Dynamic and Assembly Characteristics of Deep-Cavity Basket Acting as a Host for Inclusion Complexation of Mitoxantrone in Biotic and Abiotic Systems.

We describe the preparation, dynamic, assembly characteristics of vase-shaped basket 13- along with its ability to form an inclusion complex with anticancer drug mitoxantrone in abiotic and biotic systems. This novel cavitand has a deep nonpolar pocket consisting of three naphthalimide sides fused to a bicyclic platform at the bottom while carrying polar glycines at the top. The results of 1 H Nuclear Magnetic Resonance (NMR), 1 H NMR Chemical Exchange Saturation Transfer (CEST), Calorimetry, Hybrid Replica Exchange Molecular Dynamics (REMD), and Microcrystal Electron Diffraction (MicroED) measurements are in line with 1 forming dimer [12 ]6- , to be in equilibrium with monomers 1(R) 3- (relaxed) and 1(S) 3- (squeezed). Through simultaneous line-shape analysis of 1 H NMR data, kinetic and thermodynamic parameters characterizing these equilibria were quantified. Basket 1(R) 3- includes anticancer drug mitoxantrone (MTO2+ ) in its pocket to give stable binary complex [MTO⊂1]- (Kd =2.1 µM) that can be precipitated in vitro with UV light or pH as stimuli. Both in vitro and in vivo studies showed that the basket is nontoxic, while at a higher proportion with respect to MTO it reduced its cytotoxicity in vitro. With well-characterized internal dynamics and dimerization, the ability to include mitoxantrone, and biocompatibility, the stage is set to develop sequestering agents from deep-cavity baskets.

ARCHE-NOAH: NMR supersequence with five different CEST experiments for studying protein conformational dynamics.

An NMR NOAH-supersequence is presented consisting of five CEST experiments for studying protein backbone and side-chain dynamics by 15N-CEST, carbonyl-13CO-CEST, aromatic-13Car-CEST, 13Cα-CEST, and methyl-13Cmet-CEST. The new sequence acquires the data for these experiments in a fraction of the time required for the individual experiments, saving over four days of NMR time per sample.

Fundamental and practical aspects of machine learning for the peak picking of biomolecular NMR spectra.

Rapid progress in machine learning offers new opportunities for the automated analysis of multidimensional NMR spectra ranging from protein NMR to metabolomics applications. Most recently, it has been demonstrated how deep neural networks (DNN) designed for spectral peak picking are capable of deconvoluting highly crowded NMR spectra rivaling the facilities of human experts. Superior DNN-based peak picking is one of a series of critical steps during NMR spectral processing, analysis, and interpretation where machine learning is expected to have a major impact. In this perspective, we lay out some of the unique strengths as well as challenges of machine learning approaches in this new era of automated NMR spectral analysis. Such a discussion seems timely and should help define common goals for the NMR community, the sharing of software tools, standardization of protocols, and calibrate expectations. It will also help prepare for an NMR future where machine learning and artificial intelligence tools will be common place.

Observation of Sub-Microsecond Protein Methyl-Side Chain Dynamics by Nanoparticle-Assisted NMR Spin Relaxation.

Amino-acid side-chain properties in proteins are key determinants of protein function. NMR spin relaxation of side chains is an important source of information about local protein dynamics and flexibility. However, traditional solution NMR relaxation methods are most sensitive to sub-nanosecond dynamics lacking information on slower ns-µs time-scale motions. Nanoparticle-assisted NMR spin relaxation (NASR) of methyl-side chains is introduced here as a window into these ns-µs dynamics. NASR utilizes the transient and nonspecific interactions between folded proteins and slowly tumbling spherical nanoparticles (NPs), whereby the increase of the relaxation rates reflects motions on time scales from ps all the way to the overall tumbling correlation time of the NPs ranging from hundreds of ns to µs. The observed motional amplitude of each methyl group can then be expressed by a model-free NASR S2 order parameter. The method is demonstrated for 2H-relaxation of CH2D methyl moieties and cross-correlated relaxation of CH3 groups for proteins Im7 and ubiquitin in the presence of anionic silica-nanoparticles. Both types of relaxation experiments, dominated by either quadrupolar or dipolar interactions, yield highly consistent results. Im7 shows additional dynamics on the intermediate time scales taking place in a functionally important loop, whereas ubiquitin visits the majority of its conformational substates on the sub-ns time scale. These experimental observations are in good agreement with 4-10 µs all-atom molecular dynamics trajectories. NASR probes side-chain dynamics on a much wider range of motional time scales than previously possible, thereby providing new insights into the interplay between protein structure, dynamics, and molecular interactions that govern protein function.

2D NMR-Based Metabolomics with HSQC/TOCSY NOAH Supersequences.

Sensitivity-improved versions of two-dimensional (2D) 13C-1H HSQC (heteronuclear single quantum coherence) and HSQC-TOCSY (HSQC-total correlation spectroscopy) NMR experiments optimized for small biological molecules and their complex mixtures encountered in metabolomics are presented that preserve the magnetization of 1H spins not directly attached to 13C spins. This allows (i) the application of rapid acquisition techniques to substantially shorten measurement time and (ii) their incorporation into supersequences (NOAH-NMR by ordered acquisition using 1H detection) for the compact acquisition of multiple 2D NMR data sets with significant gains in sensitivity, resolution, and/or time. The new pulse sequences, which are demonstrated for both metabolite model mixtures and mouse urine, offer an attractive approach for the efficient measurement of multiple 2D NMR spectra (HSQCsi and/or HSQCsi-TOCSY and TOCSY) of metabolomics samples in a single experiment for the accurate and comprehensive identification and quantitation of metabolites. These new methods bring to bear the advantages of 2D NMR to metabolomics studies with larger cohorts of samples.

From Selection to Instruction and Back: Competing Conformational Selection and Induced Fit Pathways in Abiotic Hosts.

Two limiting cases of molecular recognition, induced fit (IF) and conformational selection (CS), play a central role in allosteric regulation of natural systems. The IF paradigm states that a substrate "instructs" the host to change its shape after complexation, while CS asserts that a guest "selects" the optimal fit from an ensemble of preexisting host conformations. With no studies that quantitatively address the interplay of two limiting pathways in abiotic systems, we herein and for the first time describe the way by which twisted capsule M-1, encompassing two conformers M-1(+) and M-1(-), trap CX4 (X=Cl, Br) to give CX4 ⊂M-1(+) and CX4 ⊂M-1(-), with all four states being in thermal equilibrium. With the assistance of 2D EXSY, we found that CBr4 would, at its lower concentrations, bind M-1 via a M-1(+)→M-1(-)→CBr4 ⊂M-1(-) pathway corresponding to conformational selection. For M-1 complexing CCl4 though, data from 2D EXSY measurements and 1D NMR line-shape analysis suggested that lower CCl4 concentrations would favor CS while the IF pathway prevailed at higher proportions of the guest. Since CS and IF are not mutually exclusive, we reason that our work sets the stage for characterizing the dynamics of a wide range of already existing hosts to broaden our fundamental understanding of their action. The objective is to master the way in which encapsulation takes place for designing novel and allosteric sequestering agents, catalysts and chemosensors akin to those found in nature.

Extreme Nonuniform Sampling for Protein NMR Dynamics Studies in Minimal Time.

NMR spectroscopy is an extraordinarily rich source of quantitative dynamics of proteins in solution using spin relaxation or chemical exchange saturation transfer (CEST) experiments. However, 15N-CEST measurements require prolonged multidimensional, so-called pseudo-3D HSQC experiments where the pseudo dimension is a radio frequency offset Δω of a weak 15N saturation field. Nonuniform sampling (NUS) approaches have the potential to significantly speed up these measurements, but they also carry the risk of introducing serious artifacts and the systematic optimization of nonuniform sampling schedules has remained elusive. It is demonstrated here how this challenge can be addressed by using fitted cross-peaks of a reference 2D HSQC experiment as footprints, which are subsequently used to reconstruct cross-peak amplitudes of a pseudo-3D data set as a function of Δω by a linear least-squares fit. It is shown for protein Im7 how the approach can yield highly accurate CEST profiles based on an absolutely minimally sampled (AMS) data set allowing a speed-up of a factor 20-30. Spectrum-specific optimized nonuniform sampling (SONUS) schemes based on the Cramer-Rao lower bound metric were critical to achieve such a performance, revealing also more general properties of optimal sampling schedules. This is the first systematic exploration and optimization of NUS schedules for the dramatic speed-up of quantitative multidimensional NMR measurements that minimize unwanted errors.

Real-Time Pure Shift HSQC NMR for Untargeted Metabolomics.

