Optimization of TROSY- and anti-TROSY-based 15N CPMG relaxation dispersion experiments through phase cycling.

CPMG relaxation dispersion studies of biomolecular dynamics on the µs-ms timescale can provide detailed kinetic, thermodynamic, and structural insights into function. Frequently, the 15N spin serves as the probe of choice, as uniform incorporation of the 15N isotope is facile and cost-effective, and the interpretation of the resulting data is often relatively straightforward. In conventional CPMG relaxation dispersion experiments the application of CPMG pulses with constant radiofrequency (RF) phase can lead to artifactual dispersion profiles that result from off-resonance effects, RF field inhomogeneity, and pulse miscalibration. The development of CPMG experiments with the [0013]-phase cycle has significantly reduced the impact of pulse imperfections over a greater bandwidth of frequency offsets in comparison to constant phase experiments. Application of 15N-TROSY-based CPMG schemes to studies of the dynamics of large molecules is necessary for high sensitivity, yet the correct incorporation of the [0013]-phase cycle is non-trivial. Here we present TROSY- and anti-TROSY-based 15N CPMG experiments with the [0013]-phase cycling scheme and demonstrate, through comprehensive numerical simulations and experimental validation, enhanced resistance to pulse imperfections relative to traditional schemes utilizing constant phase CPMG pulses. Notably, exchange parameters derived from the new experiments are in good agreement with those obtained using other, more established, 15N-based CPMG approaches.

Protein Conformational Exchanges Modulated by the Environment of Outer Membrane Vesicles.

Protein function, in many cases, is strongly coupled to the dynamics and conformational equilibria of the protein. The environment surrounding proteins is critical for their dynamics and can dramatically affect the conformational equilibria and subsequently the activities of proteins. However, it is unclear how protein conformational equilibria are modulated by their crowded native environments. Here we reveal that outer membrane vesicle (OMV) environments modulate the conformational exchanges of Im7 protein at its local frustrated sites and shift the conformation toward its ground state. Further experiments show both macromolecular crowding and quinary interactions with the periplasmic components stabilize the ground state of Im7. Our study highlights the key role that the OMV environment plays in the protein conformational equilibria and subsequently the conformation-related protein functions. Furthermore, the long-lasting nuclear magnetic resonance measurement time of proteins within OMVs indicates that they could serve as a promising system for investigating protein structures and dynamics in situ via nuclear magnetic spectroscopy.

PDTAC: Targeted Photodegradation of GPX4 Triggers Ferroptosis and Potent Antitumor Immunity.

Targeted degradation of proteins, especially those regarded as undruggable or difficult to drug, attracts wide attention to develop novel therapeutic strategies. Glutathione peroxidase 4 (GPX4), the key enzyme regulating ferroptosis, is currently a target with just covalent inhibitors. Here, we developed a targeted photolysis approach and achieved efficient degradation of GPX4. The photodegradation-targeting chimeras (PDTACs) were synthesized by conjugating a clinically approved photosensitizer (verteporfin) to noninhibitory GPX4-targeting peptides. These chimeras selectively degraded the target protein in both cell lysates and living cells upon red-light irradiation. The targeted photolysis of GPX4 resulted in dominant ferroptotic cell death in malignant cancer cells. Moreover, the dying cells resulting from the PDTACs exhibited potent immunogenicity in vitro and efficiently elicited antitumor immunity in vivo. Our approach therefore provides a novel method to induce GPX4 dysfunction based on noncovalent binding and specifically trigger immunogenic ferroptosis, which may boost the application of ferroptosis in cancer immunotherapy.

Towards autonomous analysis of chemical exchange saturation transfer experiments using deep neural networks.

