Virtual Homonuclear Decoupling in Direct Detection Nuclear Magnetic Resonance Experiments Using Deep Neural Networks.

Nuclear magnetic resonance (NMR) experiments are frequently complicated by the presence of homonuclear scalar couplings. For the growing body of biomolecular 13C-detected NMR methods, one-bond 13C-13C couplings significantly reduce sensitivity and resolution. The solution to this problem has typically been to perform virtual decoupling by recording multiple spectra and taking linear combinations. Here, we propose an alternative method of virtual decoupling using deep neural networks, which only requires a single spectrum and gives a significant boost in resolution while reducing the minimum effective phase cycles of the experiments by at least a factor of 2. We successfully apply this methodology to virtually decouple in-phase CON (13CO-15N) protein NMR spectra, 13C-13C correlation spectra of protein side chains, and 13Cα-detected protein 13Cα-13CO spectra where two large homonuclear couplings are present. The deep neural network approach effectively decouples spectra with a high degree of flexibility, including in cases where existing methods fail, and facilitates the use of simpler pulse sequences.

Intra-residue methyl-methyl correlations for valine and leucine residues in large proteins from a 3D-HMBC-HMQC experiment.

Methyl-TROSY based NMR experiments have over the last two decades become one of the most important means to characterise dynamics and functional mechanisms of large proteins and macromolecular machines in solution. The chemical shift assignment of methyl groups in large proteins is, however, still not trivial and it is typically performed using backbone-dependent experiments in a 'divide and conquer' approach, mutations, structure-based assignments or a combination of these. Structure-based assignment of methyl groups is an emerging strategy, which reduces the time and cost required as well as providing a method that is independent of a backbone assignment. One crucial step in available structure-based assignment protocols is linking the two prochiral methyl groups of leucine and valine residues. This has previously been achieved by recording NOESY spectra with short mixing times or by comparing NOESY spectra. Herein, we present a method based on through-bond scalar coupling transfers, a 3D-HMBC-HMQC experiment, to link the intra-residue methyl groups of leucine and valine. It is shown that the HMBC-HMQC method has several advantages over solely using NOESY spectra since a unique intra-residue cross-peak is observed. Moreover, overlap in the methyl-TROSY HMQC spectrum can easily be identified with the HMBC-HMQC experiment, thereby removing possible ambiguities in the assignment.

Arginine Side-Chain Hydrogen Exchange: Quantifying Arginine Side-Chain Interactions in Solution.

The rate with which labile backbone hydrogen atoms in proteins exchange with the solvent has long been used to probe protein interactions in aqueous solutions. Arginine, an essential amino acid found in many interaction interfaces, is capable of an impressive range of interactions via its guanidinium group. The hydrogen exchange rate of the guanidinium hydrogens therefore becomes an important measure to quantify side-chain interactions. Herein we present an NMR method to quantify the hydrogen exchange rates of arginine side-chain 1 Hϵ protons and thus present a method to gauge the strength of arginine side-chain interactions. The method employs 13 C-detection and the one-bond deuterium isotope shift observed for 15 Nϵ to generate two exchanging species in 1 H2 O/2 H2 O mixtures. An application to the protein T4 Lysozyme is shown, where protection factors calculated from the obtained exchange rates correlate well with the interactions observed in the crystal structure. The methodology presented provides an important step towards characterising interactions of arginine side-chains in enzymes, in phase separation, and in protein interaction interfaces in general.

An Environmentally Responsive Reciprocal Replicating Network.

A reciprocal replication system is constructed from four building blocks, A, B, C, and D, which react in a pairwise manner through either a 1,3-dipolar cycloaddition or the condensation reaction between an amine and an aldehyde to create two templates, trans-TAB and TCD. These templates are equipped with complementary recognition sites-two carboxylic acids ( trans-TAB) or two 4,6-dimethylamidopyridines (TCD)-that enable each template to direct the formation of its complementary partner through two mutually reinforcing cross-catalytic pathways, in which the templates trans-TAB or TCD preorganize the appropriate building blocks within two catalytically active ternary complexes: [C•D• trans-TAB] and [A•B•TCD]. The template-directed processes within these complexes generate a heteroduplex [ trans-TAB•TCD], which is shown to possess significant stability through kinetic simulations and fitting. As a consequence, the individual cross-catalytic pathways perform more efficiently in template-directed experiments when the concentration of the template being formed is below that of the template added as instruction. Comprehensive analysis of the system in which A, B, C, and D are mixed together directly, using a series of 1H NMR spectroscopic kinetic experiments, demonstrates that the behavior of the reciprocal system is more than the simple sum of its parts-as part of the interconnected network, the product of each reaction clearly directs the fabrication of its reciprocal partner, facilitating both higher rates of formation for both templates and improved diastereoselectivity for trans-TAB. A simple change in experimental conditions (from dry to "wet" CDCl3) demonstrates the sensitivity of the replication pathways within the network to the reaction environment, which leads to a >10-fold increase in the contribution of a new minimal self-replicator, trans-TAB*, to the replication network.

A 13C-detected 15N double-quantum NMR experiment to probe arginine side-chain guanidinium 15Nη chemical shifts.

Arginine side-chains are often key for enzyme catalysis, protein-ligand and protein-protein interactions. The importance of arginine stems from the ability of the terminal guanidinium group to form many key interactions, such as hydrogen bonds and salt bridges, as well as its perpetual positive charge. We present here an arginine 13Cζ-detected NMR experiment in which a double-quantum coherence involving the two 15Nη nuclei is evolved during the indirect chemical shift evolution period. As the precession frequency of the double-quantum coherence is insensitive to exchange of the two 15Nη; this new approach is shown to eliminate the previously deleterious line broadenings of 15Nη resonances caused by the partially restricted rotation about the Cζ-Nε bond. Consequently, sharp and well-resolved 15Nη resonances can be observed. The utility of the presented method is demonstrated on the L99A mutant of the 19 kDa protein T4 lysozyme, where the measurement of small chemical shift perturbations, such as one-bond deuterium isotope shifts, of the arginine amine 15Nη nuclei becomes possible using the double-quantum experiment.