Tien M. Phan

All-atom molecular dynamics (MD) simulations based on physics-based force fields serve as an essential complement to experiments for investigating protein structure, dynamics, and interactions. Despite significant advances in force field parameterization, achieving a consistent balance of molecular interactions that stabilize folded proteins while accurately capturing the conformational dynamics of intrinsically disordered polypeptides in solution remains challenging. In this work, we introduce two refined force fields which incorporate either a selective upscaling of protein-water interactions or targeted improvements to backbone torsional sampling: (i) amber ff03w-sc, and (ii) amber ff99SBws-STQ′. Extensive validation against small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy observables revealed that both force fields accurately reproduced the chain dimensions and secondary structure propensities of IDPs. Importantly, both force fields also maintained the stability of single-chain folded proteins and protein-protein complexes over microsecond-timescale simulations. Overall, our refinement strategies result in transferable force fields with improved accuracy for simulating diverse protein systems, ranging from folded domains to IDPs and protein-protein complexes.

Small heat shock proteins (sHSPs), including HSPB1, are essential regulators of cellular proteostasis that interact with unfolded and partially folded proteins to prevent aberrant misfolding and aggregation. These proteins fulfill a similar role in biological condensates, where they interact with intrinsically disordered proteins to modulate their liquid–liquid and liquid-to-solid phase transitions. Characterizing the sHSP structure, dynamics, and client interactions is challenging due to their partially disordered nature, their tendency to form polydisperse oligomers, and their diverse range of clients. In this work, we leverage various biophysical methods, including fast 1H-based magic angle spinning (MAS) NMR spectroscopy, molecular dynamics (MD) simulations, and modeling, to shed new light on the structure and dynamics of HSPB1 oligomers. Using split-intein-mediated segmental labeling, we provide unambiguous evidence that in the oligomer context, the N-terminal domain (NTD) of HSPB1 is rigid and adopts an ensemble of heterogeneous conformations, the α-Crystallin domain (ACD) forms dimers and experiences multiple distinct local environments, while the C-terminal domain (CTD) remains highly dynamic. Our computational models suggest that the NTDs participate in extensive NTD–NTD and NTD–ACD interactions and are sequestered within the oligomer interior. We further demonstrate that HSPB1 higher order oligomers disassemble into smaller oligomeric species in the presence of a client protein and that an accessible NTD is essential for HSPB1 partitioning into condensates and interactions with client proteins. Our integrated approach provides a high-resolution view of the complex oligomeric landscape of HSPB1 and sheds light on the elusive network of interactions that underlies the function of HSPB1 in biological condensates.

The formation of β-sheet-rich amyloid fibrils in Alzheimer’s disease and other neurodegenerative disorders is limited by a slow nucleation event. To understand the initial formation of β-sheets from disordered peptides, we used all-atom simulations to parameterize a lattice model that treats each amino acid as a binary variable with β- and non-β-sheet states. We show that translational and conformational entropy give the nascent β-sheet an anisotropic surface tension that can be used to describe the nucleus with 2D classical nucleation theory. Since translational entropy depends on concentration, the aspect ratio of the critical β-sheet changes with protein concentration. Our model explains the transition from the nucleation phase to elongation as the point where the β-sheet core becomes large enough to overcome the conformational entropy cost to straighten the terminal molecule. At this point the β-strands in the nucleus spontaneously elongate, which results in a larger binding surface to capture new molecules. These results suggest that nucleation is relatively insensitive to sequence differences in coaggregation experiments because the nucleus only involves a small portion of the peptide.

Heterochromatin protein 1α (HP1α) is a crucial element of chromatin organization. It has been proposed that HP1α functions through liquid-liquid phase separation (LLPS), which allows it to compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1α can undergo phase separation upon phosphorylation of its N-terminus extension (NTE) and/or through interactions with DNA and chromatin. Here, we combine computational and experimental approaches to elucidate the molecular interactions that drive these processes. In phosphorylation-driven LLPS, HP1α can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1α dimer conformation. This process can be enhanced by the presence of positively charged HP1α peptide ligands and disrupted by the addition of negatively charged or neutral peptides. In DNA-driven LLPS, both positively and negatively charged peptide ligands can perturb phase separation. Our findings demonstrate the importance of electrostatic interactions in HP1α LLPS where binding partners can modulate the overall charge of the droplets and screen or enhance hinge region interactions through specific and non-specific effects. Our study illuminates the complex molecular framework that can fine-tune the properties of HP1α and that can contribute to heterochromatin regulation and function.

Biomolecular condensates are increasingly recognized as key regulators of chromatin organization, yet how their formation and properties arise from protein sequences remains incompletely understood. Cross-species comparisons can reveal both conserved functions and significant evolutionary differences. Here, we integrate in vitro reconstitution, molecular dynamics simulations, and cell-based assays to examine how Drosophila and human variants of Polyhomeotic (Ph)—a subunit of the PRC1 chromatin regulatory complex—drive condensate formation through their sterile alpha motif (SAM) oligomerization domains. We identify divergent interactions between SAM and the disordered linker connecting it to the rest of Ph. These interactions enhance oligomerization and modulate both the formation and properties of reconstituted condensates. Oligomerization influences condensate dynamics but minimally impacts condensate formation. Linker-SAM interactions also affect condensate formation in Drosophila and human cells and growth in Drosophila imaginal discs. Our findings show how evolutionary changes in disordered linkers can fine-tune condensate properties, providing insights into sequence-function relationships.