Resolving Protein Conformational Plasticity and Substrate Binding Through the Lens of Machine-Learning

A long-standing target in elucidating the biomolecular recognition process is the identification of binding-competent conformations of the receptor protein. However, protein conformational plasticity and the stochastic nature of the recognition processes often preclude the assignment of a specific protein conformation to an individual ligand-bound pose. In particular, we consider multi-microsecond long Molecular dynamics simulation trajectories of ligand recognition process in solvent-inaccessible cavity of two archtypal systems: L99A mutant of T4 Lysozyme and Cytochrome P450. We first show that if the substrate-recognition occurs via long-lived intermediate, the protein conformations can be automatically classified into substrate-bound and unbound state through an unsupervised dimensionality reduction technique. On the contrary, if the recognition process is mediated by selection of transient protein conformation by the ligand, a clear correspondence between protein conformation and binding-competent macrostates can only be established via a combination of supervised machine learning (ML) and unsupervised dimension reduction approach. In such scenario, we demonstrate that a priori random forest based supervised classification of the simulated trajectories recognition process would help characterize key amino-acid residue-pairs of the protein that are deemed sensitive for ligand binding. A subsequent unsupervised dimensional reduction via time-lagged independent component analysis of the selected residue-pairs would delineate a conformational landscape of protein which is able to demarcate ligand-bound pose from the unbound ones. As a key breakthrough, the ML-based protocol would identify distal protein locations which would be allosterically important for ligand binding and characterise their roles in recognition pathways.

Codon-specific Ramachandran plots show amino acid backbone conformation depends on identity of the translated codon.

Synonymous codons translate into chemically identical amino acids. Once considered inconsequential to the formation of the protein product, there is now significant evidence to suggest that codon usage affects co-translational protein folding and the final structure of the expressed protein. Here we develop a method for computing and comparing codon-specific Ramachandran plots and demonstrate that the backbone dihedral angle distributions of some synonymous codons are distinguishable with statistical significance for some secondary structures. This shows that there exists a dependence between codon identity and backbone torsion of the translated amino acid. Although these findings cannot pinpoint the causal direction of this dependence, we discuss the vast biological implications should coding be shown to directly shape protein conformation and demonstrate the usefulness of this method as a tool for probing associations between codon usage and protein structure. Finally, we urge for the inclusion of exact genetic information into structural databases.

Manipulating Sodium Caseinate Behaviour at the Interface: Applications for Concentrated Emulsion Formulation

<p>The effect of ionic strength, pH and droplet size distribution on the stability and rheological properties of concentrated emulsions formed using sodium caseinate was investigated. The emulsions were formulated with soybean oil concentration between 50 and 70 wt% and 1 wt% protein. In order to understand the role and response of the sodium caseinate interfacial thin film to physicochemical changes to the continuous phase the behaviour of sodium caseinate at the air-water and oil-water interfaces, as a function of pH and ionic strength, was studied using Langmuir trough, surface potential and pendant drop methods. Changes in measured system response can be explained by considering changes to protein conformation. Upon increasing ionic strength the data fit with the protein conformation changing from those states where the protein extends into the aqueous phase to those where it essentially lies flat on the interface. Aggregation and dispersion of the protein at the interfaces were detected at different pH values. Also, the buffer capacity of sodium caseinate was evaluated by preparing protein solutions at different pH and ionic strengths. Bridging flocculation and creaming occurred in the emulsions investigated, evaluated via static light scattering and Cryo-SEM. Emulsions with the appearance and texture of liquid-like through to gel-like were formulated by seemingly small changes to the ionic strength and pH of the aqueous phase. Shear-thinning was the flow behaviour of the emulsions with a shear dependent flow response that was function of the parameters evaluated. Time-dependent flow behaviour was detected for the emulsions at a low shear rate and they showed rheopexy behaviour. Viscoelastic properties of the emulsions and the interaction between the droplets were evaluated by strain sweep and creep-recovery tests.</p>

Manipulating Sodium Caseinate Behaviour at the Interface: Applications for Concentrated Emulsion Formulation

