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An NMR supersequence is introduced for the rapid acquisition of 15 N-CEST and methyl- 13 C-CEST experiments in the same pulse sequence for applications to proteins. The high sensitivity and accuracy allows the simultaneous quantitative characterization of backbone and side-chain dynamics on the millisecond timescale ideal for routine screening for alternative protein states. 

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.

 

Protein function depends critically on intrinsic internal dynamics, which is manifested in distinct ways, such as loop motions that regulate protein recognition and catalysis. Under physiological conditions, dynamic processes occur on a wide range of time scales from subpicoseconds to seconds. Commonly used NMR spin relaxation in solution provides valuable information on very fast and slow motions but is insensitive to the intermediate nanosecond to microsecond range that exceeds the protein tumbling correlation time. Presently, very little is known about the nature and functional role of these motions. It is demonstrated here how transverse spin relaxation becomes exquisitely sensitive to these motions at atomic resolution when studying proteins in the presence of nanoparticles. Application of this novel cross-disciplinary approach reveals large-scale dynamics of loops involved in functionally critical protein-protein interactions and protein-calcium ion recognition that were previously unobservable. 

We describe the preparation, dynamic, assembly characteristics of vase‐shaped basket1³⁻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 of¹H Nuclear Magnetic Resonance (NMR),¹H NMR Chemical Exchange Saturation Transfer (CEST), Calorimetry, Hybrid Replica Exchange Molecular Dynamics (REMD), and Microcrystal Electron Diffraction (MicroED) measurements are in line with1forming dimer [1₂]⁶⁻, to be in equilibrium with monomers1_(R)³⁻(relaxed) and1_(S)³⁻(squeezed). Through simultaneous line‐shape analysis of¹H NMR data, kinetic and thermodynamic parameters characterizing these equilibria were quantified. Basket1_(R)³⁻includes anticancer drug mitoxantrone (MTO²⁺) in its pocket to give stable binary complex [MTO⊂1]⁻(K_d=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.

 

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 capsuleM‐1, encompassing two conformersM‐1(+) andM‐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 CBr₄would, at its lower concentrations, bindM‐1via aM‐1(+)→M‐1(−)→CBr₄⊂M‐1(−) pathway corresponding to conformational selection. ForM‐1complexing CCl₄though, data from 2D EXSY measurements and 1D NMR line‐shape analysis suggested that lower CCl₄concentrations 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.

 

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 capsuleM‐1, encompassing two conformersM‐1(+) andM‐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 CBr₄would, at its lower concentrations, bindM‐1via aM‐1(+)→M‐1(−)→CBr₄⊂M‐1(−) pathway corresponding to conformational selection. ForM‐1complexing CCl₄though, data from 2D EXSY measurements and 1D NMR line‐shape analysis suggested that lower CCl₄concentrations 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.