately the last 15years the electron paramagnetic resonance (EPR) technique of double electron electron resonance (DEER) has attracted considerable attention since it allows for the precise measurement of the dipole–dipole coupling between radicals and thus can lead to distance information between pairs of radicals separated by up to ca. 8nm. The “deadtime free” 4-pulse DEER sequence is widely used but can suffer from poor sensitivity if the electron spin-echo decays too quickly to allow collection of a sufficiently long time trace. In this paper we present a method which takes advantage of the much greater sensitivity that the 3-pulse sequence offers over the 4-pulse sequence since the measured electron spin-echo intensity (for equal sequence lengths) is greater. By combining 3- and 4-pulse DEER time traces using a method coined DEER-Stitch (DEERS) accurate dipole–dipole coupling measurements can be made which combine the sensitivity of the 3-pulse DEER sequence with the deadtime free advantage of the 4-pulse DEER sequence. To develop the DEER-Stitch method three systems were measured: a semi-rigid bis-nitroxide labeled nanowire, the bis-nitroxide labeled protein CD55 with a distance between labels of almost 8nm and a dimeric copper amine oxidase from Arthrobacter globiformis (AGAO).

The global analysis of DEER data

Double Electron–Electron Resonance (DEER) has emerged as a powerful technique for measuring long range distances and distance distributions between paramagnetic centers in biomolecules. This information can then be used to characterize functionally relevant structural and dynamic properties of biological molecules and their macromolecular assemblies. Approaches have been developed for analyzing experimental data from standard four-pulse DEER experiments to extract distance distributions. However, these methods typically use an a priori baseline correction to account for background signals. In the current work an approach is described for direct fitting of the DEER signal using a model for the distance distribution which permits a rigorous error analysis of the fitting parameters. Moreover, this approach does not require a priori background correction of the experimental data and can take into account excluded volume effects on the background signal when necessary. The global analysis of multiple DEER data sets is also demonstrated. Global analysis has the potential to provide new capabilities for extracting distance distributions and additional structural parameters in a wide range of studies.

Dead-time free measurement of dipole–dipole interactions between electron spins

A four-pulse version of the pulse double electron–electron resonance (DEER) experiment is presented, which is designed for the determination of interradical distances on a nanoscopic lengthscale. With the new pulse sequence electron–electron couplings can be studied without dead-time artifacts, so that even broad distributions of electron–electron distances can be characterized. A version of the experiment that uses a pulse train in the detection period exhibits improved signal-to-noise ratio. Tests on two nitroxide biradicals with known length indicate that the accessible range of distances extends from about 1.5 to 8 nm. The four-pulse DEER spectra of an ionic spin probe in an ionomer exhibit features due to probe molecules situated both on the same and on different ion clusters. The former feature provides information on the cluster size and is inaccessible with previous methods.

Nanometer-Scale Distance Measurements in Proteins Using Gd3+ Spin Labeling

Methods for measuring nanometer-scale distances between specific sites in proteins are essential for analysis of their structure and function. In this work we introduce Gd(3+) spin labeling for nanometer-range distance measurements in proteins by high-field pulse electron paramagnetic resonance (EPR). To evaluate the performance of such measurements, we carried out four-pulse double-electron electron resonance (DEER) measurements on two proteins, p75ICD and tau(C)14, labeled at strategically selected sites with either two nitroxides or two Gd(3+) spin labels. In analogy to conventional site-directed spin labeling using nitroxides, Gd(3+) tags that are derivatives of dipicolinic acid were covalently attached to cysteine thiol groups. Measurements were carried out on X-band (approximately 9.5 GHz, 0.35 T) and W-band (95 GHz, 3.5 T) spectrometers for the nitroxide-labeled proteins and at W-band for the Gd(3+)-labeled proteins. In the protein p75ICD, the orientations of the two nitroxides were found to be practically uncorrelated, and therefore the distance distribution could as readily be obtained at W-band as at X-band. The measured Gd(3+)-Gd(3+) distance distribution had a maximum at 2.9 nm, as compared to 2.5 nm for the nitroxides. In the protein tau(C)14, however, the orientations of the nitroxides were correlated, and the W-band measurements exhibited strong orientation selection that prevented a straightforward extraction of the distance distribution. The X-band measurements gave a nitroxide-nitroxide distance distribution with a maximum at 2.5 nm, and the W-band measurements gave a Gd(3+)-Gd(3+) distance distribution with a maximum at 3.4 nm. The Gd(3+)-Gd(3+) distance distributions obtained are in good agreement with expectations from structural models that take into account the flexibility of the tags and their tethers to the cysteine residues. These results show that Gd(3+) labeling is a viable technique for distance measurements at high fields that features an order of magnitude sensitivity improvement, in terms of protein quantity, over X-band pulse EPR measurements using nitroxide spin labels. Its advantage over W-band distance measurements using nitroxides stems from an intrinsic absence of orientation selection.

Significantly Improved Sensitivity of Q-Band PELDOR/DEER Experiments Relative to X-Band Is Observed in Measuring the Intercoil Distance of a Leucine Zipper Motif Peptide (GCN4-LZ)

Pulsed electron double resonance (PELDOR)/double electron-electron resonance (DEER) spectroscopy is a very powerful structural biology tool in which the dipolar coupling between two unpaired electron spins (site-directed nitroxide spin-labels) is measured. These measurements are typically conducted at X-band (9.4 GHz) microwave excitation using the four-pulse DEER sequence and can often require up to 12 h of signal averaging for biological samples (depending on the spin-label concentration). In this work, we present for the first time a substantial increase in DEER sensitivity obtained by collecting DEER spectra at Q-band (34 GHz), when compared to X-band. The huge boost in sensitivity (factor of 13) demonstrated at Q-band represents a 169-fold decrease in data collection time, reveals a greatly improved frequency spectrum and higher-quality distance data, and significantly increases sample throughput. Thus, the availability of Q-band DEER spectroscopy should have a major impact on structural biology studies using site-directed spin labeling EPR techniques.

Accurate long‐range distance measurements in a doubly spin‐labeled protein by a four‐pulse, double electron–electron resonance method

Distance determination in disordered systems by a four-pulse double electron-electron resonance method (DEER or PELDOR) is becoming increasingly popular because long distances (several nanometers) and their distributions can be measured. From the distance distributions eventual heterogeneities and dynamics can be deduced. To make full use of the method, typical distance distributions for structurally well-defined systems are needed. Here, the structurally well-characterized protein azurin is investigated by attaching two (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate spin labels (MTSL) by site-directed mutagenesis. Mutations at the surface sites of the protein Q12, K27, and N42 are combined in the double mutants Q12C/K27C and K27C/N42C. A distance of 4.3 nm is found for Q12C/K27C and 4.6 nm for K27C/N42C. For Q12C/K27C the width of the distribution (0.24 nm) is smaller than for the K27C/N42C mutant (0.36 nm). The shapes of the distributions are close to Gaussian. These distance distributions agree well with those derived from a model to determine the maximally accessible conformational space of the spin-label linker. Additionally, the expected distribution for the shorter distance variant Q12C/N42C was modeled. The width is larger than the calculated one for Q12C/K27C by 21%, revealing the effect of the different orientation and shorter distance. The widths and the shapes of the distributions are suited as a reference for two unperturbed MTSL labels at structurally well-defined sites.

DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data

Pulsed electron-electron double resonance techniques such as the four-pulse double electron-electron resonance experiment measure a dipolar evolution function of the sample. For a sample consisting of spin-carrying nanoobjects, this function is the product of a form factor, corresponding to the internal structure of the nanoobject, and a background factor, corresponding to the distribution of nanoobjects in space. The form factor contains information on the spin-to-spin distance distribution within the nanoobject and on the average number of spins per nanoobject, while the background factor depends on constraints, such as a confinement of the nanoobjects to a two-dimensional layer. Separation of the dipolar evolution function into these two contributions and extraction of the spin-to-spin distance distribution require numerically stable mathematical algorithms that can handle data for different classes of samples, e.g., spin-labelled biomacromolecules and synthetic materials. Furthermore, experimental imperfections such as the limited excitation bandwidth of microwave pulses need to be considered. The software package DeerAnalysis2006 provides access to a comprehensive set of tools for such data analysis within a common user interface. This interface allows for several tests of the reliability and precision of the extracted information. User-supplied models for the spin-to-spin distance distribution within a certain class of nanoobjects can be added to an existing library and be fitted with a universal algorithm.