<p>The effect of ionic strength, pH and droplet size distribution on the stability and rheological properties of concentrated emulsions formed using sodium caseinate was investigated. The emulsions were formulated with soybean oil concentration between 50 and 70 wt% and 1 wt% protein. In order to understand the role and response of the sodium caseinate interfacial thin film to physicochemical changes to the continuous phase the behaviour of sodium caseinate at the air-water and oil-water interfaces, as a function of pH and ionic strength, was studied using Langmuir trough, surface potential and pendant drop methods. Changes in measured system response can be explained by considering changes to protein conformation. Upon increasing ionic strength the data fit with the protein conformation changing from those states where the protein extends into the aqueous phase to those where it essentially lies flat on the interface. Aggregation and dispersion of the protein at the interfaces were detected at different pH values. Also, the buffer capacity of sodium caseinate was evaluated by preparing protein solutions at different pH and ionic strengths. Bridging flocculation and creaming occurred in the emulsions investigated, evaluated via static light scattering and Cryo-SEM. Emulsions with the appearance and texture of liquid-like through to gel-like were formulated by seemingly small changes to the ionic strength and pH of the aqueous phase. Shear-thinning was the flow behaviour of the emulsions with a shear dependent flow response that was function of the parameters evaluated. Time-dependent flow behaviour was detected for the emulsions at a low shear rate and they showed rheopexy behaviour. Viscoelastic properties of the emulsions and the interaction between the droplets were evaluated by strain sweep and creep-recovery tests.</p>

The Impact of Glycerol on an Affibody Conformation and Its Correlation to Chemical Degradation

The addition of glycerol to protein solutions is often used to hinder the aggregation and denaturation of proteins. However, it is not a generalised practice against chemical degradation reactions. The chemical degradation of proteins, such as deamidation and isomerisation, is an important deteriorative mechanism that leads to a loss of functionality of pharmaceutical proteins. Here, the influence of glycerol on the chemical degradation of a protein and its correlation to glycerol-induced conformational changes is presented. The time-dependent chemical degradation of a pharmaceutical protein, GA-Z, in the absence and presence of glycerol was investigated in a stability study. The effect of glycerol on protein conformation and oligomerisation was characterised using asymmetric field-flow fractionation and small-angle neutron scattering in a wide glycerol concentration range of 0–90% v/v. The results from the stability study were connected to the observed glycerol-induced conformational changes in the protein. A correlation between protein conformation and the protective effect of glycerol against the degradation reactions deamidation, isomerisation, and hydrolysis was found. The study reveals that glycerol induces conformational changes of the protein, which favour a more compact and chemically stable state. It is also shown that the conformation can be changed by other system properties, e.g., protein concentration, leading to increased chemical stability.

Evolutionary Conservation of Systemic and Reversible Amyloid Aggregation

In response to environmental stress, human cells have been shown to form reversible amyloid aggregates within the nucleus, termed amyloid bodies (A-bodies). These protective physiological structures share many of the biophysical characteristics associated with the pathological amyloids found in Alzheimer's and Parkinson's disease. Here, we show that A-bodies are evolutionarily conserved across the eukaryotic domain, with their detection in D. melanogaster and S. cerevisiae marking the first examples of these functional amyloids being induced outside of a cultured cell setting. The conditions triggering amyloidogenesis varied significantly among the species tested, with results indicating that A-body formation is a severe, but sub-lethal, stress response pathway that is tailored to an organism's environmental norms. RNA-sequencing analyses demonstrate that the regulatory low-complexity long non-coding RNAs that drive A-body aggregation are both conserved and essential in human, mouse, and chicken cells. Thus, the identification of these natural and reversible functional amyloids in a variety of evolutionarily diverse species, highlights the physiological significance of this protein conformation and will be informative in advancing our understanding of both functional and pathological amyloid aggregation events.

Rejuvenated Vintage Tissue Sections Highlight Individual Antigen Fate During Processing and Long-term Storage

Antigen-bearing proteins become progressively unavailable to immunodetection after prolonged storage of routine sections, exposed to a variety of agents, such as moisture, oxygen, and temperature. By proteomic analysis, the antigens are retained in the sections and definitely in the tissue block, pointing to fixation-independent, storage time–dependent protein modifications. Based on previous experience, we hypothesized that a combined exposure to a reducing agent and to chemicals favoring protein conformation changes would reverse the masking in aged sections. Disaccharides, lactose and sucrose, and a surfactant, added to a standard antigen retrieval buffer, reverse the negative changes in aged sections. Furthermore, they provide enhanced access to antigens in freshly cut sections, but not universally, revealing additional factors, besides heat and calcium chelation, required for antigen retrieval of individual proteins: