Protein conformation by EPR spectroscopy using gadolinium tags clicked to genetically encoded p-azido-L-phenylalanine

Quantitative cysteine-independent ligation of a Gd(3+) tag to genetically encoded p-azido-L-phenylalanine via Cu(I)-catalyzed click chemistry is shown to deliver an exceptionally powerful tool for Gd(3+)-Gd(3+) distance measurements by double electron-electron resonance (DEER) experiments, as the position of the Gd(3+) ion relative to the protein can be predicted with high accuracy.This work was supported by the Australian Research Council (ARC), an Australia-Weizmann Making Connections grant, and the Israel Science Foundation (ISF)

RIDME distance measurements using Gd(iii) tags with a narrow central transition

Methods based on pulse electron paramagnetic resonance allow measurement of the electron-electron dipolar coupling between two spin labels. Here we compare the most popular technique, Double Electron-Electron Resonance (DEER or PELDOR), with the dead-time free 5-pulse Relaxation-Induced Dipolar Modulation Enhancement (RIDME) method for Gd(iii)-Gd(iii) distance measurements at W-band (94.9 GHz, ≈3.5 T) using Gd(iii) tags with a small zero field splitting (ZFS). Such tags are important because of their high EPR sensitivity arising from their narrow central transition. Two systems were investigated: (i) a rigid model compound with an inter-spin distance of 2.35 nm, and (ii) two mutants of a homodimeric protein, both labeled with a DOTA-based Gd(iii) chelate and characterized by an inter-spin distance of around 6 nm, one having a narrow distance distribution and the other a broad distribution. Measurements on the model compound show that RIDME is less sensitive to the complications arising from the failure of the weak coupling approximation which affect DEER measurements on systems characterized by short inter-spin distances between Gd(iii) tags having a narrow central transition. Measurements on the protein samples, which are characterized by a long inter-spin distance, emphasize the complications due to the appearance of harmonics of the dipolar interaction frequency in the RIDME traces for S > 1/2 spin systems, as well as enhanced uncertainties in the background subtraction. In both cases the sensitivity of RIDME was found to be significantly better than DEER. The effects of the experimental parameters on the RIDME trace are discussed.This research was supported by the Israeli Science Foundation (grant 334/14) and was made possible in part by the historic generosity of the Harold Perlman Family (D. G.). We also acknowledge the Australian Research Council for a Discovery grant to G. O. and B. G. (DP150100383) and a Future Fellowship to B. G. (FT130100838). D. G. holds the Erich Klieger professorial chair in Chemical Physics

Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags

Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Δχ) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd(3+)-Gd(3+) distances measured by double electron-electron resonance (DEER) experiments to define the metal position, allowing Δχ-tensor determinations from more robust 5-parameter fits that can be performed with a relatively sparse set of PCSs. Using this approach with the 32 kDa E. coli aspartate/glutamate binding protein (DEBP), we demonstrate a structural transition between substrate-bound and substrate-free DEBP, supported by PCSs generated by C3-Tm(3+) and C3-Tb(3+) tags attached to a genetically encoded p-azidophenylalanine residue. The significance of small PCSs was magnified by considering the difference between the chemical shifts measured with Tb(3+) and Tm(3+) rather than involving a diamagnetic reference. The integrative sparse data approach developed in this work makes poorly soluble proteins of limited stability amenable to structural studies in solution, without having to rely on cysteine mutations for tag attachment.Financial support by the Australian Research Council (ARC) and an Australia-Weizmann Making Connections grant is gratefully acknowledged. B. G. thanks the ARC for a Future Fellowship

High-sensitivity Gd3+-Gd3+ EPR distance measurements that eliminate artefacts seen at short distances

