Multi-Chain Hamiltonian Replica Exchange Framework for Probing Transient Helices and Order-Disorder Transitions in Proteins

Abstract

Simulations have emerged as a pillar in biophysics to understand behavior at the molecular scale, in particular for proteins such as intrinsically disordered proteins. These often show transient folding with long-lived states that are challenging to efficiently sample using conventional simulations. Here, we show that Hamiltonian replica-exchange at the level of hydrogen bonds can be used to systematically explore the conformational landscape of proteins. Investigating the IM30 protein, a partially alpha helical protein, under the PLUM coarse-grain force-field, we show that this approach straightforwardly allows characterization of local helix stability, which would be otherwise hard to obtain. For instance, we show that some regions of the conserved coiled-coil structure are less stable than others. By combining the different replicas with principal component analysis, we are able to dissect the conformational landscape of the IM30 protein. Taken together, this indicates that enhanced sampling remains an important part of simulations, allowing insights that cannot be easily obtained in traditional simulations. Summary This work develops a multi-chain Hamiltonian Replica Exchange Molecular Dynamics (HREM) framework using hydrogen-bond strength (HBS) as a tunable parameter to systematically explore protein conformational ensembles and order-disorder transitions. A 64-chain dilute solution simulation strategy is introduced to enable efficient and statistically robust sampling of independent protein conformational ensembles within a single simulation box. An integrated structural analysis framework is established by combining helicity analysis, polymer physics-based characterization, and principal component analysis (PCA) to quantify secondary-structure formation, transient helix emergence, chain flexibility, and global ensemble organization across different structural states. Application of this framework to full-length IM30 and its individual components (H0-3 with helical hairpin and H4-6 IDR) reveals that HBS-dependent order-disorder transitions in all constructs are governed by transient helix formation. In the full-length protein, IDR-associated transient helices reshape the conformational landscape of the conserved hairpin architecture, enabling a continuum of conformational states through dynamic ensemble reorganization. This work demonstrates that IDRs and their associated transient helices act as key regulators of protein conformational organization, promoting continuous transitions between ordered and disordered states and providing a general framework for investigating conformational dynamics in modular proteins.