Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions

Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3M) salt concentrations, in dilute (<0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, GdnH⁺, and Ca²⁺), identity. The Q* decreased in the order F⁻ > Cl⁻ > Br⁻ > NO₃⁻ ∼ I⁻ > SCN⁻ > ClO₄⁻ ≫ SO₄²⁻, demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO₄²⁻ anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface.     

Characterization of a highly flexible self‐assembling protein system designed to form nanocages

The design of proteins that self-assemble into well-defined, higher order structures is an important goal that has potential applications in synthetic biology, materials science, and medicine. We previously designed a two-component protein system, designated A-(+) and A-(−), in which self-assembly is mediated by complementary electrostatic interactions between two coiled-coil sequences appended to the C-terminus of a homotrimeric enzyme with C₃ symmetry. The coiled-coil sequences are attached through a short, flexible spacer sequence providing the system with a high degree of conformational flexibility. Thus, the primary constraint guiding which structures the system may assemble into is the symmetry of the protein building block. We have now characterized the properties of the self-assembling system as a whole using native gel electrophoresis and analytical ultracentrifugation (AUC) and the properties of individual assemblies using cryo-electron microscopy (EM). We show that upon mixing, A-(+) and A-(−) form only six different complexes in significant concentrations. The three predominant complexes have hydrodynamic properties consistent with the formation of heterodimeric, tetrahedral, and octahedral protein cages. Cryo-EM of size-fractionated material shows that A-(+) and A-(−) form spherical particles with diameters appropriate for tetrahedral or octahedral protein cages. The particles varied in diameter in an almost continuous manner suggesting that their structures are extremely flexible.     

T-shaped arrangement of the recombinant agrin G3-IgG Fc protein

Agrin is a large heparin sulphate proteoglycan with multiple domains, which is located in the extracellular matrix. The C-terminal G3 domain of agrin is functionally one of the most important domains. It harbors an α-dystroglycan binding site and carries out acetylcholine receptor clustering activities. In the present study, we have fused the G3 domain of agrin to an IgG Fc domain to produce a G3-Fc fusion protein that we intend to use as a tool to investigate new binding partners of agrin. As a first step of the study, we have characterized the recombinant fusion protein using a multidisciplinary approach using dynamic light scattering, analytical ultracentrifugation and small angle X-ray scattering (SAXS). Interestingly, our SAXS analysis using the high-resolution structures of G3 and Fc domain as models indicates that the G3-Fc protein forms a T-shaped molecule with the G3 domains extruding perpendicularly from the Fc scaffold. To validate our models, we have used the program HYDROPRO to calculate the hydrodynamic properties of the solution models. The calculated values are in excellent agreement with those determined experimentally.     

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.     

Thermodynamics of an aminoglycoside modifying enzyme with low substrate promiscuity: The aminoglycoside N3 acetyltransferase‐VIa

Kinetic, thermodynamic, and structural properties of the aminoglycoside N3‐acetyltransferase‐VIa (AAC‐VIa) are determined. Among the aminoglycoside N3‐acetyltransferases, AAC‐VIa has one of the most limited substrate profiles. Kinetic studies showed that only five aminoglycosides are substrates for this enzyme with a range of fourfold difference in k_cat values. Larger differences in K_M (～40‐fold) resulted in ～30‐fold variation in k_cat/K_M. Binding of aminoglycosides to AAC‐VIa was enthalpically favored and entropically disfavored with a net result of favorable Gibbs energy (ΔG < 0). A net deprotonation of the enzyme, ligand, or both accompanied the formation of binary and ternary complexes. This is opposite of what was observed with several other aminoglycoside N3‐acetyltransferases, where ligand binding causes more protonation. The change in heat capacity (ΔCp) was different in H₂O and D₂O for the binary enzyme–sisomicin complex but remained the same in both solvents for the ternary enzyme–CoASH–sisomicin complex. Unlike, most other aminoglycoside‐modifying enzymes, the values of ΔCp were within the expected range of protein‐carbohydrate interactions. Solution behavior of AAC‐VIa was also different from the more promiscuous aminoglycoside N3‐acetyltransferases and showed a monomer‐dimer equilibrium as detected by analytical ultracentrifugation (AUC). Binding of ligands shifted the enzyme to monomeric state. Data also showed that polar interactions were the most dominant factor in dimer formation. Overall, thermodynamics of ligand‐protein interactions and differences in protein behavior in solution provide few clues on the limited substrate profile of this enzyme despite its >55% sequence similarity to the highly promiscuous aminoglycoside N3‐acetyltransferase. Proteins 2017; 85:1258–1265. © 2017 Wiley Periodicals, Inc.     

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

Membrane‐less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin‐3‐RING ubiquitin ligase (CRL3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL3^SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.     

Quaternary conformational stability: The effect of reversible self‐association on the fibrillation of two insulin analogs

Under conditions relevant to the manufacturing of insulin (e.g., pH 3, room temperature), biosynthetic human insulin (BHI), and Lispro insulin (Lispro) require a nucleation step to initiate aggregation. However, upon seeding with preformed aggregates, both insulins rapidly aggregate into nonnative fibrils. Far ultraviolet circular dichroism (far‐UV CD) and second derivative Fourier transform infrared (2D‐FTIR) spectroscopic analyses show that the fibrillation process involves a change in protein secondary structure from α‐helical in native insulin to predominantly β‐sheet in the nonnative fibrils. After seeding, Lispro aggregates faster than BHI, likely because of a reduced propensity to reversibly self‐associate. Composition gradient multi‐angle light scattering (CG‐MALS) analyses show that Lispro is more monomeric than BHI, whereas their conformational stabilities measured by denaturant‐induced unfolding are statistically indistinguishable. For both BHI and Lispro, as the protein concentration increases, the apparent first‐order rate constant for soluble protein loss decreases. To explain these phenomena, we propose an aggregation model that assumes fibril growth through monomer addition with competitive inhibition by insulin dimers. