Insertion and Presence of Fine-wire Intramuscular Electrodes to the Lumbar Paraspinal Muscles Do Not Affect Muscle Performance and Activation during Highexertion Spinal Extension Activities

Background Low back pain (LBP) is commonly associated with paraspinal muscle dysfunctions. A method to study deep lumbar paraspinal (i.e. multifidus) muscle function and neuromuscular activation pattern is intramuscular electromyography (EMG). Previous studies have shown that the procedure does not significantly impact muscle function during activities involving low-level muscle contractions. However, it is currently unknown how muscular function and activation are affected during high-exertion contractions. Objective To examine the effects of insertion and presence of fine-wire EMG electrodes in the lumbar multifidus on muscle strength, endurance, and activation profiles during high-exertion spinal extension muscle contractions. Design Single-blinded, repeated measures intervention trial. Setting University clinical research laboratory Participants Twenty individuals between the ages of 18-40 free of recent and current back pain. Methods Muscle performance was assessed during 3 conditions (with [WI] and without [WO] presence of intramuscular electrodes, and insertion followed by removal [IO]). Isometric spinal extension strength was assessed with a motorized dynamometer. Muscle endurance was assessed using the Sorensen test with neuromuscular activation profiles analyzed during the endurance test. Main Outcome Measurements Spinal extensor muscle strength, endurance, and activation. Results Our data showed no significant difference in isometric strength (p=.20) between the 3 conditions. A significant difference in muscle endurance was found (p=.03). Post-hoc analysis showed that the muscle endurance in the IO condition was significantly higher than the WO condition (161.3±58.3 vs. 142.1±48.2 sec, p=.04), likely due to a learning effect. All 3 conditions elicited minimal pain (range 0-4/10) and comparable muscle activation profiles. Conclusion Our findings suggested the sonographically guided insertion and presence of fine-wire intramuscular EMG electrodes in the lumbar multifidus muscles had no significant impact on spinal extension muscle function. This study provides evidence that implementing intramuscular EMG does not affect muscle performance during high-exertion contractions in individuals with no current back pain. Level of Evidence I

Trimethyllysine reader proteins exhibit widespread charge-agnostic binding via different mechanisms to cationic and neutral ligands

In the last 40 years, cation−π interactions have become part of the lexicon of noncovalent forces that drive protein binding. Indeed, tetraalkylammoniums are universally bound by aromatic cages in proteins, suggesting that cation−π interactions are a privileged mechanism for binding these ligands. A prominent example is the recognition of histone trimethyllysine (Kme3) by the conserved aromatic cage of reader proteins, dictating gene expression. However, two proteins have recently been sug-gested as possible exceptions to conventional understanding of tetraalkylammonium recognition. To broadly interrogate the role of cation−π interactions in protein binding interactions, we report the first large-scale comparative evaluation of reader proteins for a neutral Kme3 isostere, experimental and computational mechanistic studies, and structural analysis. We find unexpected widespread binding of readers to a neutral isostere, with no single factor dictating charge selectivity, demonstrat-ing the challenge to predict such interactions. Further, readers that bind both cationic and neutral ligands display an unprece-dented change in mechanism: binding Kme3 via cation−π interactions and the neutral isostere through the hydrophobic effect in the same aromatic cage. This discovery challenges traditional understanding of molecular recognition of tetraalkylammo-niums by aromatic cages in myriad protein-ligand interactions and establishes a new framework for selective inhibitor design by exploiting differences in charge-dependence

Systematic Variation of Both the Aromatic Cage and Dialkyllysine via GCE-SAR Reveal Mechanistic Insights in CBX5 Reader Protein Binding

Development of inhibitors for histone methyllysine reader proteins is an active area of research due to the importance of reader protein-methyllysine interactions in transcriptional regulation and disease. Optimized peptide-based chemical probes targeting methyllysine readers favor larger alkyllysine residues in place of methyllysine. However, the mechanism by which these larger substituents drive tighter binding is not well understood. This study describes the development of a two-pronged approach combining genetic code expansion (GCE) and structure-activity relationships (SAR) through systematic variation of both the aromatic binding pocket in the protein and the alkyllysine residues in the peptide to probe inhibitor recognition in the CBX5 chromodomain. We demonstrate a novel change in driving force for larger alkyllysines, which weaken cation-π interactions but increases dispersion forces, resulting in tighter binding. This GCE-SAR approach establishes discrete energetic contributions to binding from both ligand and protein, providing a powerful tool to gain mechanistic understanding of SAR trends

Trimethyllysine Reader Proteins Exhibit Widespread Charge-Agnostic Binding via Different Mechanisms to Cationic and Neutral Ligands

In the last 40 years, cation−π interactions have become part of the lexicon of noncovalent forces that drive protein binding. Indeed, tetraalkylammoniums are universally bound by aromatic cages in proteins, suggesting that cation−π interactions are a privileged mechanism for binding these ligands. A prominent example is the recognition of histone trimethyllysine (Kme3) by the conserved aromatic cage of reader proteins, dictating gene expression. However, two proteins have recently been suggested as possible exceptions to the conventional understanding of tetraalkylammonium recognition. To broadly interrogate the role of cation−π interactions in protein binding interactions, we report the first large-scale comparative evaluation of reader proteins for a neutral Kme3 isostere, experimental and computational mechanistic studies, and structural analysis. We find unexpected widespread binding of readers to a neutral isostere with the first examples of readers that bind the neutral isostere more tightly than Kme3. We find that no single factor dictates the charge selectivity, demonstrating the challenge of predicting such interactions. Further, readers that bind both cationic and neutral ligands differ in mechanism: binding Kme3 via cation−π interactions and the neutral isostere through the hydrophobic effect in the same aromatic cage. This discovery explains apparently contradictory results in previous studies, challenges traditional understanding of molecular recognition of tetraalkylammoniums by aromatic cages in myriad protein–ligand interactions, and establishes a new framework for selective inhibitor design by exploiting differences in charge dependence

Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon-carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction