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Many proteins have slow folding times in vitro that are physiologically untenable. To combat this challenge, ATP-dependent chaperonins are thought to possess the unique ability to catalyze protein folding. Performing quantitative model selection using protein folding and unfolding data, we here show that short nucleic acids containing G-quadruplex (G4) structure can also catalyze protein folding. Performing the experiments as a function of temperature demonstrates that the G4 reshapes the underlying driving forces of protein folding. As short nucleic acids can catalyze protein folding without the input of ATP, the ability of the cell to fold proteins is far higher than previously anticipated. 

Intrinsically disordered proteins and regions (IDPs) are involved in vital biological processes. To understand the IDP function, often controlled by conformation, we need to find the link between sequence and conformation. We decode this link by integrating theory, simulation, and machine learning (ML) where sequence-dependent electrostatics is modeled analytically while nonelectrostatic interaction is extracted from simulations for many sequences and subsequently trained using ML. The resulting Hamiltonian, combining physics-based electrostatics and machine-learned nonelectrostatics, accurately predicts sequence-specific global and local measures of conformations beyond the original observable used from the simulation. This is in contrast to traditional ML approaches that train and predict a specific observable, not a Hamiltonian. Our formalism reproduces experimental measurements, predicts multiple conformational features directly from sequence with high throughput that will give insights into IDP design and evolution, and illustrates the broad utility of using physics-based ML to train unknown parts of a Hamiltonian, rather than a specific observable, in combination with known physics. 

Abstract Conformations and dynamics of an intrinsically disordered protein (IDP) depend on its composition of charged and uncharged amino acids, and their specific placement in the protein sequence. In general, the charge (positive or negative) on an amino acid residue in the protein is not a fixed quantity. Each of the ionizable groups can exist in an equilibrated distribution of fully ionized state (monopole) and an ion-pair (dipole) state formed between the ionizing group and its counterion from the background electrolyte solution. The dipole formation (counterion condensation) depends on the protein conformation, which in turn depends on the distribution of charges and dipoles on the molecule. Consequently, effective charges of ionizable groups in the IDP backbone may differ from their chemical charges in isolation—a phenomenon termed charge-regulation. Accounting for the inevitable dipolar interactions, that have so far been ignored, and using a self-consistent procedure, we present a theory of charge-regulation as a function of sequence, temperature, and ionic strength. The theory quantitatively agrees with both charge reduction and salt-dependent conformation data of Prothymosin-alpha and makes several testable predictions. We predict charged groups are less ionized in sequences where opposite charges are well mixed compared to sequences where they are strongly segregated. Emergence of dipolar interactions from charge-regulation allows spontaneous coexistence of two phases having different conformations and charge states, sensitively depending on the charge patterning. These findings highlight sequence dependent charge-regulation and its potential exploitation by biological regulators such as phosphorylation and mutations in controlling protein conformation and function. 

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. 

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 singleXenopusembryos, 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.