Interresidual Distance Determination by Four-Pulse Double Electron-Electron Resonance in an Integral Membrane Protein: The Na +/Proline Transporter PutP of Escherichia coli

Proximity relationships within three doubly spin-labeled variants of the Na +/proline transporter PutP of Escherichia coli were studied by means of four-pulse double electron-electron resonance spectroscopy. The large value of 4.8 nm for the interspin distance determined between positions 107 in loop 4 and 223 in loop 7 strongly supports the idea of these positions being located on opposite sides of the membrane. Significant smaller values of between 1.8 and 2.5 nm were found for the average interspin distances between spin labels attached to the cytoplasmic loops 2 and 4 (position 37 and 107) and loops 2 and 6 (position 37 and 187). The large distance distribution widths visible in the pair correlation functions reveal a high flexibility of the studied loop regions. An increase of the distance between positions 37 and 187 upon Na + binding suggests ligand-induced structural alterations of PutP. The results demonstrate that four-pulse double electron-electron resonance spectroscopy is a powerful means to investigate the structure and conformational changes of integral membrane proteins reconstituted in proteoliposomes.

Applications of pi-photon-induced transparency in two-frequency pulse electron paramagnetic resonance experiments.

An approach to pulse electron paramagnetic resonance (EPR) experiments which are based on two different resonance fields is introduced. Instead of using two microwave (mw) sources or a magnetic field jump, bichromatic pulses consisting of a transverse microwave field with frequency omega(mw) and a longitudinal radio frequency field with frequency omega(rf) are employed. Such bichromatic pulses excite a number of multiple photon transitions at frequencies omega(mw)+komega(rf) (k in Z). The pi-photon-induced transparency phenomenon is used to select the required transitions. This approach is used in the stimulated soft electron spin echo envelope modulation and the four-pulse double electron-electron resonance experiments. The results obtained using the bichromatic pulse approach are in agreement with those obtained with the standard pulse EPR techniques. It is shown that applying bichromatic pulses is straightforward and advantageous in several respects.

Comparison of Electron Paramagnetic Resonance Methods to Determine Distances between Spin Labels on Human Carbonic Anhydrase II

Four doubly spin-labeled variants of human carbonic anhydrase II and corresponding singly labeled variants were prepared by site-directed spin labeling. The distances between the spin labels were obtained from continuous-wave electron paramagnetic resonance spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of line-shape broadening, and computer simulation of line-shape changes. Distances also were determined by four-pulse double electron-electron resonance. For each variant, at least two methods were applicable and reasonable agreement between methods was obtained. Distances ranged from 7 to 24 Å. The doubly spin-labeled samples contained some singly labeled protein due to incomplete labeling. The sensitivity of each of the distance determination methods to the non-interacting component was compared.

Dead-Time Free Measurement of Dipole–Dipole Interactions between Electron Spins

A four-pulse version of the pulse double electron–electron resonance (DEER) experiment is presented, which is designed for the determination of interradical distances on a nanoscopic lengthscale. With the new pulse sequence electron–electron couplings can be studied without dead-time artifacts, so that even broad distributions of electron–electron distances can be characterized. A version of the experiment that uses a pulse train in the detection period exhibits improved signal-to-noise ratio. Tests on two nitroxide biradicals with known length indicate that the accessible range of distances extends from about 1.5 to 8 nm. The four-pulse DEER spectra of an ionic spin probe in an ionomer exhibit features due to probe molecules situated both on the same and on different ion clusters. The former feature provides information on the cluster size and is inaccessible with previous methods.