Disordered Proteins and Network Disorder in Network Descriptions of Protein Structure, Dynamics and Function: Hypotheses and a Comprehensive Review

Much of our understanding of protein structure and mechanistic function has been derived from static high-resolution structures. As structural biology has continued to evolve it has become clear that high-resolution structures alone are unable to fully capture the mechanistic basis for protein structure and function in solution. Recently Hydrogen/Deuterium-exchange Mass Spectrometry (HDX-MS) has developed into a powerful and versatile tool for structural biologists that provides novel insights into protein structure and function. HDX-MS enables direct monitoring of a protein's structural fluctuations and conformational changes under native conditions in solution even as it is carrying out its functions. In this review, we focus on the use of HDX-MS to monitor these dynamic changes in proteins. We examine how HDX-MS has been applied to study protein structure and function in systems ranging from large, complex assemblies to intrinsically disordered proteins, and we discuss its use in probing conformational changes during protein folding and catalytic function. STATEMENT FOR A BROAD AUDIENCE: The biophysical and structural characterization of proteins provides novel insight into their functionalities. Protein motions, ranging from small scale local fluctuations to larger concerted structural rearrangements, often determine protein function. Hydrogen/Deuterium-exchange Mass Spectrometry (HDX-MS) has proven a powerful biophysical tool capable of probing changes in protein structure and dynamic protein motions that are often invisible to most other techniques.

Changes in protein structure are closely related to gel strength. Dense phase carbon dioxide (DPCD) treatment is an excellent non-thermal food processing method that can be used to induce gel formation in surimi. The sensory, water holding capacity and gel strength of DPCD induced gels are superior to heat-induced gels. Fourier-transform infrared spectroscopy was used to investigate the role of DPCD in the quality of golden pompano surimi gels and changes in protein structure. The intermolecular forces of surimi gels were analyzed in terms of ionic and hydrogen bonds, disulfide covalent and non-disulfide covalent bonds, as well as hydrophobic interactions. Correlation analysis was used to investigate the relationship between the changes in advanced protein structure and gel strength during DPCD-induced gel formation in golden pompano surimi. The results showed that the α-helix and random coil levels of surimi gel were significantly decreased (p < 0.05), while the β-sheet and β-turn content was significantly increased (p < 0.05). The number of ionic and hydrogen bonds in gel proteins decreased significantly (p < 0.05), while the hydrophobic interactions, and disulfide and non-disulfide covalent bonds increased significantly (p < 0.05) after DPCD treatment. Correlation analysis showed that β-sheets, β-turns, hydrophobic interactions, and disulfide and non-disulfide covalent bonds were strongly positively correlated with gel strength, whereas α-helices, random coils, and ionic and hydrogen bonds were strongly negatively correlated with gel strength. Therefore, the α-helix and random coil structures of surimi gels were transformed into β-sheet and β-turn structures after DPCD treatment. Hydrophobic interactions, and disulfide and non-disulfide covalent bonds were the main intermolecular forces during the DPCD-induced gel formation of surimi. Ionic and hydrogen bonds were not the main intermolecular forces. The results provide fundamental data for elucidating the mechanism of DPCD-induced protein gel formation.

Hydrogen exchange rates at peptide backbone amide linkages of proteins are highly sensitive to changes in protein structure and dynamics. Hydrogen exchange-mass spectrometry (HX-MS) can be used to measure hydrogen/deuterium exchange in protein fragments and allows the study of large proteins with high sensitivity and at very low concentrations as well as determination of the intermolecular distribution of deuterium. This unit provides a brief discussion of the interplay between protein dynamics and hydrogen exchange, a detailed prescription for designing and performing typical HX-MS experiments, and common procedures for processing and interpreting HX-MS data.

The ability of proteins to regulate their own enzymatic activity can be facilitated by changes in structure or protein dynamics in response to external regulators. Because many proteins contain SH2 and SH3 domains, transmission of information between the domains is a potential method of allosteric regulation. To determine if ligand binding to one modular domain may alter structural dynamics in an adjacent domain, allowing potential transmission of information through the protein, we used hydrogen exchange and mass spectrometry to measure changes in protein dynamics in the SH3 and SH2 domains of hematopoietic cell kinase (Hck). Ligand binding to either domain had little or no effect on hydrogen exchange in the adjacent domain, suggesting that changes in protein structure or dynamics are not a means of SH2/SH3 crosstalk. Furthermore, ligands of varying affinity covalently attached to SH3/SH2 altered dynamics only in the domain to which they bind. Such results demonstrate that ligand binding may not structurally alter adjacent SH3/SH2 domains and implies that other aspects of protein architecture contribute to the multiple levels of regulation in proteins containing SH3 and SH2 domains.