We would like to acknowledge EPSRC (EP/R)13705/1) for current funding on the HiPER project, and the Wellcome Trust for a multi-user equipment grant (099149/Z/12/Z) for upgrades on the Q-band system. We thank the Royal Society for an International Exchanges Grant and The Weizmann-UK Joint Research Program for allowing bilateral travel and research between the University of St Andrews and the Weizmann Institute of Science. JEL thanks the Royal Society for a University Research Fellowship. MJT thanks EPSRC for a CM-CDT studentship (EP/LO15110/1). MQ and AG thank the Deutsche Forschungsgemeinschaft (DFG) for funding within SPP 1601 (GO555/6-2).Gadolinium complexes are attracting increasing attention as spin labels for EPR dipolar distance measurements in biomolecules and particularly for in-cell measurements. It has been shown that flip-flop transitions within the central transition of the high spin Gd3+ ion can introduce artefacts in dipolar distance measurements, particularly when measuring distances less than 3–4 nm. Previous work has shown some reduction of these artefacts through increasing the frequency separation between the two frequencies required for the Double Electron-Electron Resonance (DEER) experiment. Here we use a high power (1 kW), wideband, non-resonant, system operating at 94 GHz to evaluate DEER measurement protocols using two rigid Gd(III)-rulers, consisting of two [GdIII(PyMTA)] complexes, with separations of 2.1 nm and 6.0 nm, respectively. We show that by avoiding the |−1/2⟩ → |1/2⟩ central transition completely, and placing both the pump and the observer pulses on either side of the central transition, we can now observe apparently artefact-free spectra and narrow distance distributions, even for a Gd-Gd distance of 2.1 nm. Importantly we still maintain excellent signal-to-noise ratio and relatively high modulation depths. These results have implications for in-cell EPR measurements at naturally occurring biomolecule concentrations.Publisher PDFPeer reviewe

Overcoming artificial broadening in Gd³⁺–Gd³⁺ distance distributions arising from dipolar pseudo-secular terms in DEER experiments

By providing accurate distance measurements between spin labels site-specifically attached to bio-macromolecules, double electron–electron resonance (DEER) spectroscopy provides a unique tool to probe the structural and conformational changes in these molecules. Gd3+-tags present an important family of spin-labels for such purposes, as they feature high chemical stability and high sensitivity in high-field DEER measurements. The high sensitivity of the Gd3+ ion is associated with its high spin (S = 7/2) and small zero field splitting (ZFS), resulting in a narrow spectral width of its central transition at high fields. However, under the conditions of short distances and exceptionally small ZFS, the weak coupling approximation, which is essential for straightforward DEER data analysis, becomes invalid and the pseudo-secular terms of the dipolar Hamiltonian can no longer be ignored. This work further explores the effects of pseudo-secular terms on Gd3+–Gd3+ DEER measurements using a specifically designed ruler molecule; a rigid bis-Gd3+-DOTA model compound with an expected Gd3+–Gd3+ distance of 2.35 nm and a very narrow central transition at the W-band (95 GHz). We show that the DEER dipolar modulations are damped under the standard W-band DEER measurement conditions with a frequency separation, Δν, of 100 MHz between the pump and observe pulses. Consequently, the DEER spectrum deviates considerably from the expected Pake pattern. We show that the Pake pattern and the associated dipolar modulations can be restored with the aid of a dual mode cavity by increasing Δν from 100 MHz to 1.09 GHz, allowing for a straightforward measurement of a Gd3+–Gd3+ distance of 2.35 nm. The increase in Δν increases the contribution of the |−5/2〉 → |−3/2〉 and |−7/2〉 → |−5/2〉 transitions to the signal at the expense of the |−3/2 〉 → |−1/2〉 transition, thus minimizing the effect of dipolar pseudo-secular terms and restoring the validity of the weak coupling approximation. We apply this approach to the A93C/N140C mutant of T4 lysozyme labeled with two different Gd3+ tags that have narrow central transitions and show that even for a distance of 4 nm there is still a significant (about two-fold) broadening that is removed by increasing Δν to 636 MHz and 898 MHz.This research was supported by the Israeli Science Foundation (grant 334/14) and made possible in part by the historic generosity of the Harold Perlman Family. D. G. holds the Erich Klieger professorial chair in Chemical Physic

Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme

Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including propargyl-DO3A-Gd(III) tagging and double electron-electron resonance (DEER) to study the rigid body protein dynamics of reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states, including a wide-open state, are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.J.A.K. acknowledges financial support from an Australian Government Research Training Program Scholarship. B.E.C. acknowledges financial support from a Rod Rickards PhD Scholarship. Funding by the Australian Research Council, including a Laureate Fellowship to G.O., is gratefully acknowledged. D.G. acknowledges the support of the Minerva Foundation, and this research was made possible in part by the historic generosity of the Harold Perlman Family (D.G.)

