HYPK: A marginally disordered protein sensitive to charge decoration.

Intrinsically disordered proteins (IDPs) that lie close to the empirical boundary separating IDPs and folded proteins in Uversky's charge-hydropathy plot may behave as "marginal IDPs" and sensitively switch conformation upon changes in environment (temperature, crowding, and charge screening), sequence, or both. In our search for such a marginal IDP, we selected Huntingtin-interacting protein K (HYPK) near that boundary as a candidate; PKIα, also near that boundary, has lower secondary structure propensity; and Crk1, just across the boundary on the folded side, has higher secondary structure propensity. We used a qualitative Förster resonance energy transfer-based assay together with circular dichroism to simultaneously probe global and local conformation. HYPK shows several unique features indicating marginality: a cooperative transition in end-to-end distance with temperature, like Crk1 and folded proteins, but unlike PKIα; enhanced secondary structure upon crowding, in contrast to Crk1 and PKIα; and a cross-over from salt-induced expansion to compaction at high temperature, likely due to a structure-to-disorder transition not seen in Crk1 and PKIα. We then tested HYPK's sensitivity to charge patterning by designing charge-flipped variants including two specific sequences with identical amino acid composition that markedly differ in their predicted size and response to salt. The experimentally observed trends, also including mutants of PKIα, verify the predictions from sequence charge decoration metrics. Marginal proteins like HYPK show features of both folded and disordered proteins that make them sensitive to physicochemical perturbations and structural control by charge patterning.

A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation.

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation.

Critical Assessment of Self-Consistency Checks in the All-Atom Molecular Dynamics Simulation of Intrinsically Disordered Proteins.

All atom simulations can be used to quantify conformational properties of Intrinsically Disordered Proteins (IDP). However, simulations must satisfy convergence checks to ensure observables computed from simulation are reliable and reproducible. While absolute convergence is purely a theoretical concept requiring infinitely long simulation, a more practical, yet rigorous, approach is to impose Self Consistency Checks (SCCs) to gain confidence in the simulated data. Currently there is no study of SCCs in IDPs, unlike their folded counterparts. In this paper, we introduce different criteria for self-consistency checks for IDPs. Next, we impose these SCCs to critically assess the performance of different simulation protocols using the N terminal domain of HIV Integrase and the linker region of SARS-CoV-2 Nucleoprotein as two model IDPs. All simulation protocols begin with all-atom implicit solvent Monte Carlo (MC) simulation and subsequent clustering of MC generated conformations to create the representative structures of the IDPs. These representative structures serve as the initial structure for subsequent molecular dynamics (MD) runs with explicit solvent. We conclude that generating multiple short (∼3 µs) MD simulation trajectories─all starting from the most representative MC generated conformation─and merging them is the protocol of choice due to (i) its ability to satisfy multiple SCCs, (ii) consistently reproducing experimental data, and (iii) the efficiency of running independent trajectories in parallel by harnessing multiple cores available in modern GPU clusters. Running one long trajectory (greater than 20 µs) can also satisfy the first two criteria but is less desirable due to prohibitive computation time. These findings help resolve the challenge of identifying a usable starting configuration, provide an objective measure of SCC, and establish rigorous criteria to determine the minimum length (for one long simulation) or number of trajectories needed in all-atom simulation of IDPs.

Rules of Physical Mathematics Govern Intrinsically Disordered Proteins.

In stark contrast to foldable proteins with a unique folded state, intrinsically disordered proteins and regions (IDPs) persist in perpetually disordered ensembles. Yet an IDP ensemble has conformational features-even when averaged-that are specific to its sequence. In fact, subtle changes in an IDP sequence can modulate its conformational features and its function. Recent advances in theoretical physics reveal a set of elegant mathematical expressions that describe the intricate relationships among IDP sequences, their ensemble conformations, and the regulation of their biological functions. These equations also describe the molecular properties of IDP sequences that predict similarities and dissimilarities in their functions and facilitate classification of sequences by function, an unmet challenge to traditional bioinformatics. These physical sequence-patterning metrics offer a promising new avenue for advancing synthetic biology at a time when multiple novel functional modes mediated by IDPs are emerging.