Biology subjects

Mulder, F. A.

Publications and source records attributed to Mulder, F. A..

3 recordsLinked to original sources

bioRxiv2025

Phase separation of protein kinase A regulatory subunits is driven by similar inter- and intra-molecular interactions involving the inhibitory segment

Phase separation of biological macromolecules has emerged as a widespread mechanism for cellular organization, governing the spatiotemporal regulation of biochemical processes including signaling pathways. The regulatory subunit of the cAMP-dependent protein kinase A (PKA) RI phase separates following cAMP-induced kinase activation, thereby restricting and enhancing PKA activity in space and time. The N-terminal dimerization/docking (D/D) domain and the linker region of RI have been identified as necessary and sufficient for phase separation, but their interactions at atomic resolution have remained elusive. We report here a structural investigation of the minimal phase-separating region of RI by nuclear magnetic resonance spectroscopy (NMR) and molecular dynamics (MD) simulations. The intrinsically disordered regions of RI cause broadening of NMR signals throughout the protein indicative of widespread dynamic interactions. Mutation of arginine residues in the RI inhibitory sequence leads to a reduction in signal broadening accompanied by loss of phase separation. Independently, binding of RI to an A-kinase anchoring protein (AKAP) leads to spectral changes that are indicative of a loss of interactions between the D/D domain and the linker region, suggestive of a competition between intramolecular interactions and partner binding. Corroborating the experimental observations, multiscale MD simulations detect pervasive interactions of the folded D/D domain of RI with the intrinsically disordered linker, and the intermolecular interactions in the condensate mirror those found internally in RI dimers. Our results support the view that for PKA RI, homotypic phase separation is underpinned by intermolecular interactions that are similar to intramolecular interactions.

biophysics↗

bioRxiv2021

The contribution of electrostatics to hydrogen exchange in the unfolded protein state

Although electrostatics have long been recognized to play an important role in hydrogen exchange (HX) with solvent, the quantitative assessment of its magnitude in the unfolded state has hitherto been lacking. This limits the utility of HX as a quantitative method to study protein stability, folding and dynamics. Using the intrinsically disordered human protein -synuclein as a proxy for the unfolded state, we show that a hybrid mean-field approach can effectively compute the electrostatic potential at all backbone amide positions along the chain. From the electrochemical potential a fourfold reduction in hydroxide concentration near the protein backbone is predicted for the C-terminal domain, a prognosis that is in direct agreement with experimentally-derived protection factors from NMR spectroscopy. Thus, impeded HX for the C-terminal region of -synuclein is not the result of intramolecular hydrogen bonding and/or structure formation.

biophysics↗

bioRxiv2021

CheSPI: Chemical shift Secondary structure Population Inference

NMR chemical shifts (CSs) are delicate reporters of local protein structure, and recent advances in random coil CS (RCCS) prediction and interpretation now offer the compelling prospect of inferring small populations of structure from small deviations from RCCSs. Here, we present CheSPI, a simple and efficient method that provides unbiased and sensitive aggregate measures of local structure and disorder. It is demonstrated that CheSPI can predict even very small amounts of residual structure and robustly delineate subtle differences into four structural classes for intrinsically disordered proteins. For structured regions and proteins, CheSPI can assign up to eight structural classes, which coincide with the well-known DSSP classification. The program is freely available, and can either be invoked from URL www.protein-nmr.org as a web implementation, or run locally from command line as a python program. CheSPI generates comprehensive numeric and graphical output for intuitive annotation and visualization of protein structures. A number of examples are provided.

biochemistry↗