Solvent-exposed Side Chains Research Articles

Dimethylthiourea as a Quencher in Hydroxyl Radical Protein Footprinting Experiments.

Hydroxyl radical protein footprinting (HRPF) is a mass-spectrometry-based method for studying protein structures, interactions, conformations, and folding. This method is based on the irreversible labeling of solvent-exposed amino acid side chains by hydroxyl radicals. While catalase is commonly used as a quencher after the labeling of a protein by the hydroxyl radicals to efficiently remove the remaining hydrogen peroxide, it has some disadvantages. Catalase quenching adds a relatively high amount of protein to the sample, limiting the sensitivity of the method due to dynamic range issues and causing significant issues when dealing with more complex samples. We evaluated dimethylthiourea (DMTU) as a replacement for catalase in the quenching HRPF reactions. We observed that DMTU is highly effective at quenching HRPF oxidation. DMTU does not cause the background protein issues that catalase does, resulting in an increased number of protein identifications from complex mixtures. We recommend the replacement of catalase quenching with DMTU for all HRPF experiments.

In silico investigation of the structural stability as the origin of the pathogenicity of α-synuclein protofibrils

α-Synuclein is a presynaptic neuronal protein. The fibril form of α-synuclein is a major constituent of the intraneuronal inclusion called Lewy body, a characteristic hallmark of Parkinson’s disease. Recent ssNMR and cryo-EM experiments of wild-type α-synuclein fibrils have shown polymorphism and observed two major polymorphs, rod and twister. To associate the cytotoxicity of α-synuclein fibrils with their structural features, it is essential to understand the origins of their structural stability. In this study, we performed molecular dynamics simulations of the two major polymorphs of wild-type α-synuclein fibrils. The predominance of specific fibril polymorphs was rationalized in terms of relative structural stability in aqueous environments, which was attributed to the cooperative contributions of various stabilizing features. The results of the simulations indicated that highly stable structures in aqueous environments could be maintained by the cooperation of compact sidechain packing in the hydrophobic core, backbone geometry of the maximal β-sheet content wrapping the hydrophobic core, and solvent-exposed sidechains with large fluctuations maximizing the solvation entropy. The paired structure of the two protofilaments provides additional stability, especially at the interface region, by forming steric zipper interactions and hiding the hydrophobic residues from exposure to water. The sidechain interaction analyses and pulling simulations showed that the rod polymorph has stronger sidechain interactions and exhibits higher dissociation energy than the twister polymorph. It is expected that our study will provide a basis for understanding the pathogenic behaviors of diverse amyloid strains in terms of their structural properties.Communicated by Ramaswamy H. Sarma

Early Stage UV-B Induced Molecular Modifications of Human Eye Lens γD-Crystallin.

In the human eye lenses, the crystallin proteins facilitate transparency, light refraction, as well as UV light protection. A deregulated balanced interplay between α-, β-, and γ-crystallin can cause cataract. γD-crystallin (hγD) is involved in the energy dissipation of absorbed UV light by energy transfer between aromatic side chains. Early UV-B induced damage of hγD with molecular resolution is studied by solution NMR and fluorescence spectroscopy. hγD modifications are restricted to Tyr 17 and Tyr 29 in the N-terminal domain, where a local unfolding of the hydrophobic core is observed. None of the tryptophan residues assisting fluorescence energy transfer is modified and hγD is remained soluble over month. Investigating isotope-labeled hγD surrounded by eye lens extracts from cataract patients reveals very week interactions of solvent-exposed side chains in the C-terminal hγD domain and some remaining photoprotective properties of the extracts. Hereditary E107A hγD found in the eye lens core of infants developing cataract shows under the here used conditions a thermodynamic stability comparable to the wild type but an increased sensitivity toward UV-B irradiation.

Stimuli responsive hierarchical assembly of supramolecular conductive fibers from coiled-coil building blocks.

Proteins and peptides provide inspiration for synthetic stimuli-responsive nanomaterials. In this work, the assembly mechanism of a de novo designed peptide that forms α-helical coiled-coil hexamer fibers was probed through point mutations of the amino acid sequence and varying assembly solution pH. Buffer pH affects multi-scale aspects of supramolecular assembly, including peptide monomer secondary structure and interactions between α-helical coiled-coil hexamer building blocks. These higher order assembly interfaces are formed by electrostatic networks between solvent-exposed polarizable side chains, as observed by cryo-electron microscopy. One of the resulting hierarchical structures was a novel assembly of α-helical cross coiled-coils that exhibits electrical conductivity. Here, we show that pH-gated assembly can be achieved with only one amino acid mutation. This work opens materials design for more stimuli-responsive and functional materials with simple pH-gating structural motifs.

Laser-free flash oxidation (Fox) hydroxyl radical protein footprinting system accurately maps the paratope and epitope of TNFα bound to adalimumab

The Fox Protein Footprinting System is a novel hydroxyl radical protein footprinting (HRPF) method that uses a proprietary flash oxidation lamp to photolyze hydrogen peroxide to form hydroxyl radicals (⋅OH). The ⋅OH irreversibly modify solvent exposed side chains of amino acids. As solvent accessibility changes, the ⋅OH modification concordantly changes. With the addition of real time radical dosimetry, the effective ·OH concentration can be determined and easily adjusted, allowing conditions with varying scavenging effects to be easily compared.

Correlation between surface friction and the hydrophobicity of structure-related side-chain exposure of albumin on contact lens

The management of contact lens discomfort remains a challenge leading to the increased contact lens dropout rates. Tear protein accumulates on the lens surfaces with different configurations observed are correlated to the lens friction, with the improved comfort experienced by reduced surface friction in the eye. However, protein adsorption is a complex process with the combined protein-protein interactions (PPI) and protein-surface interactions (PSI) involved, which is difficult to explain the complicated tribological behavior in terms of protein structural shifts alone on lens surfaces. On the other hand, the type of solvent-exposed side chains from specific protein conformations on lens surfaces should be more important to the lens friction involved. We aim to investigate the correlation between the structure-related side-chain exposure and corresponding lens friction of adsorbed tear proteins on lens surfaces under varied PPI and PSI. Albumin was the model protein adsorbed onto the conventional lens material. Such as polymethylmethacrylate (PMMA) or the poly-2-hydroxyethyl methacrylate (PHEMA) surfaces applied here. Adsorption was conducted under varying protein solution concentrations to saturate the model surface to change the PPI effects over a wide range. Our results indicate that PPI effects help stabilize protein structures on both surfaces. When PPI is minimized, a distinct correlation was observed between the surface friction and the hydrophobicity of structure-related side-chain exposure of albumin on lens surfaces depending on the different PSI involved. At a fundamental understanding, our results would provide insights for developing new lens materials or the lens care solution designs to reduce the lens discomfort.

3D Interaction Homology: Hydropathic Analyses of the "π-Cation" and "π-π" Interaction Motifs in Phenylalanine, Tyrosine, and Tryptophan Residues.

Three-dimensional (3D) maps of the hydropathic environments of protein amino acid residues are information-rich descriptors of preferred conformations, interaction types and energetics, and solvent accessibility. The interactions made by each residue are the primary factor for rotamer selection and the secondary, tertiary, and even quaternary protein structure. Our evolving basis set of environmental data for each residue type can be used to understand the protein structure. This work focuses on the aromatic residues phenylalanine, tyrosine, and tryptophan and their structural roles. We calculated and analyzed side chain-to-environment 3D maps for over 70,000 residues of these three types that reveal, with respect to hydrophobic and polar interactions, the environment around each. After binning with backbone ϕ/ψ and side chain χ1, we clustered each bin by 3D similarities between map-map pairs. For each of the three residue types, four bins were examined in detail: one in the β-pleat, two in the right-hand α-helix, and one in the left-hand α-helix regions of the Ramachandran plot. For high degrees of side chain burial, encapsulation of the side chain by hydrophobic interactions is ubiquitous. The more solvent-exposed side chains are more likely to be involved in polar interactions with their environments. Evidence for π-π interactions was observed in about half of the residues surveyed [phenylalanine (PHE): 53.3%, tyrosine (TYR): 34.1%, and tryptophan (TRP): 55.7%], but on an energy basis, this contributed to only ∼4% of the total. Evidence for π-cation interactions was observed in 14.1% of PHE, 8.3% of TYR, and 26.8% of TRP residues, but on an energy basis, this contributed to only ∼1%. This recognition of even these subtle interactions in the 3D hydropathic environment maps is key support for our interaction homology paradigm of protein structure elucidation and possibly prediction.

Benefits of Ion Mobility Separation and Parallel Accumulation-Serial Fragmentation Technology on timsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis.

Fast photochemical oxidation of proteins (FPOP) is a recently developed technique for studying protein folding, conformations, interactions, etc. In this method, hydroxyl radicals, usually generated by KrF laser photolysis of H2O2, are used for irreversible labeling of solvent-exposed side chains of amino acids. Mapping of the oxidized residues to the protein’s structure requires pinpointing of modifications using a bottom-up proteomic approach. In this work, a quadrupole time-of-flight (QTOF) mass spectrometer coupled with trapped ion mobility spectrometry (timsTOF Pro) was used for identification of oxidative modifications in a model protein. Multiple modifications on the same residues, including six modifications of histidine, were successfully resolved. Moreover, parallel accumulation–serial fragmentation (PASEF) technology allows successful sequencing of even minor populations of modified peptides. The data obtained indicate a clear improvement of the quality of the FPOP analysis from the viewpoint of the number of identified peptides bearing oxidative modifications and their precise localization. Data are available via ProteomeXchange with identifier PXD020509.

Structure–activity relationship of ipglycermide binding to phosphoglycerate mutases

Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex.

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins.

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein's solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.

Visible-light-driven photocontrol of the Trp-cage protein fold by a diazocine cross-linker.

Diazocines are characterized by extraordinary photochemical properties rendering them of particular interest for switching the conformation of biomolecules with visible light. Current developments afford synthetic access to unprecedented diazocine derivatives promising particular opportunities in photocontrol of proteins and biological systems. In this work, the well-established approach of photocontrolling the secondary structure of α-helices was exploited using a diazocine to reversibly fold and unfold the tertiary structure of a small protein. The protein of choice was the globulary folded Trp-cage, a widely used model system for the elucidation of protein folding pathways. A specifically designed, short and rigid dicarboxy-functionalized diazocine-based cross-linker was attached to two solvent-exposed side chains at the α-helix of the miniprotein through the use of a primary amine-selective active ester. This cross-linking strategy is orthogonal to the common cysteine-based chemistry. The cross-linked Trp-cage was successfully photoisomerized and exhibited a strong correlation between protein fold and diazocine isomeric state. As determined by NMR spectroscopy, the cis-isomer stabilized the fold, while the trans-isomer led to complete protein unfolding. The successful switching of the protein fold in principle demonstrates the ability to control protein function, as the activity depends on their structural integrity.

Mutation in a flexible linker modulates binding affinity for modular complexes.

Tandem beta zippers are modular complexes formed between repeated linear motifs and tandemly arrayed domains of partner proteins in which β-strands form upon binding. Studies of such complexes, formed by LIM domain proteins and linear motifs in their intrinsically disordered partners, revealed spacer regions between the linear motifs that are relatively flexible but may affect the overall orientation of the binding modules. We demonstrate that mutation of a solvent exposed side chain in the spacer region of an LHX4-ISL2 complex has no significant effect on the structure of the complex, but decreases binding affinity, apparently by increasing flexibility of the linker.

Rigid versus Flexible Protein Matrix: Light-Harvesting Complex II Exhibits a Temperature-Dependent Phonon Spectral Density.

Dynamics-function correlations are usually inferred when molecular mobility and protein function are simultaneously impaired at characteristic temperatures or hydration levels. In this sense, excitation energy transfer in the photosynthetic light-harvesting complex II (LHC II) is an untypical example because it remains fully functional even at cryogenic temperatures relying mainly on interactions of electronic states with protein vibrations. Here, we study the vibrational and conformational protein dynamics of monomeric and trimeric LHC II from spinach using inelastic neutron scattering (INS) in the temperature range of 20-305 K. INS spectra of trimeric LHC II reveal a distinct vibrational peak at ∼2.4 meV. At temperatures above ∼160 K, however, the inelastic peak shifts toward lower energies, which is attributed to vibrational anharmonicity. A more drastic shift is observed at about 240 K, which is interpreted in terms of a "softening" of the protein matrix along with the dynamical transition. Monomeric LHC II exhibits a higher degree of conformational mobility at physiological temperatures, which can be attributed to a higher number of solvent-exposed side chains at the protein surface. The effects of the changes in protein dynamics on the spectroscopic properties of LHC II are considered in comparative model calculations. The absorption line shapes of a pigment molecule embedded into LHC II are simulated for the cases of (i) a rigid protein matrix, (ii) a protein matrix with temperature-dependent spectral density of protein vibrations, and (iii) temperature-dependent electron-phonon coupling strength. Our findings indicate that vibrational and conformational protein dynamics affect the spectroscopic (absorption) properties of LHC II at physiological temperatures.

