Combining short- and long-range fluorescence reporters with simulations to explore the intramolecular dynamics of an intrinsically disordered protein

Intrinsically disordered proteins (IDPs) are increasingly recognized as a class of molecules that can exert essential biological functions even in the absence of a well-defined three-dimensional structure. Understanding the conformational distributions and dynamics of these highly flexible proteins is thus essential for explaining the molecular mechanisms underlying their function. Single-molecule fluorescence spectroscopy in combination with Förster resonance energy transfer (FRET) is a powerful tool for probing intramolecular distances and the rapid long-range distance dynamics in IDPs. To complement the information from FRET, we combine it with photoinduced electron transfer (PET) quenching to monitor local loop-closure kinetics at the same time and in the same molecule. Here we employed this combination to investigate the intrinsically disordered N-terminal domain of HIV-1 integrase. The results show that both long-range dynamics and loop closure kinetics on the sub-microsecond time scale can be obtained reliably from a single set of measurements by the analysis with a comprehensive model of the underlying photon statistics including both FRET and PET. A more detailed molecular interpretation of the results is enabled by direct comparison with a recent extensive atomistic molecular dynamics simulation of integrase. The simulations are in good agreement with experiment and can explain the deviation from simple models of chain dynamics by the formation of persistent local secondary structure. The results illustrate the power of a close combination of single-molecule spectroscopy and simulations for advancing our understanding of the dynamics and detailed mechanisms in unfolded and intrinsically disordered proteins

A multi-step nucleation process determines the kinetics of prion-like domain phase separation

The nucleation mechanisms of biological protein phase separation are poorly understood. Here, the authors perform time-resolved SAXS experiments with the low-complexity domain (LCD) of hnRNPA1 and uncover multiple kinetic regimes on the micro- to millisecond timescale. Initially, individual proteins collapse. Nucleation then occurs via two steps distinguished by their protein cluster size distributions

The disordered N-terminal tail of SARS-CoV-2 Nucleocapsid protein forms a dynamic complex with RNA

The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Characterizing the mechanisms controlling nucleic acid binding is a key step in understanding how condensation is realized. Here, we focus on the role of the RNA binding domain (RBD) and its flanking disordered N-terminal domain (NTD) tail, using single-molecule Förster Resonance Energy Transfer and coarse-grained simulations. We quantified contact site size and binding affinity for nucleic acids and concomitant conformational changes occurring in the disordered region. We found that the disordered NTD increases the affinity of the RBD for RNA by about 50-fold. Binding of both nonspecific and specific RNA results in a modulation of the tail configurations, which respond in an RNA length-dependent manner. Not only does the disordered NTD increase affinity for RNA, but mutations that occur in the Omicron variant modulate the interactions, indicating a functional role of the disordered tail. Finally, we found that the NTD-RBD preferentially interacts with single-stranded RNA and that the resulting protein:RNA complexes are flexible and dynamic. We speculate that this mechanism of interaction enables the Nucleocapsid protein to search the viral genome for and bind to high-affinity motifs

Apolipoprotein E4 has extensive conformational heterogeneity in lipid-free and lipid-bound forms

The ε4-allele variant of apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer\u27s disease, although it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of pathogenicity remain undetermined due to limited structural information. Previous studies have led to conflicting models of the C-terminal region positioning with respect to the N-terminal domain across isoforms largely because the data are potentially confounded by the presence of heterogeneous oligomers. Here, we apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically detailed model of monomeric ApoE4 and probe the effect of lipid association. Importantly, our approach overcomes previous limitations by allowing us to work at picomolar concentrations where only the monomer is present. Our data reveal that ApoE4 is far more disordered and extended than previously thought and retains significant conformational heterogeneity after binding lipids. Comparing the proximity of the N- and C-terminal domains across the three major isoforms (ApoE4, ApoE3, and ApoE2) suggests that all maintain heterogeneous conformations in their monomeric form, with ApoE2 adopting a slightly more compact ensemble. Overall, these data provide a foundation for understanding how ApoE4 differs from nonpathogenic and protective variants of the protein

The Neglected Price of Pediatric Acute Kidney Injury: Non-renal Implications

Preclinical models and emerging translational data suggest that acute kidney injury (AKI) has far reaching effects on all other major organ systems in the body. Common in critically ill children and adults, AKI is independently associated with worse short and long term morbidity, as well as mortality, in these vulnerable populations. Evidence exists in adult populations regarding the impact AKI has on life course. Recently, non-renal organ effects of AKI have been highlighted in pediatric AKI survivors. Given the unique pediatric considerations related to somatic growth and neurodevelopmental consequences, pediatric AKI has the potential to fundamentally alter life course outcomes. In this article, we highlight the challenging and complex interplay between AKI and the brain, heart, lungs, immune system, growth, functional status, and longitudinal outcomes. Specifically, we discuss the biologic basis for how AKI may contribute to neurologic injury and neurodevelopment, cardiac dysfunction, acute lung injury, immunoparalysis and increased risk of infections, diminished somatic growth, worsened functional status and health related quality of life, and finally the impact on young adult health and life course outcomes

Extreme disorder in an ultrahigh-affinity protein complex

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes

Depletion interactions modulate the binding between disordered proteins in crowded environments

Intrinsically disordered proteins (IDPs) abound in cellular regulation. Their interactions are often transitory and highly sensitive to salt concentration and posttranslational modifications. However, little is known about the effect of macromolecular crowding on the interactions of IDPs with their cellular targets. Here, we investigate the influence of crowding on the interaction between two IDPs that fold upon binding, with polyethylene glycol as a crowding agent. Single-molecule spectroscopy allows us to quantify the effects of crowding on a comprehensive set of observables simultaneously: the equilibrium stability of the complex, the association and dissociation kinetics, and the microviscosity, which governs translational diffusion. We show that a quantitative and coherent explanation of all observables is possible within the framework of depletion interactions if the polymeric nature of IDPs and crowders is incorporated based on recent theoretical developments. The resulting integrated framework can also rationalize important functional consequences, for example, that the interaction between the two IDPs is less enhanced by crowding than expected for folded proteins of the same size