Phenolic compounds can undergo auto-oxidation, especially at alkaline pH, forming reactive o -quinones that bind covalently to proteins and may affect the protein structure, solubility, and functional properties. Yet, it is still unclear how the phenolic compounds' structure and properties affect their reaction with proteins, and how they influence the resulting changes in protein structure and functionality. Therefore, the model protein β-lactoglobulin (BLG) was incubated with ten common phenolic compounds at pH 8.5 for 24 h at a phenolic-to-protein molar ratio of 5:1. RP-HPLC was used to screen for covalent modifications. Protein structural changes were investigated using MALDI-TOF-MS, OPA and Ellman's assays, ATR-FTIR, tryptophan fluorescence quenching, SDS-PAGE, and SEC. Protein functionality changes were determined by oil droplet size measurement after emulsion formation via high pressure homogenization. Only phenolic compounds with a di or-trihydroxybenzene moiety resulted in noteworthy BLG modification (>40 %), with an average of one phenolic compound bound per protein molecule, primarily on the thiol groups of the cysteine residues. Modification decreased the protein's α-helices and shifted the intramolecular β-sheets to higher wavelengths. Protein modification resulted in the formation of smaller oil droplets (from 1.38 to 2 μm) at low homogenization pressure. A positive relationship was found between the presence of a carboxyl group in the phenolic compounds, the unfolding of the protein's tertiary structure (R 2 = 0.95), and smaller oil droplet size. The insights from this study support the selection of phenolic compounds for targeted protein modifications or for avoiding undesired protein modifications. • Only phenolics with a catechol or pyrogallol moiety modify proteins at alkaline pH • At pH 8.5, phenolic compounds primarily bind at the free thiol groups of cysteines • Modification results in a decrease and shift in intramolecular β-sheets • Phenolic compounds with a carboxyl group induced protein tertiary structure changes • Protein tertiary structure changes lead to a decrease in emulsion droplet size

Protein three-dimensional structure dynamically changes in solution depending on the presence of ligands and interacting proteins. Methods for detecting these changes in protein conformation include ‘protein footprinting,’ using mass spectrometry. We describe herein a new technique, PLIMB (Plasma Induced Modification of Biomolecules), that generates µs bursts of hydroxyl radicals from water, to measure changes in protein structure via altered solvent accessibility of amino acid side chains. PLIMB was first benchmarked with model compounds, and then applied to a biological problem, i.e., ligand (EGF) induced changes in the conformation of the external (ecto) domain of Epidermal Growth Factor Receptor (EGFR). Regions in which oxidation decreased upon adding EGF fall along the dimerization interface, consistent with models derived from crystal structures. These results demonstrate that plasma-generated hydroxyl radicals from water can be used to map protein conformational changes, and provide a readily accessible means of studying protein structure in solution.

Prediction of the Effects of the Val66Met Polymorphism on the Conformational Ensemble of an Intrinsically Disordered Protein, Brain-Derived Neurotrophic Factor

Although structure-based drug design (SBDD) has become an indispensable tool in drug discovery for a long time, it continues to pose major challenges to date. With the advancement of "omics" techniques, systems biology has enriched SBDD into a new era, called polypharmacology, in which multi-targets drug or drug combination is designed to fight complex diseases. As a preliminary tool in systems biology, protein structure networks (PSNs) treat a protein as a set of residues linked by edges corresponding to the intramolecular interactions existing in folded structures between the residues. The PSN offers a computationally efficient tool to study the structure and function of proteins, and thus may facilitate structurebased drug design. Herein, we provide an overview of recent advances in PSNs, from predicting functionally important residues, to charactering protein-protein interactions and allosteric communication paths. Furthermore, we discuss potential pharmacological applications of PSN concepts and tools, and highlight the application to two families of drug targets, GPCRs and Hsp90. Although the application of PSNs as a framework for computer-aided drug discovery has been limited to date, we put forward the potential utility value in the near future and propose the PSNs could also serve as a new tool for polypharmacology research.

Protein dynamics play a crucial role in their diverse functions. The intracellular environment significantly influences protein dynamics, particularly for intrinsically disordered proteins (IDPs). To comprehensively capture structural information from various proteins wihtin cells and characterize protein dynamics, chemical cross-linking mass spectrometry was employed. In this study, we introduce a hierarchical decoding strategy that enables the investigation of protein dynamics in vivo. Computational analysis based on distance restraints derived from cross-links is used to infer protein dynamics in cells. To facilitate this analysis, we leverage the prior structure obtained from AlphaFold2. By employing this strategy, we can characterize the full-length structure of multi-domain proteins taking into account their distinct dynamic features. Furthermore, by combining restraint sampling with an unbiased sampling and evaluation approach, we can provide a comprehensive description of the intrinsic motion of IDPs. Consequently, the hierarchical strategy we propose holds significant potential in advancing our understanding of the molecular mechanisms that undelie protein functions in cells.

1. Quantitative analysis of structural proteins from a minute amount of myocardial tissue was performed from 10 mu thick cardiac tissue slices weighing 2 to 5 mg by the extraction in glycerol solution and by sodium dodecylsulfate (SDS) gel electrophoresis, and these changes were compared with the histologic alterations in the striated structure of the adjacent cardiac slices in the experimental myocardial infarction in the dog. 2. Approximately 69 micrograms of structural proteins were obtained from 1 mg of the normal heart muscle. In the central portions of the myocardial infarction, reductions in myosin heavy chain (HC), light chain (LC) 1 and alpha-actinin were observed at 12 to 24 hours after the coronary occlusion followed by the decrease in myosin LC 2 at 48 hours. Those changes became intense at 72 hours to 7 days, but restored gradually at 14 to 28 days. 3. Alterations in the striated structure of cardiac muscle fibers of the adjacent tissues slices were found simultaneously with the changes in structural proteins. At 12 to 24 hours after the coronary ligation increase in eosinophilia and overstretch of cross-striation were observed. The findings of coagulation necrosis, loss of striation, fragmentation, swelling of A-band, etc. of the infarcted fibers were markedly observed at 48 hours to 7 days, but the histologic restoration of cardiac fibers was found with the recovery of the infarcted tissue at 14 to 28 days after the coronary ligation. 4. Changes in the compositions of structural proteins corresponded well to the alterations in the striated structure in chronology and in quantity.

Complex carbohydrates linked to glycoproteins are recently being implicated to play a variety of biological roles. The lack of well-resolved crystallographic coordinates of the carbohydrates makes it difficult to assess the contributions of the glycan chain on protein structure and dynamics. We have modeled two different oligosaccharides NeuNAc2Gal3Man3GlcNAc5Fuc and Man3GlcNAc4 to generate two glycosylation variants of major histocompatibility complex (MHC) class I glycoprotein. Molecular dynamics simulations of the isolated fourteen- and seven-residue oligosaccharides have been done in vacuo and in solution. The dynamics of the two glycoforms of MHC class I protein have been simulated in solution in the free as well as in the peptide-bound form. Good agreement between the calculated solution conformations of the oligosaccharides in isolated and conjugated forms and the average conformations obtained from x-ray or NMR data was observed for most of the glycosidic linkages. These molecular dynamics simulations of the isolated glycan chains and the glycoconjugates reveal the details of the conformational flexibility of the glycan chains; they also provide atomic level details of protein-carbohydrate interactions and the effect of the ligand binding on the carbohydrate structure and dynamics. It was found that though there is some flexibility in some of the glycosidic linkages in the isolated oligosaccharides, in the protein-conjugated form the linkages adopt more restricted conformations. The glycan chains protrude out into the solvent and might hinder the lateral association of the proteins. The presence of the bulky glycan chains does not affect the average backbone fold of the protein but induces local changes in protein structure and dynamics. It has been noted that the extent of the changes depends upon the nature of the attached glycan chain. The glycan chains do not appear to influence the peptide binding property of the protein directly, but may stabilize the protein residues that are involved in ligand binding.

Dynamical structure of glutamate dehydrogenase as monitored by tryptophan phosphorescence: Signal transmission following binding of allosteric effectors

The effect of dough mixing speed and work input on the structure, digestibility and celiac immunogenicity of the gluten macropolymer within bread

Assessing protein–surface interactions with a series of multi-labeled BSA using fluorescence lifetime microscopy and Förster Energy Resonance Transfer

The conformational ensemble and function of intrinsically disordered proteins (IDPs) are sensitive to their solution environment. The inherent malleability of disordered proteins combined with the exposure of their residues accounts for this sensitivity. One context in which IDPs play important roles that is concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has long been linked to the accumulation of high levels of cosolutes such as trehalose or sucrose as well as the enrichment of IDPs, such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat soluble (CAHS) proteins. Despite knowing that IDPs play important roles and are co-enriched alongside endogenous, species-specific cosolutes during desiccation, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance. Here, we test the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by endogenous cosolutes. We find that desiccation-related IDPs derived from four different organisms spanning two LEA protein families and the CAHS protein family, synergize best with endogenous cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. We further demonstrate that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel. Our results demonstrate that functional synergy between IDPs and endogenous cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet, the mechanisms underlying this synergy differ between IDP families.