Loss of Protein Stability due to Formation of Intermolecular Disulfide Bonds under the Effect of Oxidative Stress: Case Study of the RRM2 Domain from Neuropathological Protein TDP-43

In the presence of reactive oxygen species, proteins with solvent-exposed cysteine side chains readily undergo disulfide-mediated dimerization (oligomerization). Disulfide bridges are fairly short linkages that bring into close contact protein surfaces which have not evolved to complement each other. The resulting random pairing of surface residues as occurs at the protein-protein interfaces is likely to have an appreciable destabilizing effect on the protein structure. To test this hypothesis we have studied the effect of oxidative stress (modeled by hydrogen peroxide treatment) on the second RNA-recognition motif from the neuropathological protein TDP-43. 1H-15N HSQC titration and H/D exchange experiments confirm that formation of disulfide-bonded dimers leads to a loss of protein stability. In addition, temperature-dependent 1H spectroscopy indicates that dimeric RRM2 has lower melting temperature. As a negative control we have characterized the sample of RRM2 with C244S mutation, which does not form intermolecular disulfide bonds. The destabilizing effect of adventitious disulfide bridges has also been investigated by MD simulations of the RRM2 dimers. The simulations were conducted in explicit solvent under Amber 14SB force field; the data were used to predict HN solvent exchange protection factors, thus paving the way for comparison with the experimental results. The loss of structural stability caused by disulfide bonding under the conditions of oxidative stress may lead to increased proteolytic degradation of the RRM2 domain, resulting in formation of C-terminal fragments of TDP-43, which have been viewed as one of the causative factors in genesis of the neuronal inclusion bodies.1[1] Zhang, Y. J. et al. Proc. Natl. Acad. Sci. USA 106:7607-7612 (2009).

Evaluating the dynamics and electrostatic interactions of folded proteins in implicit solvents.

Three implicit solvent models, namely GBMVII, FACTS, and SCPISM, were evaluated for their abilities to emulate an explicit solvent environment by comparing the simulated conformational ensembles, dynamics, and electrostatic interactions of the Src SH2 domain and the Lyn kinase domain. This assessment in terms of structural features in folded proteins expands upon the use of hydration energy as a metric for comparison. All-against-all rms coordinate deviation, average positional fluctuations, and ion-pair distance distribution were used to compare the implicit solvent models with the TIP3P explicit solvent model. Our study shows that the Src SH2 domains solvated with TIP3P, GBMVII, and FACTS sample similar global conformations. Additionally, the Src SH2 ion-pair distance distributions of solvent-exposed side chains corresponding to TIP3P, GBMVII, and FACTS do not differ substantially, indicating that GBMVII and FACTS are capable of modeling these electrostatic interactions. The ion-pair distance distributions of SCPISM are distinct from others, demonstrating that these electrostatic interactions are not adequately reproduced with the SCPISM model. On the other hand, for the Lyn kinase domain, a non-globular protein with bilobal structure and a large concavity on the surface, implicit solvent does not accurately model solvation to faithfully reproduce partially buried electrostatic interactions and lobe-lobe conformations. Our work reveals that local structure and dynamics of small, globular proteins are modeled well using FACTS and GBMVII. Nonetheless, global conformations and electrostatic interactions in concavities of multi-lobal proteins resulting from simulations with implicit solvent models do not match those obtained from explicit water simulations.

Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression.

We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials.

Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting.

To infect a cell, the Paramyxoviridae family of enveloped viruses relies on the coordinated action of a receptor-binding protein (variably HN, H, or G) and a more conserved metastable fusion protein (F) to effect membrane fusion and allow genomic transfer. Upon receptor binding, HN (H or G) triggers F to undergo an extensive refolding event to form a stable postfusion state. Little is known about the intermediate states of the F refolding process. Here, a soluble form of parainfluenza virus 5 F was triggered to refold using temperature and was footprinted along the refolding pathway using fast photochemical oxidation of proteins (FPOP). Localization of the oxidative label to solvent-exposed side chains was determined by high-resolution MS/MS. Globally, metastable prefusion F is oxidized more extensively than postfusion F, indicating that the prefusion state is more exposed to solvent and is more flexible. Among the first peptides to be oxidatively labeled after temperature-induced triggering is the hydrophobic fusion peptide. A comparison of peptide oxidation levels with the values of solvent-accessible surface area calculated from molecular dynamics simulations of available structural data reveals regions of the F protein that lie at the heart of its prefusion metastability. The strong correlation between the regions of F that experience greater-than-expected oxidative labeling and epitopes for neutralizing antibodies suggests that FPOP has a role in guiding the development of targeted therapeutics. Analysis of the residue levels of labeled F intermediates provides detailed insights into the mechanics of this critical refolding event.

Surface-Catalyzed Nucleation of Amyloidogenic Peptides by Peptide-Specific Templates

Many soluble proteins self-assemble into fibrillar, β-sheet rich, stable amyloid aggregates, many of which are implicated in diseases. Peptide segments within these proteins form the core amyloidogenic region that drives fibrillation of the protein, and it has been shown that these segments, alone, form fibrillar structures. Amyloid fibers are nucleated via a fiber-dependent pathway in which additional protein adds onto the ends and extends existing fibers. However, it is hypothesized that the walls of existing fibers can also nucleate amyloid fibers in a peptide-specific manner using the solvent-exposed side chains as a template. Using a naturally occurring protein that presents side chains of beta sheet structures but no fiber ends, we have engineered templates specific to amyloidogenic peptides to test this hypothesis. We have found that introduction of these templates drastically alter peptide fiber formation kinetics. These findings suggest that amino acid side chains on the surface of amyloid fibers, and not just fiber ends, play an important role in the formation of amyloid fibers.

Medicago truncatula histidine‐containing phosphotransfer protein

Histidine-containing phosphotransfer proteins (HPts) take part in hormone signal transduction in higher plants. The overall pathway of this process is reminiscent of the two-component system initially identified in prokaryotes. HPts function in histidine-aspartate phosphorelays in which they mediate the signal from sensory kinases (usually membrane proteins) to RRs in the nucleus. Here, we report the crystal structure of an HPt protein from Medicago truncatula (MtHPt1) determined at 1.45 Å resolution and refined to an R-factor of 16.7% using low-temperature synchrotron-radiation X-ray diffraction data. There is one MtHPt1 molecule in the asymmetric unit of the crystal lattice with P2(1)2(1)2(1) symmetry. The protein fold consists of six α helices, four of which form a C-terminal helix bundle. The coiled-coil structure of the bundle is stabilized by a network of S-aromatic interactions involving highly conserved sulfur-containing residues. The structure reveals a solvent-exposed side chain of His79, which is the phosphorylation site, as demonstrated by autoradiography combined with site-directed mutation. It is surrounded by highly conserved residues present in all plant HPts. These residues form a putative docking interface for either the receiver domain of the sensory kinase, or for the RR. The biological activity of MtHPt1 was tested by autoradiography. It demonstrated phosphorylation by the intracellular kinase domain of the cytokinin receptor MtCRE1. Complex formation between MtHPt1 and the intracellular fragment of MtCRE1 was confirmed by thermophoresis, with a dissociation constant K(d) of 14 μM.

Geometry and Mechanism of Fiber Formation: A Study of Peptide Segments of Amyloid Proteins

Many soluble proteins self-assemble into fibrillar, β-sheet rich, stable aggregates that are each associated with a particular disease. It is thought that peptide segments within these proteins form the core amyloidogenic region that drives fibrillization of the protein, and it has been shown that these segments, alone, form fibrillar structures. Here, we seek to determine the nature of fiber nucleation of these peptides. Amyloid fibers can be nucleated via a fiber-dependent pathway in which additional protein adds onto the ends and extends existing fibers. However, it is hypothesized that the walls of existing fibers can also nucleate amyloid fibers in a peptide-specific manner using the solvent-exposed side chains as a template. Conditions necessary for aggregation have been investigated for a set of amyloidogenic peptides. We have found that aggregation depends on peptide and salt concentration and occurs on the timescale of hours. We have also employed a novel approach for presenting an amyloid fiber wall without presenting fiber ends. These studies illustrate the conditions and geometry important for novel nucleation mechanisms of fiber formation.