Comparative Assessment of NMR Probes for the Experimental Description of Protein Folding Pathways with High-Pressure NMR

Multidimensional NMR intrinsically provides multiple probes that can be used for deciphering the folding pathways of proteins: NH amide and CH groups are strategically located on the backbone of the protein, while CH3 groups, on the side-chain of methylated residues, are involved in important stabilizing interactions in the hydrophobic core. Combined with high hydrostatic pressure, these observables provide a powerful tool to explore the conformational landscapes of proteins. In the present study, we made a comparative assessment of the NH, CH, and CH3 groups for analyzing the unfolding pathway of ∆+PHS Staphylococcal Nuclease. These probes yield a similar description of the folding pathway, with virtually identical thermodynamic parameters for the unfolding reaction, despite some notable differences. Thus, if partial unfolding begins at identical pressure for these observables (especially in the case of backbone probes) and concerns similar regions of the molecule, the residues involved in contact losses are not necessarily the same. In addition, an unexpected slight shift toward higher pressure was observed in the sequence of the scenario of unfolding with CH when compared to amide groups.

Titr-DMD – A Rapid, Coarse-Grained Quasi-All-Atom Constant pH Molecular Dynamics Framework

The pH dependence of enzyme fold stability and catalytic activity is a fundamentally dynamic, structural property which is difficult to study. Computational methods, particularly constant pH molecular dynamics (CpHMD), are the best situated tools for this. However, these often struggle with affordable sampling of sufficiently long timescales, accuracy of pKa prediction, and verification of the structures they generate. We introduce Titr-DMD, an affordable CpHMD method with a protonation state sampler that can be systematically improved, to circumvent these issues. We benchmark the method on a set of proteins with experimentally attested pKa and on the pH triggered conformational change in a staphylococcal nuclease mutant, a rare experimental study of such behavior. Our results show Titr-DMD to be an effective method to study pH coupled protein dynamics. <br>

Titr-DMD – A Rapid, Coarse-Grained Quasi-All-Atom Constant pH Molecular Dynamics Framework

The pH dependence of enzyme fold stability and catalytic activity is a fundamentally dynamic, structural property which is difficult to study. Computational methods, particularly constant pH molecular dynamics (CpHMD), are the best situated tools for this. However, these often struggle with affordable sampling of sufficiently long timescales, accuracy of pKa prediction, and verification of the structures they generate. We introduce Titr-DMD, an affordable CpHMD method with a protonation state sampler that can be systematically improved, to circumvent these issues. We benchmark the method on a set of proteins with experimentally attested pKa and on the pH triggered conformational change in a staphylococcal nuclease mutant, a rare experimental study of such behavior. Our results show Titr-DMD to be an effective method to study pH coupled protein dynamics. <br>

Titr-DMD – A Rapid Constant pH Molecular Dynamics Framework

The pH dependence of enzyme fold stability and catalytic activity is a fundamentally dynamic, structural property which is difficult to study. Computational methods, particularly constant pH molecular dynamics (CpHMD), are the best situated tools for this. However, these often struggle with affordable sampling of sufficiently long timescales, accuracy of pKa prediction, and verification of the structures they generate. We introduce Titr-DMD, an affordable CpHMD method with a protonation state sampler that can be systematically improved, to circumvent these issues. We benchmark the method on a set of proteins with experimentally attested pKa and on the pH triggered conformational change in a staphylococcal nuclease mutant, a rare experimental study of such behavior. Our results show Titr-DMD to be an effective method to study pH coupled protein dynamics. <br>

The Role of the PRMT5–SND1 Axis in Hepatocellular Carcinoma

Arginine methylation is an essential post-translational modification (PTM) deposited by protein arginine methyltransferases (PRMTs) and recognized by Tudor domain-containing proteins. Of the nine mammalian PRMTs, PRMT5 is the primary enzyme responsible for the deposition of symmetric arginine methylation marks in cells. The staphylococcal nuclease and Tudor domain-containing 1 (SND1) effector protein is a key reader of the marks deposited by PRMT5. Both PRMT5 and SND1 are broadly expressed and their deregulation is reported to be associated with a range of disease phenotypes, including cancer. Hepatocellular carcinoma (HCC) is an example of a cancer type that often displays elevated PRMT5 and SND1 levels, and there is evidence that hyperactivation of this axis is oncogenic. Importantly, this pathway can be tempered with small-molecule inhibitors that target PRMT5, offering a therapeutic node for cancer, such as HCC, that display high PRMT5–SND1 axis activity. Here we summarize the known activities of this writer–reader pair, with a focus on their biological roles in HCC. This will help establish a foundation for treating HCC with PRMT5 inhibitors and also identify potential biomarkers that could predict sensitivity to this type of therapy.