Macromolecules often exchange between functional states on timescales that can be accessed with NMR spectroscopy and many NMR tools have been developed to characterise the kinetics and thermodynamics of the exchange processes, as well as the structure of the conformers that are involved. However, analysis of the NMR data that report on exchanging macromolecules often hinges on complex least-squares fitting procedures as well as human experience and intuition, which, in some cases, limits the widespread use of the methods. The applications of deep neural networks (DNNs) and artificial intelligence have increased significantly in the sciences, and recently, specifically, within the field of biomolecular NMR, where DNNs are now available for tasks such as the reconstruction of sparsely sampled spectra, peak picking, and virtual decoupling. Here we present a DNN for the analysis of chemical exchange saturation transfer (CEST) data reporting on two- or three-site chemical exchange involving sparse state lifetimes of between approximately 3-60 ms, the range most frequently observed via experiment. The work presented here focuses on the 1H CEST class of methods that are further complicated, in relation to applications to other nuclei, by anti-phase features. The developed DNNs accurately predict the chemical shifts of nuclei in the exchanging species directly from anti-phase 1HN CEST profiles, along with an uncertainty associated with the predictions. The performance of the DNN was quantitatively assessed using both synthetic and experimental anti-phase CEST profiles. The assessments show that the DNN accurately determines chemical shifts and their associated uncertainties. The DNNs developed here do not contain any parameters for the end-user to adjust and the method therefore allows for autonomous analysis of complex NMR data that report on conformational exchange.

Molecular Insight into the Extracellular Chaperone Serum Albumin in Modifying the Folding Free Energy Landscape of Client Proteins.

Serum albumin (SA) is the most abundant extracellular chaperone protein presenting in various bodily fluids. Recently, several studies have revealed molecular mechanisms of SA in preventing the amyloid formation of amyloidogenic proteins. However, our insight into the mechanism SA employed to sense and regulate the folding states of full-length native proteins is still limited. Addressing this question is technically challenging due to the intrinsic dynamic nature of both chaperones and clients. Here using nuclear magnetic resonance spectroscopy, we show SA modifies the folding free energy landscape of clients and subsequently alters the equilibria between different compact conformations of its clients, resulting in the increased populations of excited states of client proteins. This modulation of client protein conformation by SA can change the client protein activity in a way that cannot be interpreted on the basis of its ground state structure; therefore, our work provides an alternative insight of SA in retaining a balanced functional proteome.

Opening of a cryptic pocket in ß-lactamase increases penicillinase activity.

Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in ß-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol-labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across ß-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM ß-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR chemical exchange saturation transfer experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket's functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.

Chemical shift prediction of RNA imino groups: application toward characterizing RNA excited states.

NH groups in proteins or nucleic acids are the most challenging target for chemical shift prediction. Here we show that the RNA base pair triplet motif dictates imino chemical shifts in its central base pair. A lookup table is established that links each type of base pair triplet to experimental chemical shifts of the central base pair, and can be used to predict imino chemical shifts of RNAs to remarkable accuracy. Strikingly, the semiempirical method can well interpret the variations of chemical shifts for different base pair triplets, and is even applicable to non-canonical motifs. This finding opens an avenue for predicting chemical shifts of more complicated RNA motifs. Furthermore, we combine the imino chemical shift prediction with NMR relaxation dispersion experiments targeting both 15N and 1HN of the imino group, and verify a previously characterized excited state of P5abc subdomain including an earlier speculated non-native G•G mismatch.

Removal of 2H-decoupling sidebands in 13CHD2 13C-CEST profiles.

A unique aspect of NMR is its capacity to provide integrated insight into both the structure and intrinsic dynamics of biomolecules. Chemical exchange phenomena that often serve as probes of dynamic processes in biological macromolecules can be quantitatively investigated with chemical exchange saturation transfer (CEST) experiments. 2H-decoupling sidebands, however, always occur in the profiles of 13CHD2 13C-CEST experiments when using the simple CW (continuous wave) method, which may obscure the detection of minor dips of excited states. Traditionally, these sidebands are manually eliminated from the profiles before data analysis by removing experimental points in the range of 2H-decoupling field strength ±50 Hz away from the major dips of the ground state on either side of the dips. Unfortunately, this may also eliminate potential minor dips if they overlap with the decoupling sidebands. Here, we developed methods that use pseudo-continuous waves with variable RF amplitudes distributed onto ramps for 2H decoupling. The new methods were thoroughly validated on Bruker spectrometers at a range of fields (1H frequencies of 600, 700, and 850 MHz, and 1.1 GHz). By using these methods, we successfully removed the sidebands from the NMR profiles of 13CHD2 13C-CEST experiments.

Understanding and solving abnormal peak splitting in 3D HCCH-TOCSY and HCC(CO)NH-TOCSY.

The 3D HCCH-TOCSY and HCC(CO)NH-TOCSY experiments provide through bond connectivity and are used for side-chain chemical shift assignment by solution-state NMR. Careful design and implementation of the pulse sequence are key to the successful application of the technique particularly when trying to extract the maximum information out of challenging biomolecules. Here we investigate the source of and propose solutions for abnormal peak splitting ranging from 152 to 80 Hz and below that were found in three popular TOCSY-based experiment types: H(F1)-C(F2)-DIPSI-H(F3), C(F1)-DIPSI-C(F2)-H(F3), and C(F1)-DIPSI-N(F2)-HN(F3). Peak splitting occurs in the indirect C(F1) or C(F2) dimension before DIPSI and analyses indicate that the artifacts are resulted mainly from the DIPSI transforming a double spin order [Formula: see text] from 13C-13C scalar 1JCC coupling during t1 into observable megnetization. The splitting is recapitulated by numerical simulation and approaches are proposed to remove it. Adding a pure delay of 3.7 ms immediately before DIPSI is a simple and effective strategy to achieve 3D HCCH-TOCSY and HCC(CO)NH-TOCSY spectra free of splitting with full crosspeak intensity.

Revisiting 1HN CPMG relaxation dispersion experiments: a simple modification can eliminate large artifacts.

Carr-Purcell-Meiboom-Gill relaxation dispersion experiments are commonly used to probe biomolecular dynamics on the millisecond timescale. The simplest experiment involves using backbone 15N spins as probes of motion and pulse sequences are now available for providing accurate dispersion profiles in this case. In contrast, 1H-based experiments recorded on fully protonated samples are less common because of difficulties associated with homonuclear scalar couplings that can result in transfer of magnetization between coupled spins, leading to significant artifacts. Herein we examine a version of the 1HN CPMG experiment that has been used in our laboratory where a pair of CPMG pulse trains comprising non-selective, high power 1H refocusing pulses sandwich an amide selective pulse that serves to refocus scalar-coupled evolution by the end of the train. The origin of the artifacts in our original scheme is explained and a new, significantly improved sequence is presented. The utility of the new experiment is demonstrated by obtaining flat 1HN dispersion profiles in a protonated protein system that is not expected to undergo millisecond timescale dynamics, and subsequently by measuring profiles on a cavity mutant of T4 lysozyme that exchanges between a pair of distinct states, establishing that high quality data can be generated even for fully protonated samples.

A Methyl-TROSY-Based 1 H Relaxation Dispersion Experiment for Studies of Conformational Exchange in High Molecular Weight Proteins.

Molecular complexes often sample conformational states that direct them to specific functions. These states can be difficult to observe through traditional biophysical approaches but they can be studied using a variety of different NMR spin relaxation experiments. However, these applications, when focused on moderate to high molecular weight proteins, are complicated by fast relaxing signals that negatively affect the sensitivity and resolution of spectra. Here a methyl 1 H CPMG-based experiment for studies of excited conformational states of protein machines is described that exploits a TROSY-effect to increase signal-to-noise. Complexities from the multiplicity of methyl 1 H transitions are addressed to generate a robust pulse scheme that is applied to a 320 kDa homeostasis protein, p97.

A methyl 1H double quantum CPMG experiment to study protein conformational exchange.

Protein conformational changes play crucial roles in enabling function. The Carr-Purcell-Meiboom-Gill (CPMG) experiment forms the basis for studying such dynamics when they involve the interconversion between highly populated and sparsely formed states, the latter having lifetimes ranging from ~ 0.5 to ~ 5 ms. Among the suite of experiments that have been developed are those that exploit methyl group probes by recording methyl 1H single quantum (Tugarinov and Kay in J Am Chem Soc 129:9514-9521, 2007) and triple quantum (Yuwen et al. in Angew Chem Int Ed Engl 55:11490-11494, 2016) relaxation dispersion profiles. Here we build upon these by developing a third experiment in which methyl 1H double quantum coherences evolve during a CPMG relaxation element. By fitting single, double, and triple quantum datasets, akin to recording the single quantum dataset at static magnetic fields of Bo, 2Bo and 3Bo, we show that accurate exchange values can be obtained even in cases where exchange rates exceed 10,000 s-1. The utility of the double quantum experiment is demonstrated with a pair of cavity mutants of T4 lysozyme (T4L) with ground and excited states interchanged and with exchange rates differing by fourfold (~ 900 s-1 and ~ 3600 s-1), as well as with a fast-folding domain where the unfolded state lifetime is ~ 80 µs.

Measuring Diffusion Constants of Invisible Protein Conformers by Triple-Quantum 1 H CPMG Relaxation Dispersion.

