Solution properties of γ‐crystallins: Hydration of fish and mammal γ‐crystallins

Lens γ crystallins are found at the highest protein concentration of any tissue, ranging from 300 mg/mL in some mammals to over 1000 mg/mL in fish. Such high concentrations are necessary for the refraction of light, but impose extreme requirements for protein stability and solubility. γ‐crystallins, small stable monomeric proteins, are particularly associated with the lowest hydration regions of the lens. Here, we examine the solvation of selected γ‐crystallins from mammals (human γD and mouse γS) and fish (zebrafish γM2b and γM7). The thermodynamic water binding coefficient B₁ could be probed by sucrose expulsion, and the hydrodynamic hydration shell of tightly bound water was probed by translational diffusion and structure‐based hydrodynamic boundary element modeling. While the amount of tightly bound water of human γD was consistent with that of average proteins, the water binding of mouse γS was found to be relatively low. γM2b and γM7 crystallins were found to exhibit extremely low degrees hydration, consistent with their role in the fish lens. γM crystallins have a very high methionine content, in some species up to 15%. Structure‐based modeling of hydration in γM7 crystallin suggests low hydration is associated with the large number of surface methionine residues, likely in adaptation to the extremely high concentration and low hydration environment in fish lenses. Overall, the degree of hydration appears to balance stability and tissue density requirements required to produce and maintain the optical properties of the lens in different vertebrate species.     

Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions

A reversible green fluorogenic protein‐fragment complementation assay was developed based on the crystal structure of UnaG, a recently discovered fluorescent protein. In living mammalian cells, the nonfluorescent fragments complemented and rapidly became fluorescent upon rapamycin‐induced FKBP and Frb protein interaction, and lost fluorescence when the protein interaction was inhibited. This reversible fluorogenic reporter, named uPPI [UnaG‐based protein‐protein interaction (PPI) reporter], uses bilirubin (BR) as the chromophore and requires no exogenous cofactor. BR is an endogenous molecule in mammalian cells and is not fluorescent by itself. uPPI may have many potential applications in visualizing spatiotemporal dynamics of PPIs.     

TSGIT: An N‐ and C‐terminal tandem tag system for purification of native and intein‐mediated ligation‐ready proteins

A large variety of fusion tags have been developed to improve protein expression, solubilization, and purification. Nevertheless, these tags have been combined in a rather limited number of composite tags and usually these composite tags have been dictated by traditional commercially‐available expression vectors. Moreover, most commercially‐available expression vectors include either N‐ or C‐terminal fusion tags but not both. Here, we introduce TSGIT, a fusion‐tag system composed of both N‐ and a C‐terminal composite fusion tags. The system includes two affinity tags, two solubilization tags and two cleavable tags distributed at both termini of the protein of interest. Therefore, the N‐ and the C‐terminal composite fusion tags in TSGIT are fully orthogonal in terms of both affinity selection and cleavage. For using TSGIT, we streamlined the cloning, expression, and purification procedures. Each component tag is selected to maximize its benefits toward the final construct. By expressing and partially purifying the protein of interest between the components of the TSGIT fusion, the full‐length protein is selected over truncated forms, which has been a long‐standing problem in protein purification. Moreover, due to the nature of the cleavable tags in TSGIT, the protein of interest is obtained in its native form without any additional undesired N‐ or C‐terminal amino acids. Finally, the resulting purified protein is ready for efficient ligation with other proteins or peptides for downstream applications. We demonstrate the use of this system by purifying a large amount of native fluorescent mRuby3 protein and bacteriophage T7 gp2.5 ssDNA‐binding protein.     

Rules for connectivity of secondary structure elements in protein: Two–layer αβ sandwiches

In protein structures, the fold is described according to the spatial arrangement of secondary structure elements (SSEs: α‐helices and β‐strands) and their connectivity. The connectivity or the pattern of links among SSEs is one of the most important factors for understanding the variety of protein folds. In this study, we introduced the connectivity strings that encode the connectivities by using the types, positions, and connections of SSEs, and computationally enumerated all the connectivities of two‐layer αβ sandwiches. The calculated connectivities were compared with those in natural proteins determined using MICAN, a nonsequential structure comparison method. For 2α‐4β, among 23,000 of all connectivities, only 48 were free from irregular connectivities such as loop crossing. Of these, only 20 were found in natural proteins and the superfamilies were biased toward certain types of connectivities. A similar disproportional distribution was confirmed for most of other spatial arrangements of SSEs in the two‐layer αβ sandwiches. We found two connectivity rules that explain the bias well: the abundances of interlayer connecting loops that bridge SSEs in the distinct layers; and nonlocal β‐strand pairs, two spatially adjacent β‐strands located at discontinuous positions in the amino acid sequence. A two‐dimensional plot of these two properties indicated that the two connectivity rules are not independent, which may be interpreted as a rule for the cooperativity of proteins.     

