The Effect of Multisite Phosphorylation on the Conformational Properties of Intrinsically Disordered Proteins.

Intrinsically disordered proteins are involved in many biological processes such as signaling, regulation, and recognition. A common strategy to regulate their function is through phosphorylation, as it can induce changes in conformation, dynamics, and interactions with binding partners. Although phosphorylated intrinsically disordered proteins have received increased attention in recent years, a full understanding of the conformational and structural implications of phosphorylation has not yet been achieved. Here, we present all-atom molecular dynamics simulations of five disordered peptides originated from tau, statherin, and ß-casein, in both phosphorylated and non-phosphorylated state, to compare changes in global dimensions and structural elements, in an attempt to gain more insight into the controlling factors. The changes are in qualitative agreement with experimental data, and we observe that the net charge is not enough to predict the impact of phosphorylation on the global dimensions. Instead, the distribution of phosphorylated and positively charged residues throughout the sequence has great impact due to the formation of salt bridges. In statherin, a preference for arginine-phosphoserine interaction over arginine-tyrosine accounts for a global expansion, despite a local contraction of the phosphorylated region, which implies that also non-charged residues can influence the effect of phosphorylation.

Molecular Dynamics Simulations of Phosphorylated Intrinsically Disordered Proteins: A Force Field Comparison.

Phosphorylation is a common post-translational modification among intrinsically disordered proteins and regions, which helps regulate function by changing the protein conformations, dynamics, and interactions with binding partners. To fully comprehend the effects of phosphorylation, computer simulations are a helpful tool, although they are dependent on the accuracy of the force field used. Here, we compared the conformational ensembles produced by Amber ff99SB-ILDN+TIP4P-D and CHARMM36m, for four phosphorylated disordered peptides ranging in length from 14-43 residues. CHARMM36m consistently produced more compact conformations with a higher content of bends, mainly due to more stable salt bridges. Based on comparisons with experimental size estimates for the shortest and longest peptide, CHARMM36m appeared to overestimate the compactness. The difference between the force fields was largest for the peptide showing the greatest separation between positively charged and phosphorylated residues, in line with the importance of charge distribution. For this peptide, the conformational ensemble did not change significantly upon increasing the ionic strength from 0 mM to 150 mM, despite a reduction of the salt-bridging probability in the CHARMM36m simulations, implying that salt concentration has negligible effects in this study.

Determining Rg of IDPs from SAXS Data.

There is a great interest within the research community to understand the structure-function relationship for intrinsically disordered proteins (IDPs); however, the heterogeneous distribution of conformations that IDPs can adopt limits the applicability of conventional structural biology methods. Here, scattering techniques, such as small-angle X-ray scattering, can contribute. In this chapter, we will describe how to make a model-free determination of the radius of gyration by using two different approaches, the Guinier analysis and the pair distance distribution function. The ATSAS package (Franke et al., J Appl Crystallogr 50:1212-1225, 2017) has been used for the evaluation, and throughout the chapter, different examples will be given to illustrate the discussed phenomena, as well as the pros and cons of using the different approaches.

Phosphorylation of a Disordered Peptide-Structural Effects and Force Field Inconsistencies.

Phosphorylation is one of the most abundant types of post-translational modifications of intrinsically disordered proteins (IDPs). This study examines the conformational changes in the 15-residue-long N-terminal fragment of the IDP statherin upon phosphorylation, using computer simulations with two different force fields: AMBER ff99SB-ILDN and CHARMM36m. The results from the simulations are compared with experimental small-angle X-ray scattering (SAXS) and circular dichroism data. In the unphosphorylated state, the two force fields are in excellent agreement regarding global structural properties such as size and shape. However, they exhibit some differences in the extent and type of the secondary structure. In the phosphorylated state, neither of the force fields performs well compared to the experimental data. Both force fields show a compaction of the peptide upon phosphorylation, greater than what is seen in SAXS experiments, although they differ in the local structure. While the CHARMM force field increases the fraction of bends in the peptide as a response to strong interactions between the phosphorylated residues and arginines, the AMBER force field shows an increase of the helical content in the N-terminal part of the peptide, where the phosphorylated residues reside, in better agreement with circular dichroism results.

Assembling responsive microgels at responsive lipid membranes.

Directed colloidal self-assembly at fluid interfaces can have a large impact in the fields of nanotechnology, materials, and biomedical sciences. The ability to control interfacial self-assembly relies on the fine interplay between bulk and surface interactions. Here, we investigate the interfacial assembly of thermoresponsive microgels and lipogels at the surface of giant unilamellar vesicles (GUVs) consisting of phospholipids bilayers with different compositions. By altering the properties of the lipid membrane and the microgel particles, it is possible to control the adsorption/desorption processes as well as the organization and dynamics of the colloids at the vesicle surface. No translocation of the microgels and lipogels through the membrane was observed for any of the membrane compositions and temperatures investigated. The lipid membranes with fluid chains provide highly dynamic interfaces that can host and mediate long-range ordering into 2D hexagonal crystals. This is in clear contrast to the conditions when the membranes are composed of lipids with solid chains, where there is no crystalline arrangement, and most of the particles desorb from the membrane. Likewise, we show that in segregated membranes, the soft microgel colloids form closely packed 2D crystals on the fluid bilayer domains, while hardly any particles adhere to the more solid bilayer domains. These findings thus present an approach for selective and controlled colloidal assembly at lipid membranes, opening routes toward the development of tunable soft materials.

Assessing the Intricate Balance of Intermolecular Interactions upon Self-Association of Intrinsically Disordered Proteins.

Attractive interactions between intrinsically disordered proteins can be crucial for the functionality or, on the contrary, lead to the formation of harmful aggregates. For obtaining a molecular understanding of intrinsically disordered proteins and their interactions, computer simulations have proven to be a valuable complement to experiments. In this study, we present a coarse-grained model and its applications to a system dominated by attractive interactions, namely, the self-association of the saliva protein Statherin. SAXS experiments show that Statherin self-associates with increased protein concentration, and that both an increased temperature and a lower ionic strength decrease the size of the formed complexes. The model captures the observed trends and provides insight into the size distribution. Hydrophobic interaction is considered to be the major driving force of the self-association, while electrostatic repulsion represses the growth. In addition, the model suggests that the decrease of association number with increased temperature is of entropic origin.

Utilizing Coarse-Grained Modeling and Monte Carlo Simulations to Evaluate the Conformational Ensemble of Intrinsically Disordered Proteins and Regions.

In this study, we have used the coarse-grained model developed for the intrinsically disordered saliva protein (IDP) Histatin 5, on an experimental selection of monomeric IDPs, and we show that the model is generally applicable when electrostatic interactions dominate the intra-molecular interactions. Experimental and theoretically calculated small-angle X-ray scattering data are presented in the form of Kratky plots, and discussions are made with respect to polymer theory and the self-avoiding walk model. Furthermore, the impact of electrostatic interactions is shown and related to estimations of the conformational ensembles obtained from computer simulations and "Flexible-meccano." Special attention is given to the form factor and how it is affected by the salt concentration, as well as the approximation of using the form factor obtained under physiological conditions to obtain the structure factor.

Structural Characterization of Bubbles Formed in DNA Melting: A Monte Carlo Simulation Study.

Bubbles in DNA are involved in many important biological processes. In this work, a coarse-grained model is used for characterizing bubbles formed in DNA melting. The model resorts only to electrostatic interactions at the Debye-Hückel level, in combination with a short-ranged attractive interaction within a base pair. In spite of also omitting atomistic details, the model is able to capture experimentally established trends in persistence length and radius of gyration. By applying it on different systems, it is possible to conclude that there is a minimum size for stable bubbles, in the interval between 12 and 20 bp, which agrees well with previously published experimental findings. Simulated scattering data distinguishes between different bubbles and detects conformational changes in the melting process. Therefore, SAXS is regarded as useful for bubble formation studies, while simulations provide a molecular understanding.