Sensitivity and resolution are key considerations for NMR applications in general and for metabolomics in particular, where complex mixtures containing hundreds of metabolites over a large range of concentrations are commonly encountered. There is a strong demand for advanced methods that can provide maximal information in the shortest possible time frame. Here, we present the optimization and application of the recently introduced 2D real-time BIRD 1H-13C HSQC experiment for NMR-based metabolomics of aqueous samples at 13C natural abundance. For mouse urine samples, it is demonstrated how this real-time pure shift sensitivity-improved heteronuclear single quantum correlation method provides broadband homonuclear decoupling along the proton detection dimension and thereby significantly improves spectral resolution in regions that are affected by spectral overlap. Moreover, the collapse of the scalar multiplet structure of cross-peaks leads to a sensitivity gain of about 40-50% over a traditional 2D HSQC-SI experiment. The experiment works well over a range of magnetic field strengths and is particularly useful when resonance overlap in crowded regions of the HSQC spectra hampers accurate metabolite identification and quantitation.

Differential Conformational Dynamics Encoded by the Linker between Quasi RNA Recognition Motifs of Heterogeneous Nuclear Ribonucleoprotein H.

Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) F/H family are multipurpose RNA binding proteins that participate in most stages of RNA metabolism. Despite having similar RNA sequence preferences, hnRNP F/H proteins function in overlapping and, in some cases, distinct cellular processes. The domain organization of hnRNP F/H proteins is modular, consisting of N-terminal tandem quasi-RNA recognition motifs (F/HqRRM1,2) and a third C-terminal qRRM3 embedded between glycine-rich repeats. The tandem qRRMs are connected through a 10-residue linker, with several amino acids strictly conserved between hnRNP H and F. A significant difference occurs at position 105 of the linker, where hnRNP H contains a proline and hnRNP F an alanine. To investigate the influence of P105 on the conformational properties of hnRNP H, we probed the structural dynamics of its HqRRM1,2 domain with X-ray crystallography, NMR spectroscopy, and small-angle X-ray scattering. The collective results best describe that HqRRM1,2 exists in a conformational equilibrium between compact and extended structures. The compact structure displays an electropositive surface formed at the qRRM1-qRRM2 interface. Comparison of NMR relaxation parameters, including Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion, between HqRRM1,2 and FqRRM1,2 indicates that FqRRM1,2 primarily adopts a more extended and flexible conformation. Introducing the P105A mutation into HqRRM1,2 alters its conformational dynamics to favor an extended structure. Thus, our work demonstrates that the linker compositions confer different structural properties between hnRNP F/H family members that might contribute to their functional diversity.

Non-Uniform and Absolute Minimal Sampling for High-Throughput Multidimensional NMR Applications.

Many biomolecular NMR applications can benefit from the faster acquisition of multidimensional NMR data with high resolution and their automated analysis and interpretation. In recent years, a number of non-uniform sampling (NUS) approaches have been introduced for the reconstruction of multidimensional NMR spectra, such as compressed sensing, thereby bypassing traditional Fourier-transform processing. Such approaches are applicable to both biomacromolecules and small molecules and their complex mixtures and can be combined with homonuclear decoupling (pure shift) and covariance processing. For homonuclear 2D TOCSY experiments, absolute minimal sampling (AMS) permits the drastic shortening of measurement times necessary for high-throughput applications for identification and quantification of components in complex biological mixtures in the field of metabolomics. Such TOCSY spectra can be comprehensively represented by graphic theoretical maximal cliques for the identification of entire spin systems and their subsequent query against NMR databases. Integration of these methods in webservers permits the rapid and reliable identification of mixture components. Recent progress is reviewed in this Minireview.

Quantitative Binding Behavior of Intrinsically Disordered Proteins to Nanoparticle Surfaces at Individual Residue Level.

The quantitative and predictive understanding how intrinsically disordered proteins (IDPs) interact with engineered nanoparticles has potentially important implications for new therapeutics as well as nanotoxicology. Based on a recently developed solution 15 N NMR relaxation approach, the interactions between four representative IDPs with silica nanoparticles are reported at atomic detail. Each IDP possesses distinct binding modes, which can be quantitatively explained by the local amino-acid residue composition using a "free residue interaction model". The model was parameterized using the binding affinities of free proteinogenic amino acids along with long-range effects, derived by site-specific mutagenesis, that exponentially scale with distance along the primary sequence. The model, which is accessible through a web server, can be applied to predict the residue-specific binding affinities of a large number of IDPs.

The Michaelis Complex of Arginine Kinase Samples the Transition State at a Frequency That Matches the Catalytic Rate.

Arginine kinase (AK), which is a member of the phosphagen kinase family, serves as a model system for studying the structural and dynamic determinants of biomolecular enzyme catalysis of all major states involved of the enzymatic cycle. These states are the apo state (substrate free), the Michaelis complex analogue AK:Arg:Mg·AMPPNP (MCA), a product complex analogue AK:pAIE:Mg·ADP (PCA), and the transition state analogue AK:Arg:Mg·ADP:NO3- (TSA). The conformational dynamics of these states have been studied by NMR relaxation dispersion measurements of the methyl groups of the Ile, Leu, and Val residues at two static magnetic fields. Although all states undergo significant amounts of µs-ms time scale dynamics, only the MCA samples a dominant excited state that resembles the TSA, as evidenced by the strong correlation between the relaxation dispersion derived chemical shift differences Δω and the equilibrium chemical shift differences Δδ of these states. The average lifetime of the MCA is 36 ms and the free energy difference to the TSA-like form is 8.5 kJ/mol. It is shown that the conformational energy landscape of the Michaelis complex analogue is shaped in a way that at room temperature it channels passage to the transition state, thereby determining the rate-limiting step of the phosphorylation reaction of arginine. Conversely, relaxation dispersion experiments of the TSA reveal that it samples the structures of the Michaelis complex analogue or the apo state as its dominant excited state. This reciprocal behavior shows that the free energy of the TSA, with all ligands bound, is lower by only about 8.9 kJ/mol than that of the Michaelis or apo complex conformations with the TSA ligands present.

Measurement of histidine pKa values and tautomer populations in invisible protein states.

The histidine imidazole side chain plays a critical role in protein function and stability. Its importance for catalysis is underscored by the fact that histidines are localized to active sites in ∼ 50% of all enzymes. NMR spectroscopy has become an important tool for studies of histidine side chains through the measurement of site-specific pK(a)s and tautomer populations. To date, such studies have been confined to observable protein ground states; however, a complete understanding of the role of histidine electrostatics in protein function and stability requires that similar investigations be extended to rare, transiently formed conformers that populate the energy landscape, yet are often "invisible" in standard NMR spectra. Here we present NMR experiments and a simple strategy for studies of such conformationally excited states based on measurement of histidine (13)Cγ, (13)Cδ2 chemical shifts and (1)Hε-(13)Cε one-bond scalar couplings. The methodology is first validated and then used to obtain pKa values and tautomer distributions for histidine residues of an invisible on-pathway folding intermediate of the colicin E7 immunity protein. Our results imply that the side chains of H40 and H47 are exposed in the intermediate state and undergo significant conformational rearrangements during folding to the native structure. Further, the pKa values explain the pH-dependent stability differences between native and intermediate states over the pH range 5.5-6.5 and they suggest that imidazole deprotonation is not a barrier to the folding of this protein.

Absolute Minimal Sampling of Homonuclear 2D NMR TOCSY Spectra for High-Throughput Applications of Complex Mixtures.

Modern applications of 2D NMR spectroscopy to diagnostic screening, metabolomics, quality control, and other high-throughput applications are often limited by the time-consuming sampling requirements along the indirect time domain t1 . 2D total correlation spectroscopy (TOCSY) provides unique spin connectivity information for the analysis of a large number of compounds in complex mixtures, but standard methods typically require >100 t1 increments for an accurate spectral reconstruction, rendering these experiments ineffective for high-throughput applications. For a complex metabolite mixture it is demonstrated that absolute minimal sampling (AMS), based on direct fitting of resonance frequencies and amplitudes in the time domain, yields an accurate spectral reconstruction of TOCSY spectra using as few as 16 t1 points. This permits the rapid collection of homonuclear 2D NMR experiments at high resolution with measurement times that previously were only the realm of 1D experiments.

Absolute Minimal Sampling in High-Dimensional NMR Spectroscopy.

Standard three-dimensional Fourier transform (FT) NMR experiments of molecular systems often involve prolonged measurement times due to extensive sampling required along the indirect time domains to obtain adequate spectral resolution. In recent years, a wealth of alternative sampling methods has been proposed to ease this bottleneck. However, due to their algorithmic complexity, for a given sample and experiment it is often hard to determine the minimal sampling requirement, and hence the maximal achievable experimental speed up. Herein we introduce an absolute minimal sampling (AMS) method that can be applied to common 3D NMR experiments. We show for the proteins ubiquitin and arginine kinase that for widely used experiments, such as 3D HNCO, accurate carbon frequencies can be obtained with a single time increment, while for others, such as 3D HN(CA)CO, all relevant information is obtained with as few as 6 increments amounting to a speed up of a factor 7-50.

Rapid Determination of Fast Protein Dynamics from NMR Chemical Exchange Saturation Transfer Data.

Functional motions of (15)N-labeled proteins can be monitored by solution NMR spin relaxation experiments over a broad range of timescales. These experiments however typically take of the order of several days to a week per protein. Recently, NMR chemical exchange saturation transfer (CEST) experiments have emerged to probe slow millisecond motions complementing R1ρ and CPMG-type experiments. CEST also simultaneously reports on site-specific R1 and R2 parameters. It is shown here how CEST-derived R1 and R2 relaxation parameters can be measured within a few hours at an accuracy comparable to traditional relaxation experiments. Using a "lean" version of the model-free approach S(2) order parameters can be determined that match those from the standard model-free approach applied to (15)N R1, R2 , and {(1)H}-(15)N NOE data. The new methodology, which is demonstrated for ubiquitin and arginine kinase (42 kDa), should serve as an effective screening tool of protein dynamics from picosecond-to-millisecond timescales.

Kinetic Cooperativity in Human Pancreatic Glucokinase Originates from Millisecond Dynamics of the Small Domain.

The hallmark of glucokinase (GCK), which catalyzes the phosphorylation of glucose during glycolysis, is its kinetic cooperativity, whose understanding at atomic detail has remained open since its discovery over 40 years ago. Herein, by using kinetic CPMG NMR spectroscopic data for 17 isoleucine side chains distributed over all parts of GCK, we show that the origin of kinetic cooperativity is rooted in intramolecular protein dynamics. Residues of glucose-free GCK located in the small domain displayed distinct exchange behavior involving multiple conformers that are substantially populated (p>17 %) with a kex  value of 509±51 s(-1) , whereas in the glucose-bound form these exchange processes were quenched. This exchange behavior directly competes with the enzymatic turnover rate at physiological glucose concentrations, thereby generating the sigmoidal rate dependence that defines kinetic cooperativity.

Characterizing slow chemical exchange in nucleic acids by carbon CEST and low spin-lock field R(1ρ) NMR spectroscopy.

Quantitative characterization of dynamic exchange between various conformational states provides essential insights into the molecular basis of many regulatory RNA functions. Here, we present an application of nucleic-acid-optimized carbon chemical exchange saturation transfer (CEST) and low spin-lock field R(1ρ) relaxation dispersion (RD) NMR experiments in characterizing slow chemical exchange in nucleic acids that is otherwise difficult if not impossible to be quantified by the ZZ-exchange NMR experiment. We demonstrated the application on a 47-nucleotide fluoride riboswitch in the ligand-free state, for which CEST and R(1ρ) RD profiles of base and sugar carbons revealed slow exchange dynamics involving a sparsely populated (p ~ 10%) and shortly lived (τ ~ 10 ms) NMR "invisible" state. The utility of CEST and low spin-lock field R(1ρ) RD experiments in studying slow exchange was further validated in characterizing an exchange as slow as ~60 s(-1).

Characterizing RNA dynamics at atomic resolution using solution-state NMR spectroscopy.

Many recently discovered noncoding RNAs do not fold into a single native conformation but sample many different conformations along their free-energy landscape to carry out their biological function. Here we review solution-state NMR techniques that measure the structural, kinetic and thermodynamic characteristics of RNA motions spanning picosecond to second timescales at atomic resolution, allowing unprecedented insights into the RNA dynamic structure landscape. From these studies a basic description of the RNA dynamic structure landscape is emerging, bringing new insights into how RNA structures change to carry out their function as well as applications in RNA-targeted drug discovery and RNA bioengineering.

Thermodynamic coupling of the tandem RRM domains of hnRNP A1 underlie its pleiotropic RNA binding functions.

The functional properties of RNA binding proteins (RBPs) require allosteric regulation through interdomain communication. Despite the importance of allostery to biological regulation, only a few studies have been conducted to describe the biophysical nature by which interdomain communication manifests in RBPs. Here, we show for hnRNP A1 that interdomain communication is vital for the unique stability of its amino-terminal domain, which consists of two RNA recognition motifs (RRMs). These RRMs exhibit drastically different stability under pressure. RRM2 unfolds as an individual domain but remains stable when appended to RRM1. Variants that disrupt interdomain communication between the tandem RRMs show a significant decrease in stability. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered carboxyl-terminal domain to engage in protein-protein interactions and influenced the protein's RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions.