Integrating Electron Paramagnetic Resonance Spectroscopy and Computational Modeling to Measure Protein Structure and Dynamics.

Electron paramagnetic resonance (EPR) has become a powerful probe of conformational heterogeneity and dynamics of biomolecules. In this Review, we discuss different computational modeling techniques that enrich the interpretation of EPR measurements of dynamics or distance restraints. A variety of spin labels are surveyed to provide a background for the discussion of modeling tools. Molecular dynamics (MD) simulations of models containing spin labels provide dynamical properties of biomolecules and their labels. These simulations can be used to predict EPR spectra, sample stable conformations and sample rotameric preferences of label sidechains. For molecular motions longer than milliseconds, enhanced sampling strategies and de novo prediction software incorporating or validated by EPR measurements are able to efficiently refine or predict protein conformations, respectively. To sample large-amplitude conformational transition, a coarse-grained or an atomistic weighted ensemble (WE) strategy can be guided with EPR insights. Looking forward, we anticipate an integrative strategy for efficient sampling of alternate conformations by de novo predictions, followed by validations by systematic EPR measurements and MD simulations. Continuous pathways between alternate states can be further sampled by WE-MD including all intermediate states.

Direct observation of negative cooperativity in a detoxification enzyme at the atomic level by Electron Paramagnetic Resonance spectroscopy and simulation.

The catalytic activity of human glutathione S-transferase A1-1 (hGSTA1-1), a homodimeric detoxification enzyme, is dependent on the conformational dynamics of a key C-terminal helix α9 in each monomer. However, the structural details of how the two monomers interact upon binding of substrates is not well understood and the structure of the ligand-free state of the hGSTA1-1 homodimer has not been resolved. Here, we used a combination of electron paramagnetic resonance (EPR) distance measurements and weighted ensemble (WE) simulations to characterize the conformational ensemble of the ligand-free state at the atomic level. EPR measurements reveal a broad distance distribution between a pair of Cu(II) labels in the ligand-free state that gradually shifts and narrows as a function of increasing ligand concentration. These shifts suggest changes in the relative positioning of the two α9 helices upon ligand binding. WE simulations generated unbiased pathways for the seconds-timescale transition between alternate states of the enzyme, leading to the generation of atomically detailed structures of the ligand-free state. Notably, the simulations provide direct observations of negative cooperativity between the monomers of hGSTA1-1, which involve the mutually exclusive docking of α9 in each monomer as a lid over the active site. We identify key interactions between residues that lead to this negative cooperativity. Negative cooperativity may be essential for interaction of hGSTA1-1 with a wide variety of toxic substrates and their subsequent neutralization. More broadly, this work demonstrates the power of integrating EPR distances with WE rare-events sampling strategy to gain mechanistic information on protein function at the atomic level.

"Store-bought is fine": Sensitivity considerations using shaped pulses for DEER measurements on Cu(II) labels.

The narrow excitation bandwidth of monochromic pulses is a sensitivity limitation for pulsed dipolar spectroscopy on Cu(II)-based measurements. In response, frequency-swept pulses with large excitation bandwidths have been adopted to probe a greater range of the EPR spectrum. However, much of the work utilizing frequency-swept pulses in Cu(II) distance measurements has been carried out on home-built spectrometers and equipment. Herein, we carry out systematic Cu(II) based distance measurements to demonstrate the capability of chirp pulses on commercial instrumentation. More importantly we delineate sensitivity considerations under acquisition schemes that are necessary for robust distance measurements using Cu(II) labels for proteins. We show that a 200 MHz sweeping bandwidth chirp pulse can improve the sensitivity of long-range distance measurements by factors of three to four. The sensitivity of short-range distances only increases slightly due to special considerations for the chirp pulse duration relative to the period length of the modulated dipolar signal. Enhancements in sensitivity also dramatically reduce measurement collection times enabling rapid collection of orientationally averaged Cu(II) distance measurements in under two hours.

Synthesis of Square Planar Cu4 Clusters.

Template-assisted synthesis of well-defined polynuclear clusters remains a challenge for [M4 ] square planar topologies. Herein, we present a tetraamine scaffold R L(NH2 )4 , where L is a rigidified resorcin[4]arene, to direct the formation of C4 -symmetric R L(NH)4 Cu4 clusters with Cu-Cu distances around 2.7 Å, suggesting metal-metal direct interactions are operative since the sum of copper's van der Waals radii is 2.8 Å. DFT calculations display HOMO to HOMO-3 residing all within a 0.1 eV gap. These four orbitals display significant electron density contribution from the Cu centers suggesting a delocalized electronic structure. The one-electron oxidized [Cu4 ]+ species was probed by variable temperature X-band continuous wave-electron paramagnetic resonance (CW-EPR), which displays a multiline spectrum at room temperature. This work presents a novel synthetic strategy for [M4 ] clusters and a new platform to investigate activation of small molecules.

An optimal acquisition scheme for Q-band EPR distance measurements using Cu2+-based protein labels.

Recent advances in site-directed Cu2+ labeling of proteins and nucleic acids have added an attractive new methodology to measure the structure-function relationship in biomolecules. Despite the promise, accessing the higher sensitivity of Q-band Double Electron Electron Resonance (DEER) has been challenging for Cu2+ labels designed for proteins. Q-band DEER experiments on this label typically require many measurements at different magnetic fields, since the pulses can excite only a few orientations at a given magnetic field. Herein, we analyze such orientational effects through simulations and show that three DEER measurements, at strategically selected magnetic fields, are generally sufficient to acquire an orientational-averaged DEER time trace for this spin label at Q-band. The modeling results are experimentally verified on Cu2+ labeled human glutathione S-transferase (hGSTA1-1). The DEER distance distribution measured at the Q-band shows good agreement with the distance distribution sampled by molecular dynamics (MD) simulations and X-band experiments. The concordance of MD sampled distances and experimentally measured distances adds growing evidence that MD simulations can accurately predict distances for the Cu2+ labels, which remains a key bottleneck for the commonly used nitroxide label. In all, this minimal collection scheme reduces data collection time by as much as six-fold and is generally applicable to many octahedrally coordinated Cu2+ systems. Furthermore, the concepts presented here may be applied to other metals and pulsed EPR experiments.

Going the dHis-tance: Site-Directed Cu2+ Labeling of Proteins and Nucleic Acids.

In this Account, we showcase site-directed Cu2+ labeling in proteins and DNA, which has opened new avenues for the measurement of the structure and dynamics of biomolecules using electron paramagnetic resonance (EPR) spectroscopy. In proteins, the spin label is assembled in situ from natural amino acid residues and a metal complex and requires no post-expression synthetic modification or purification procedures. The labeling scheme exploits a double histidine (dHis) motif, which utilizes endogenous or site-specifically mutated histidine residues to coordinate a Cu2+ complex. Pulsed EPR measurements on such Cu2+-labeled proteins potentially yield distance distributions that are up to 5 times narrower than the common protein spin label-the approach, thus, overcomes the inherent limitation of the current technology, which relies on a spin label with a highly flexible side chain. This labeling scheme provides a straightforward method that elucidates biophysical information that is costly, complicated, or simply inaccessible by traditional EPR labels. Examples include the direct measurement of protein backbone dynamics at ß-sheet sites, which are largely inaccessible through traditional spin labels, and rigid Cu2+-Cu2+ distance measurements that enable higher precision in the analysis of protein conformations, conformational changes, interactions with other biomolecules, and the relative orientations of two labeled protein subunits. Likewise, a Cu2+ label has been developed for use in DNA, which is small, is nucleotide independent, and is positioned within the DNA helix. The placement of the Cu2+ label directly reports on the biologically relevant backbone distance. Additionally, for both of these labeling techniques, we have developed models for interpretation of the EPR distance information, primarily utilizing molecular dynamics (MD) simulations. Initial results using force fields developed for both protein and DNA labels have agreed with experimental results, which has been a major bottleneck for traditional spin labels. Looking ahead, we anticipate new combinations of MD and EPR to further our understanding of protein and DNA conformational changes, as well as working synergistically to investigate protein-DNA interactions.

Orientation and dynamics of Cu2+ based DNA labels from force field parameterized MD elucidates the relationship between EPR distance constraints and DNA backbone distances.

Pulsed electron paramagnetic resonance (EPR) based distance measurements using the recently developed Cu2+-DPA label present a promising strategy for measuring DNA backbone distance constraints. Herein we develop force field parameters for Cu2+-DPA in order to understand the features of this label at an atomic level. We perform molecular dynamics (MD) simulations using the force field parameters of Cu2+-DPA on four different DNA duplexes. The distance between the Cu2+ centers, extracted from the 2 µs MD trajectories, agrees well with the experimental distance for all the duplexes. Further analyses of the trajectory provide insight into the orientation of the Cu2+-DPA inside the duplex that leads to such agreement with experiments. The MD results also illustrate the ability of the Cu2+-DPA to report on the DNA backbone distance constraints. Furthermore, measurement of fluctuations of individual residues showed that the flexibility of Cu2+-DPA in a DNA depends on the position of the label in the duplex, and a 2 µs MD simulation is not sufficient to fully capture the experimental distribution in some cases. Finally, the MD trajectories were utilized to understand the key aspects of the double electron electron resonance (DEER) results. The lack of orientational selectivity effects of the Cu2+-DPA at Q-band frequency is rationalized in terms of fluctuations in the Cu2+ coordination environment and rotameric fluctuations of the label linker. Overall, a combination of EPR and MD simulations based on the Cu2+-DPA labelling strategy can contribute towards understanding changes in DNA backbone conformations during protein-DNA interactions.

Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site-Specific Elucidation of Hidden Dynamics in Enzymes.

Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis-CuII NTA label to obtain site-specific information on dynamics for both an α-helix and ß-sheet site of GB1, the immunoglobulin binding domain of protein G. Spectral features found in our CW-EPR measurements were consistent with the overall rigid nature of GB1 and with predictions from molecular dynamics simulations. Using this information, we show the potential of this approach to elucidate the role of dynamics in substrate binding of a functionally necessary α-helix in human glutathione transferase A1-1 (hGSTA1-1). We observe two dynamical modes for the helix. The addition of the inhibitor GS-Met and GS-Hex resulted in hGSTA1-1 to favor the more rigid active state conformation, while the faster mode potentially aids the search for substrates. Together the results illustrate the remarkable potential of the dHis-based labelling approach to measure site-specific dynamics using room temperature lineshape analysis.

Development of Cu2+-Based Distance Methods and Force Field Parameters for the Determination of PNA Conformations and Dynamics by EPR and MD Simulations.

Peptide nucleic acids (PNAs) are a promising group of synthetic analogues of DNA and RNA that offer several distinct advantages over the naturally occurring nucleic acids for applications in biosensing, drug delivery, and nanoelectronics. Because of its structural differences from DNA/RNA, methods to analyze and assess the structure, conformations, and dynamics are needed. In this work, we develop synergistic techniques for the study of the PNA conformation. We use CuQ2, a Cu2+ complex with 8-hydroxyquinoline (HQ), as an alternative base pair and as a spin label in electron paramagnetic resonance (EPR) distance methods. We use molecular dynamics (MD) simulations with newly developed force field parameters for the spin labels to interpret the distance constraints determined by EPR. We complement these methods by UV-vis and circular dichroism measurements and assess the efficacy of the Cu2+ label on a PNA duplex whose backbone is based on aminoethylglycine and a duplex with a hydroxymethyl backbone modification. We show that the Cu2+ label functions efficiently within the standard PNA and the hydroxymethyl-modified PNA and that the MD parameters may be used to accurately reproduce our EPR findings. Through the combination of EPR and MD, we gain new insights into the PNA structure and conformations as well as into the mechanism of orientational selectivity in Cu2+ EPR at X-band. These results present for the first time a rigid Cu2+ spin label used for EPR distance measurements in PNA and the accompanying MD force fields for the spin label. Our studies also reveal that the spin labels have a low impact on the structure of the PNA duplexes. The combined MD and EPR approach represents an important new tool for the characterization of the PNA duplex structure and provides valuable information to aid in the rational application of PNA at large.

Molecular Dynamics Simulations Based on Newly Developed Force Field Parameters for Cu2+ Spin Labels Provide Insights into Double-Histidine-Based Double Electron-Electron Resonance.

Electron paramagnetic resonance (EPR) in combination with the recently developed double-histidine (dHis)-based Cu2+ spin labeling has provided valuable insights into protein structure and conformational dynamics. To relate sparse distance constraints measured by EPR to protein fluctuations in solution, modeling techniques are needed. In this work, we have developed force field parameters for Cu2+-nitrilotriacetic and Cu2+-iminodiacetic acid spin labels. We employed molecular dynamics (MD) simulations to capture the atomic-level details of dHis-labeled protein fluctuations. The interspin distances extracted from 200 ns MD trajectories show good agreement with the experimental results. The MD simulations also illustrate the dramatic rigidity of the Cu2+ labels compared to the standard nitroxide spin label. Further, the relative orientations between spin-labeled sites were measured to provide insight into the use of double electron-electron resonance (DEER) methods for such labels. The relative mean angles, as well as the standard deviations of the relative angles, agree well in general with the spectral simulations published previously. The fluctuations of relative orientations help rationalize why orientation selectivity effects are minimal at X-band frequencies, but observable at the Q-band for such labels. In summary, the results show that by combining the experimental results with MD simulations precise information about protein conformations as well as flexibility can be obtained.