Proteins are not locked in a single structure but often interconvert with other conformers that are critical for function. When such conformers are sparsely populated and transiently formed they become invisible to routine biophysical methods, however they can be studied in detail by NMR spin-relaxation experiments. Few experiments are available in the NMR toolkit, however, for characterizing the hydrodynamic properties of invisible states. Herein we describe a CPMG-based experiment for measuring translational diffusion constants of invisible states using a pulsed-field gradient approach that exploits methyl 1 H triple-quantum coherences. An example, involving diffusion of a sparsely populated and hence invisible unfolded protein ensemble is presented, without the need for the addition of denaturants that tend to destroy weak interactions that can be involved in stabilizing residual structure in the unfolded state.

Measuring Solvent Hydrogen Exchange Rates by Multifrequency Excitation 15N CEST: Application to Protein Phase Separation.

Solvent exchange rates provide important information about the structural and dynamical properties of biomolecules. A large number of NMR experiments have been developed to measure such rates in proteins, the great majority of which quantify the buildup of signals from backbone amides after initial perturbation of water magnetization. Here we present a different approach that circumvents the main limitations that result from these classical hydrogen exchange NMR experiments. Building on recent developments that enable rapid recording of chemical exchange saturation transfer (CEST) pseudo-3D data sets, we describe a 15N-based CEST scheme for measurement of solvent exchange in proteins that exploits the one-bond 15N deuterium isotope shift. The utility of the approach is verified with an application to a 236 residue intrinsically disordered protein domain under conditions where it phase separates and a second application involving a mutated form of the domain that does not phase separate, establishing very similar hydrogen exchange rates for both samples. The methodology is well suited for studies of hydrogen exchange in any 15N-labeled biomolecule. A discussion of the merits of the CEST experiment in relation to the popular CLEANEX-PM scheme is presented.

Reversible inhibition of the ClpP protease via an N-terminal conformational switch.

Protein homeostasis is critically important for cell viability. Key to this process is the refolding of misfolded or aggregated proteins by molecular chaperones or, alternatively, their degradation by proteases. In most prokaryotes and in chloroplasts and mitochondria, protein degradation is performed by the caseinolytic protease ClpP, a tetradecamer barrel-like proteolytic complex. Dysregulating ClpP function has shown promise in fighting antibiotic resistance and as a potential therapy for acute myeloid leukemia. Here we use methyl-transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, biochemical assays, and molecular dynamics simulations to characterize the structural dynamics of ClpP from Staphylococcus aureus (SaClpP) in wild-type and mutant forms in an effort to discover conformational hotspots that regulate its function. Wild-type SaClpP was found exclusively in the active extended form, with the N-terminal domains of its component protomers in predominantly ß-hairpin conformations that are less well-defined than other regions of the protein. A hydrophobic site was identified that, upon mutation, leads to unfolding of the N-terminal domains, loss of SaClpP activity, and formation of a previously unobserved split-ring conformation with a pair of 20-Å-wide pores in the side of the complex. The extended form of the structure and partial activity can be restored via binding of ADEP small-molecule activators. The observed structural plasticity of the N-terminal gates is shown to be a conserved feature through studies of Escherichia coli and Neisseria meningitidis ClpP, suggesting a potential avenue for the development of molecules to allosterically modulate the function of ClpP.

Exploring methods to expedite the recording of CEST datasets using selective pulse excitation.

Chemical Exchange Saturation Transfer (CEST) has emerged as a powerful tool for studies of biomolecular conformational exchange involving the interconversion between a major, visible conformer and one or more minor, invisible states. Applications typically entail recording a large number of 2D datasets, each of which differs in the position of a weak radio frequency field, so as to generate a CEST profile for each nucleus from which the chemical shifts of spins in the invisible state(s) are obtained. Here we compare a number of band-selective CEST schemes for speeding up the process using either DANTE or cosine-modulated excitation approaches. We show that while both are essentially identical for applications such as 15N CEST, in cases where the probed spins are dipolar or scalar coupled to other like spins there can be advantages for the cosine-excitation scheme.

Dramatic Decrease in CEST Measurement Times Using Multi-Site Excitation.

Chemical exchange saturation transfer (CEST) has recently evolved into a powerful approach for studying sparsely populated, "invisible" protein states in slow exchange with a major, visible conformer. Central to the technique is the use of a weak, highly selective radio-frequency field that is applied at different frequency offsets in successive experiments, "searching" for minor state resonances. The recording of CEST profiles with enough points to ensure coverage of the entire spectrum at sufficient resolution can be time-consuming, especially for applications that require high static magnetic fields or when small chemical shift differences between exchanging states must be quantified. Here, we show - with applications involving 15 N CEST - that the process can be significantly accelerated by using a multi-frequency irradiation scheme, leading in some applications to an order of magnitude savings in measurement time.

Investigating the Dynamics of Destabilized Nucleosomes Using Methyl-TROSY NMR.

The nucleosome core particle (NCP), comprised of histone proteins wrapped with ∼146 base pairs of DNA, provides both protection and controlled access to DNA so as to regulate vital cellular processes. High-resolution structures of nucleosomes and nucleosome complexes have afforded a clear understanding of the structural role of NCPs, but a detailed description of the dynamical properties that facilitate DNA-templated processes is only beginning to emerge. Using methyl-TROSY NMR approaches we evaluate the effect of point mutations designed to perturb key histone interfaces that become destabilized during nucleosome remodeling in an effort to probe NCP plasticity. Notably the NCP retains its overall structural integrity, yet relaxation experiments of mutant nucleosomes reveal significant dynamics within a central histone interface associated with alternative NCP conformations populated to as much as 15% under low salt conditions. This work highlights the inherent plasticity of NCPs and establishes methyl-TROSY NMR as a valuable compliment to current single molecule methods in quantifying NCP dynamic properties.

Probing Conformational Exchange in Weakly Interacting, Slowly Exchanging Protein Systems via Off-Resonance R1ρ Experiments: Application to Studies of Protein Phase Separation.

R1ρ relaxation dispersion experiments are increasingly used in studies of protein dynamics on the micro- to millisecond time scale. Traditional R1ρ relaxation dispersion approaches are typically predicated on changes in chemical shifts between corresponding probe spins, ΔωGE, in the interconverting states. Here, we present a new application of off-resonance 15N R1ρ relaxation dispersion that enables the quantification of slow exchange processes even in the limit where ΔωGE = 0 so long as the spins in the exchanging states have different intrinsic transverse relaxation rates (ΔR2 = R2,E - R2,G ≠ 0). In this limit, the dispersion profiles become inverted relative to those measured in the case where ΔωGE ≠ 0, ΔR2 = 0. The theoretical background to understand this effect is presented, along with a simplified exchange matrix that is valid in the limits that are germane here. An application to the study of the dynamics of the germ granule protein Ddx4 in a highly concentrated phase-separated state is described. Notably, exchange-based dispersion profiles can be obtained despite the fact that ΔωGE ≈ 0 and ΔR2 is small, ∼20-30 s-1. Our results are consistent with the formation of a significantly populated excited conformational state that displays increased contacts between adjacent protein molecules relative to the major conformer in solution, leading to a decrease in overall motion of the protein backbone. A complete set of exchange parameters is obtained from analysis of a single set of 15N off-resonance R1ρ measurements recorded at a single static magnetic field and with a single spin-lock radio frequency field strength. This new approach holds promise for studies of weakly interacting systems, especially those involving intrinsically disordered proteins that form phase-separated organelles, where little change to chemical shifts between interconverting states would be expected, but where finite ΔR2 values are observed.

A new class of CEST experiment based on selecting different magnetization components at the start and end of the CEST relaxation element: an application to 1H CEST.

Chemical exchange saturation transfer (CEST) experiments are becoming increasingly popular for investigating biomolecular exchange dynamics with rates on the order of approximately 50-500 s-1 and a rich toolkit of different methods has emerged over the past few years. Typically, experiments are based on the evolution of longitudinal magnetization, or in some cases two-spin order, during a fixed CEST relaxation delay, with the same class of magnetization prepared at the start and selected at end of the CEST period. Here we present a pair of TROSY-based pulse schemes for recording amide and methyl 1H CEST profiles where longitudinal magnetization at the start evolves to produce two-spin order that is then selected at the completion of the CEST element. This selection process subtracts out contributions from 1H-1H cross-relaxation on the fly that would otherwise complicate analysis of the data. It also obviates the need to record spin-state selective CEST profiles as an alternative to eliminating NOE effects, leading to significant improvements in sensitivity. The utility of the approach is demonstrated on a sample of a cavity mutant of T4 lysozyme that undergoes chemical exchange between conformations where the cavity is free and occupied.