Cooperative hydrophobic core interactions in the β‐trefoil architecture

Symmetric protein architectures have a compelling aesthetic that suggests a plausible evolutionary process (i.e., gene duplication/fusion) yielding complex architecture from a simpler structural motif. Furthermore, symmetry inspires a practical approach to computational protein design that substantially reduces the combinatorial explosion problem, and may provide practical solutions for structure optimization. Despite such broad relevance, the role of structural symmetry in the key area of hydrophobic core‐packing cooperativity has not been adequately studied. In the present report, the threefold rotational symmetry intrinsic to the β‐trefoil architecture is shown to form a geometric basis for highly‐cooperative core‐packing interactions that both stabilize the local repeating motif and promote oligomerization/long‐range contacts in the folding process. Symmetry in the β‐trefoil structure also permits tolerance towards mutational drift that involves a structural quasi‐equivalence at several key core positions.     

Mass-spectrometric Identification of Binding Proteins of <Emphasis Type="BoldItalic">M</Emphasis><Subscript>r</Subscript> 25,000 Protein,a Part of Vitellogenin B1, Detected in Particulate Fraction of <Emphasis Type="Italic">Xenopus laevis</Emphasis> Oocytes

A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. Our previous report showed the existence of several binding proteins of pp25 in the particulate fraction of Xenopus oocytes. In an attempt to elucidate the function of pp25, two of these binding proteins were purified, analyzed by mass-spectrometry, and identified as ribosomal proteins S13 and S14. Other binding proteins in the particulate fraction mostly corresponded to those derived from purified 40S and 60S ribosomal subunits, as shown by the overlay assay method. However, pp25 did not show any effect on protein synthesis in the rabbit reticulocyte lysate system. A model in which pp25 connects a type of serpin (serine protease inhibitor), the only pp25-binding protein detected in the cytoplasm, to the endoplasmic reticulum through two serine clusters is proposed to explain a possible function of this protein.     

Representing and comparing protein folds and fold families using three‐dimensional shape‐density representations

The question of how best to compare and classify the (three‐dimensional) structures of proteins is one of the most important unsolved problems in computational biology. To help tackle this problem, we have developed a novel shape‐density superposition algorithm called 3D‐Blast which represents and superposes the shapes of protein backbone folds using the spherical polar Fourier correlation technique originally developed by us for protein docking. The utility of this approach is compared with several well‐known protein structure alignment algorithms using receiver‐operator‐characteristic plots of queries against the “gold standard” CATH database. Despite being completely independent of protein sequences and using no information about the internal geometry of proteins, our results from searching the CATH database show that 3D‐Blast is highly competitive compared to current state‐of‐the‐art protein structure alignment algorithms. A novel and potentially very useful feature of our approach is that it allows an average or “consensus” fold to be calculated easily for a given group of protein structures. We find that using consensus shapes to represent entire fold families also gives very good database query performance. We propose that using the notion of consensus fold shapes could provide a powerful new way to index existing protein structure databases, and that it offers an objective way to cluster and classify all of the currently known folds in the protein universe. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Solution properties of γ‐crystallins: Compact structure and low frictional ratio are conserved properties of diverse γ‐crystallins

γ‐crystallins are highly specialized proteins of the vertebrate eye lens where they survive without turnover under high molecular crowding while maintaining transparency. They share a tightly folded structural template but there are striking differences among species. Their amino acid compositions are unusual. Even in mammals, γ‐crystallins have high contents of sulfur‐containing methionine and cysteine, but this reaches extremes in fish γM‐crystallins with up to 15% Met. In addition, fish γM‐crystallins do not conserve the paired tryptophan residues found in each domain in mammalian γ‐crystallins and in the related β‐crystallins. To gain insight into important, evolutionarily conserved properties and functionality of γ‐crystallins, zebrafish (Danio rerio) γM2b and γM7 were compared with mouse γS and human γD. For all four proteins, far UV CD spectra showed the expected β‐sheet secondary structure. Like the mammalian proteins, γM7 was highly soluble but γM2b was much less so. The heat and denaturant stability of both fish proteins was lower than either mammalian protein. The ability of full‐length and truncated versions of human αB‐crystallin to retard aggregation of the heat denatured proteins also showed differences. However, when solution behavior was investigated by sedimentation velocity experiments, the diverse γ‐crystallins showed remarkably similar hydrodynamic properties with low frictional ratios and partial specific volumes. The solution behavior of γ‐crystallins, with highly compact structures suited for the densely packed environment of the lens, seems to be highly conserved and appears largely independent of amino acid composition.     

Metal‐dependent assembly of a protein nano‐cage

Short, alpha‐helical coiled coils provide a simple, modular method to direct the assembly of proteins into higher order structures. We previously demonstrated that by genetically fusing de novo–designed coiled coils of the appropriate oligomerization state to a natural trimeric protein, we could direct the assembly of this protein into various geometrical cages. Here, we have extended this approach by appending a coiled coil designed to trimerize in response to binding divalent transition metal ions and thereby achieve metal ion‐dependent assembly of a tetrahedral protein cage. Ni²⁺, Co²⁺, Cu²⁺, and Zn²⁺ ions were evaluated, with Ni²⁺ proving the most effective at mediating protein assembly. Characterization of the assembled protein indicated that the metal ion–protein complex formed discrete globular structures of the diameter expected for a complex containing 12 copies of the protein monomer. Protein assembly could be reversed by removing metal ions with ethylenediaminetetraacetic acid or under mildly acidic conditions.     

A rapid fluorogenic GPCR–β‐arrestin interaction assay

Detection of protein–protein interactions involved in signal transduction in live cells and organisms has a variety of important applications. We report a fluorogenic assay for G protein‐coupled receptor (GPCR)–β‐arrestin interaction that is genetically encoded, generalizes to multiple GPCRs, and features high signal‐to‐noise because fluorescence is absent until its components interact upon GPCR activation. Fluorescence after protease‐activated receptor‐1 activation developed in minutes and required specific serine–threonine residues in the receptor carboxyl tail, consistent with a classical G protein‐coupled receptor kinase dependent β‐arrestin recruitment mechanism. This assay provides a useful complement to other in vivo assays of GPCR activation.     

Fine‐tuning sortase‐mediated immobilization of protein layers on surfaces using sequential deprotection and coupling

Increasing interest in protein immobilization on surfaces has heightened the need for techniques enabling layer‐by‐layer protein attachment. Here, we report a technique for controlling enzyme‐mediated immobilization of layers of protein on the surface using a genetically encoded protecting group. An enterokinase‐cleavable peptide sequence was inserted at the N‐terminus of bifunctional fluorescent proteins containing Sortase A substrate recognition tags at both ends to control Sortase A‐mediated protein immobilization on the surface layer‐by‐layer. Efficient, sequential immobilization of a second layer of protein using Sortase A required removal of the N‐terminal protecting group, suggesting the method enables multilayer synthesis using cyclic deprotection and coupling steps. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:824–831, 2017     

Chondroitin Sulfate Accelerates Trans‐Golgi‐to‐Surface Transport of Proteoglycan Amyloid Precursor Protein

The amyloid precursor protein (APP) is a membrane protein implicated in the pathogenesis of Alzheimer's disease. APP is a part‐time proteoglycan, as splice variants lacking exon 15 are modified by a chondroitin sulfate glycosaminoglycan (GAG) chain. Investigating the effect of the GAG chain on the trafficking of APP in non‐polarized cells, we found it to increase the steady‐state surface‐to‐intracellular distribution, to reduce the rate of endocytosis and to accelerate transport kinetics from the trans‐Golgi network (TGN) to the plasma membrane. Deletion of the cytosolic domain resulted in delayed surface arrival of GAG‐free APP, but did not affect the rapid export kinetics of the proteoglycan form. Protein‐free GAG chains showed the same TGN‐to‐cell surface transport kinetics as proteoglycan APP. Endosome ablation experiments were performed to distinguish between indirect endosomal and direct pathways to the cell surface. Surprisingly, TGN‐to‐cell surface transport of both GAG‐free and proteoglycan APP was found to be indirect via transferrin‐positive endosomes. Our results show that GAGs act as alternative sorting determinants in cellular APP transport that are dominant over cytoplasmic signals and involve distinct sorting mechanisms.      

