Polycation-π Interactions Are a Driving Force for Molecular Recognition by an Intrinsically Disordered Oncoprotein Family

Molecular recognition by intrinsically disordered proteins (IDPs) commonly involves specific localized contacts and target-induced disorder to order transitions. However, some IDPs remain disordered in the bound state, a phenomenon coined "fuzziness", often characterized by IDP polyvalency, sequence-insensitivity and a dynamic ensemble of disordered bound-state conformations. Besides the above general features, specific biophysical models for fuzzy interactions are mostly lacking. The transcriptional activation domain of the Ewing's Sarcoma oncoprotein family (EAD) is an IDP that exhibits many features of fuzziness, with multiple EAD aromatic side chains driving molecular recognition. Considering the prevalent role of cation-π interactions at various protein-protein interfaces, we hypothesized that EAD-target binding involves polycation- π contacts between a disordered EAD and basic residues on the target. Herein we evaluated the polycation-π hypothesis via functional and theoretical interrogation of EAD variants. The experimental effects of a range of EAD sequence variations, including aromatic number, aromatic density and charge perturbations, all support the cation-π model. Moreover, the activity trends observed are well captured by a coarse-grained EAD chain model and a corresponding analytical model based on interaction between EAD aromatics and surface cations of a generic globular target. EAD-target binding, in the context of pathological Ewing's Sarcoma oncoproteins, is thus seen to be driven by a balance between EAD conformational entropy and favorable EAD-target cation-π contacts. Such a highly versatile mode of molecular recognition offers a general conceptual framework for promiscuous target recognition by polyvalent IDPs. © 2013 Song et al

On the energy components governing molecular recognition in the framework of continuum approaches

Molecular recognition is a process that brings together several biological macromolecules to form a complex and one of the most important characteristics of the process is the binding free energy. Various approaches exist to model the binding free energy, provided the knowledge of the 3D structures of bound and unbound molecules. Among them, continuum approaches are quite appealing due to their computational efficiency while at the same time providing predictions with reasonable accuracy. Here we review recent developments in the field emphasizing on the importance of adopting adequate description of physical processes taking place upon the binding. In particular, we focus on the efforts aiming at capturing some of the atomistic details of the binding phenomena into the continuum framework. When possible, the energy components are reviewed independently of each other. However, it is pointed out that rigorous approaches should consider all energy contributions on the same footage. The two major schemes for utilizing the individual energy components to predict binding affinity are outlined as well

Importance of Electrostatic Interactions in the Association of Intrinsically Disordered Histone Chaperone Chz1 and Histone H2A.Z-H2B

<div><p>Histone chaperones facilitate assembly and disassembly of nucleosomes. Understanding the process of how histone chaperones associate and dissociate from the histones can help clarify their roles in chromosome metabolism. Some histone chaperones are intrinsically disordered proteins (IDPs). Recent studies of IDPs revealed that the recognition of the biomolecules is realized by the flexibility and dynamics, challenging the century-old structure-function paradigm. Here we investigate the binding between intrinsically disordered chaperone Chz1 and histone variant H2A.Z-H2B by developing a structure-based coarse-grained model, in which Debye-Hückel model is implemented for describing electrostatic interactions due to highly charged characteristic of Chz1 and H2A.Z-H2B. We find that major structural changes of Chz1 only occur after the rate-limiting electrostatic dominant transition state and Chz1 undergoes folding coupled binding through two parallel pathways. Interestingly, although the electrostatic interactions stabilize bound complex and facilitate the recognition at first stage, the rate for formation of the complex is not always accelerated due to slow escape of conformations with non-native electrostatic interactions at low salt concentrations. Our studies provide an ionic-strength-controlled binding/folding mechanism, leading to a cooperative mechanism of “local collapse or trapping” and “fly-casting” together and a new understanding of the roles of electrostatic interactions in IDPs' binding.</p> </div

Digital photocorrosion of quantum semiconductor microstructures: a method for structural diagnostics and sensing of electrically charged molecules

La fabrication de dispositifs à base de structures multicouches de semi-conducteurs exige une mesure de routine des épaisseurs et de la localisation des interfaces des couches formées. Ceci est souvent réalisé en utilisant des techniques coûteuses et compliquées telles que la microscopie à force atomique (AFM) ou la spectroscopie de photoélectrons à rayons X (XPS). Dans ce travail, la métrologie à température ambiante dans un environnement aqueux a été développée pour des tests de post-croissance des nano-hétérostructures (NHs) semi-conductrices. La méthode utilise le procédé de photocorrosion numérique (DIP) et la sensibilité de l'émission de photoluminescence (PL) aux états de surface révélés pendant la photocorrosion. Le processus de photocorrosion des NHs semi-conducteurs GaAs/AlGaAs a été étudié en présence d'une excitation la bande interdite d'échantillons immergés dans différentes solutions aqueuses. Une photo-excitation de faible intensité au-dessus de la bande interdite (<105 mW/cm2) a été appliquée en mode pulsé caractérisée par un duty cycle (DC) donné par TON/(TON + TOFF). Ceci a produit des vitesses moyennes de gravure du matériau enleves à la précision de la sous-monocouche pendant chaque cycle DIP. En utilisant les techniques d’AFM et de XPS, il a été démontré que l'émission de la PL d'une NH GaAs/AlGaAs au cours de la DIP oscille en raison des couches de GaAs et d’AlGaAs révélées. Ces oscillations sont causées par la sensibilité de l'émission PL à la vitesse de recombinaison de surface des porteurs, qui diffère considérablement pour GaAs et AlGaAs. Le processus DIP a révélé une épaisseur de 1 nm de GaAs dans une structure de GaAs/AlGaAs, mais cela ne semble pas être une limite de résolution de cette approche. Le potentiel de circuit ouvert (OCP) mesuré au cours du DIP diffère entre les jonctions d'électrolyte-GaAs et électrolyte-AlGaAs formées au cours du processus de la photocorrosion. Les différences de OCP sont interprétées comme pouvant provenir des photo-oxydes superficiels qui portent la charge électrique. Le dipôle formé par ces oxydes superficiels définit l'OCP mesuré. L'oscillation de l’OCP pourrait également être utilisée pour la métrologie des NHs. Cela ouvre la perspective d'étendre la métrologie DIP aux NHs semi-conducteurs avec un signal PL non existant ou négligeable. Enfin, les entités chargées proches du voisinage d'une surface semi-conductrice affectent le taux de DIP. Cette propriété a été utilisée pour détecter la Legionella pneumophila qui est normalement chargée négativement au pH> 4. Les mesures de FTIR ont indiqué que les monocouches auto-assemblées (SAM) d’alkanethiol restent sur la surface semi-conductrice pendant le DIP. Cela a permis la détection de la Legionella pneumophila vivante à une concentration de 105 CFU/mL avec une architecture simple à base d'anticorps. Une discussion a été proposée suggérant des protocoles de biocapteurs possibles pour atteindre des limites de détection améliorées avec le biocapteur DIP.Fabrication of devices based on semiconductor multilayer structures demands routine measurement of thicknesses and location of the interfaces of the constituent layers. This is often achieved using expensive and complicated techniques such as scanning electron microscopy, secondary ion mass spectroscopy, atomic force microscopy (AFM) or x-ray photoelectron spectroscopy (XPS). In this work, room temperature metrology in water environment has been developed for post-growth testing of semiconductor nanoheterostructures (NHs). The method utilizes the process of digital photocorrosion (DIP) and the sensitivity of photoluminescence (PL) emission to surface states revealed during photocorrosion. The photocorrosion process of GaAs/AlGaAs semiconductor NHs has been investigated in the presence of above bandgap excitation of samples immersed in different aqueous solutions. In order to achieve precise control over photocorrosion rates, the NH samples were placed in a flow cell with controlled aqueous environment. A low intensity above-bandgap photoexcitation (< 105 mW/cm2) was incident in a pulse mode and characterized by a duty cycle (DC) given by TON/(TON + TOFF). This has produced average etch rates of material removed at sub-monolayer precision during each DIP cycle. Using AFM and XPS, it has been demonstrated that the PL emission from a GaAs/AlGaAs NH during DIP oscillates owing to revealed GaAs and AlGaAs layers. These oscillations are caused by the sensitivity of the PL emission to the carrier surface recombination velocity, which drastically differs for GaAs and AlGaAs. The DIP process has revealed a 1 nm thick GaAs in a GaAs/AlGaAs NH structure, but this does not seem to be the resolution limit of this approach. Open circuit potential (OCP) measured during DIP differs amongst GaAs-electrolyte and AlGaAs-electrolyte junctions formed during the photocorrosion process. The OCP oscillations were found in-phase with the PL oscillations measured during DIP. The differences of OCP are theorized to originate from the surficial photo-oxides that carry electric charge. The dipole formed by these surficial oxides define the measured OCP. The OCP oscillation could also be used for metrology of NHs. This opens the prospect of extending the DIP metrology to semiconductor NHs with non-existing or negligible PL signal. Lastly, charged entities near the vicinity of a semiconductor surface affects the rate of DIP. This property has been utilized to detect Legionella pneumophila that normally are negatively charged at pH > 4. Fourier transform infrared spectroscopy measurements have indicated that alkanethiol self-assembled monolayers (SAMs) remain on the semiconductor surface during DIP. This has allowed for the detection of live Legionella pneumophila at 105 CFU/mL with a simple antibody-based architecture. A discussion has been provided suggesting possible biosensing protocols for achieving enhanced detection limits with the DIP biosensor

COMPUTATIONAL STUDIES ON THE STRUCTURAL ASPECTS OF PROTEIN-PEPTIDE INTERACTIONS

Ph.DDOCTOR OF PHILOSOPH

Multi-scale Simulations of Dynamic Protein Structures and Interactions

Intrinsically disordered proteins (IDPs) are functional proteins that lack stable tertiary structures in the unbound state. They frequently remain dynamic even within specific complexes and assemblies. IDPs are major components of cellular regulatory networks and have been associated with cancers, diabetes, neurodegenerative diseases, and other human diseases. Computer simulations are essential for deriving a molecular description of the disordered protein ensembles and dynamic interactions for mechanistic understanding of IDPs in biology, diseases, and therapeutics. However, accurate simulation of the heterogeneous ensembles and dynamic interactions of IDPs is extremely challenging because of both the prohibitive computational cost and demanding force field accuracy. In this dissertation, we developed a set of enhanced sampling and multi-scale simulation methods to overcome these limitations, and successfully applied them to study the structure, interaction and phase separation of IDPs. We have first applied the state-of-the-art explicit solvent atomistic simulations to study the inhibitory mechanism of the disordered N-terminal domain of Staphylococcal peroxidase inhibitor (SPIN). We performed high-temperature simulations to study the coupled binding and folding process during SPIN inhibition of the host myeloperoxidase (MPO) enzyme. The results showed that differences in dynamics may provide a physical basis of the ability of different SPIN homologs to inhibit innate immunity. Recognizing the need for enhanced sampling methods for IDP simulation, we have developed a new replica-exchange with solute tempering (REST) protocol to achieve more efficient explicit solvent sampling of disordered protein ensembles. We proposed that the scaling of protein-water interactions in REST is a free parameter that could be optimized to better control how the protein conformational properties (e.g., chain expansion) at different effective temperatures and achieve more effective sampling. Specifically, we developed a REST3 protocol that rebalances the protein-protein and protein-water interactions and eliminates the unanticipated chain collapse at high temperature conditions in the previous REST2 protocol. Application to model IDPs demonstrated that REST3 prevented the conformational segregation during exchanges, leading to an effective temperature random walk across all conditions and accelerating the simulation of the protein conformational space. Even with enhanced sampling, accurate description of disordered conformations at atomistic level remains extremely challenging for complex IDPs. Alternatively, coarse-grained simulations can provide an effective strategy for overcoming the length- and time-scale limitations. Here, we refined a hybrid-resolution coarse-grained model (HyRes) for accurate simulation of disordered protein ensembles and dynamic protein interactions. HyRes contains atomistic backbone and coarse-grained sidechain beads, to provide semi-quantitative description of residual secondary structures and long-range interactions. Specifically, we introduced a surface area-based implicit solvation energy term, and iteratively re-optimized the effective interaction strength potentials. The new model, referred as HyRes II, provides near quantitative descriptions of IDP long-range non-specific interactions and secondary structures, at a level comparable to the latest atomistic protein force fields. Applied to the disordered N-terminal transactivation domain (TAD) of tumor suppressor p53, HyRes II faithfully recapitulates various nontrivial structural properties to a level of accuracy that is comparable to a99SB-disp, one of the best atomistic protein force fields. Moreover, we demonstrate HyRes II’s efficacy in accurately simulating the dynamic interaction between TAD and the DNA-binding domain of p53, generating structural ensembles that align closely with existing NMR data. Encouraged by successes of HyRes II for probing dynamic interactions of IDPs, we further investigated its suitability for simulating IDP-mediated phase separation, which underlies the formation of biomolecular condensates and has attracted intensive interests. Compared to the popular single-bead models, HyRes has the potential to describe backbone-mediated interactions and capture the role of residual structures in phase separation. Reimplemented on GPU, our simulations showed that HyRes is efficient enough to directly simulate the spontaneous phase separation of IDPs and at the time balanced enough to capture the effects of mutational and structural perturbations. For peptide GY-23, HyRes simulations reveal increased ��-structures in condensates, which are consistent with experimental observations. For the conserved region (CR) of TDP-43, HyRes simulations successfully recapitulate the apparent correlation between helical propensities and condensate stability. In depth analysis, however, revealed that residual helices did not directly mediate interpeptide interactions to stabilize the condensed phase. Instead, it is the balance between backbone and sidechain-mediated interactions, as modulated by residual structures, that actually determines phase separation propensity. Finally, we have applied the HyRes II model to study the dynamic interaction of West Nile virus (WNV) NS2B/NS3 proteases with the ClyA protein nanopore. Nanopore tweezers provide a powerful approach for label-free detection of protein dynamics at the single-molecule level, by capturing the protein analyte in the lumen of the nanopore. From the steered-MD and standard MD simulations, we discovered that the protease could bind dynamically to a middle region of the ClyA nanopore, mediated mainly by electrostatically interactions. In particular, we identified a key Glu residue within the ClyA lumen, mutation of which to Ala or Lys could further stabilize the protease/nanopore interaction. This led to the design a modified ClyA nanopore tweezer that can stably capture the protease and resolve the dynamics between NS2B/NS3 open and closed conformations