Review Article MR technology for biological studies in mice

ABSTRACT: Mouse models are crucial for the study of genetic factors and processes that influence human disease. In addition to tools for measuring genetic expression and establishing genotype, tools to accurately and comparatively assess mouse phenotype are essential in order to characterize pathology and make comparisons with human disease. MRI provides a powerful means of evaluating various anatomical and functional changes and hence is growing in popularity as a phenotypic readout for biomedical research studies. To accommodate the large numbers of mice needed in most biological studies, mouse MRI must offer high-throughput image acquisition and efficient image analysis. This article reviews the technology of multiple-mouse MRI, a method that images multiple mice or specimens simultaneously as a means of enabling high-throughput studies. Aspects of image acquisition and computational analysis in multiple-mouse studies are also described

An animal-specific FSI model of the abdominal aorta in anesthetized mice

Recent research has revealed that angiotensin II-induced abdominal aortic aneurysm in mice can be related to medial ruptures occurring in the vicinity of abdominal side branches. Nevertheless a thorough understanding of the biomechanics near abdominal side branches in mice is lacking. In the current work we present a mouse-specific fluid-structure interaction (FSI) model of the abdominal aorta in ApoE(-/-) mice that incorporates in vivo stresses. The aortic geometry was based on contrast-enhanced in vivo micro-CT images, while aortic flow boundary conditions and material model parameters were based on in vivo high-frequency ultrasound. Flow waveforms predicted by FSI simulations corresponded better to in vivo measurements than those from CFD simulations. Peak-systolic principal stresses at the inner and outer aortic wall were locally increased caudal to the celiac and left lateral to the celiac and mesenteric arteries. Interestingly, these were also the locations at which a tear in the tunica media had been observed in previous work on angiotensin II-infused mice. Our preliminary results therefore suggest that local biomechanics play an important role in the pathophysiology of branch-related ruptures in angiotensin-II infused mice. More elaborate follow-up research is needed to demonstrate the role of biomechanics and mechanobiology in a longitudinal setting

Elevated Uptake of Plasma Macromolecules by Regions of Arterial Wall Predisposed to Plaque Instability in a Mouse Model

Atherosclerosis may be triggered by an elevated net transport of lipid-carrying macromolecules from plasma into the arterial wall. We hypothesised that whether lesions are of the thin-cap fibroatheroma (TCFA) type or are less fatty and more fibrous depends on the degree of elevation of transport, with greater uptake leading to the former. We further hypothesised that the degree of elevation can depend on haemodynamic wall shear stress characteristics and nitric oxide synthesis. Placing a tapered cuff around the carotid artery of apolipoprotein E -/- mice modifies patterns of shear stress and eNOS expression, and triggers lesion development at the upstream and downstream cuff margins; upstream but not downstream lesions resemble the TCFA. We measured wall uptake of a macromolecular tracer in the carotid artery of C57bl/6 mice after cuff placement. Uptake was elevated in the regions that develop lesions in hyperlipidaemic mice and was significantly more elevated where plaques of the TCFA type develop. Computational simulations and effects of reversing the cuff orientation indicated a role for solid as well as fluid mechanical stresses. Inhibiting NO synthesis abolished the difference in uptake between the upstream and downstream sites. The data support the hypothesis that excessively elevated wall uptake of plasma macromolecules initiates the development of the TCFA, suggest that such uptake can result from solid and fluid mechanical stresses, and are consistent with a role for NO synthesis. Modification of wall transport properties might form the basis of novel methods for reducing plaque rupture