CONFOLD: Residue‐residue contact‐guided ab initio protein folding

Predicted protein residue–residue contacts can be used to build three‐dimensional models and consequently to predict protein folds from scratch. A considerable amount of effort is currently being spent to improve contact prediction accuracy, whereas few methods are available to construct protein tertiary structures from predicted contacts. Here, we present an ab initio protein folding method to build three‐dimensional models using predicted contacts and secondary structures. Our method first translates contacts and secondary structures into distance, dihedral angle, and hydrogen bond restraints according to a set of new conversion rules, and then provides these restraints as input for a distance geometry algorithm to build tertiary structure models. The initially reconstructed models are used to regenerate a set of physically realistic contact restraints and detect secondary structure patterns, which are then used to reconstruct final structural models. This unique two‐stage modeling approach of integrating contacts and secondary structures improves the quality and accuracy of structural models and in particular generates better β‐sheets than other algorithms. We validate our method on two standard benchmark datasets using true contacts and secondary structures. Our method improves TM‐score of reconstructed protein models by 45% and 42% over the existing method on the two datasets, respectively. On the dataset for benchmarking reconstructions methods with predicted contacts and secondary structures, the average TM‐score of best models reconstructed by our method is 0.59, 5.5% higher than the existing method. The CONFOLD web server is available at http://protein.rnet.missouri.edu/confold/ . Proteins 2015; 83:1436–1449. © 2015 Wiley Periodicals, Inc.     

Structure-based functional identification of a novel heme-binding protein from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB8

The TT1485 gene from Thermus thermophilus HB8 encodes a hypothetical protein of unknown function with about 20 sequence homologs of bacterial or archaeal origin. Together they form a family of uncharacterized proteins, the cluster of orthologous group COG3253. Using a combination of amino acid sequence analysis, three-dimensional structural studies and biochemical assays, we identified TT1485 as a novel heme-binding protein. The crystal structure reveals that this protein is a pentamer and each monomer exhibits a β-barrel fold. TT1485 is structurally similar to muconolactone isomerase, but this provided no functional clues. Amino acid sequence analysis revealed remote homology to a heme enzyme, chlorite dismutase. Strikingly, amino acid residues that are highly conserved in the homologous hypothetical proteins and chlorite dismutase cluster around a deep cavity on the surface of each monomer. Molecular modeling shows that the cavity can accommodate a heme group with a strictly conserved His as a heme ligand. TT1485 reconstituted with iron protoporphyrin IX chloride gave a low chlorite dismutase activity, indicating that TT1485 catalyzes a reaction other than chlorite degradation. The presence of a possible Fe–His–Asp triad in the heme proximal site suggests that TT1485 functions as a novel heme peroxidase to detoxify hydrogen peroxide within the cell.     

Human interleukin‐23 receptor antagonists derived from an albumin‐binding domain scaffold inhibit IL‐23‐dependent ex vivo expansion of IL‐17‐producing T‐cells

Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin‐23 receptor (IL‐23R), which is a key element of proinflammatory IL‐23‐mediated signaling. A library of variants derived from the three‐helix bundle scaffold of the albumin‐binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high‐affinity binders of recombinant extracellular IL‐23R. A collection of 34 IL‐23R‐binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL‐23, or the biologically active human IL‐23 cytokine, to the recombinant IL‐23R or soluble IL‐23R‐IgG chimera. The strongest competitors for IL‐23R binding in ELISA were confirmed to recognize human IL‐23R‐IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub‐ to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K‐562, THP‐1 and Jurkat, and this binding correlated with IL‐23R cell‐surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP‐1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL‐23‐driven expansion of IL‐17‐producing primary human CD4⁺ T‐cells. Thus, we conclude that unique IL‐23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti‐inflammatory biologicals. Proteins 2014; 82:975–989. © 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Mitotic spindle association of TACC3 requires Aurora‐A‐dependent stabilization of a cryptic α‐helix

Aurora‐A regulates the recruitment of TACC3 to the mitotic spindle through a phospho‐dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora‐A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora‐A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical‐repeat region of CHC, not a recognized phospho‐reader domain. This potentially widespread mechanism of phospho‐recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding.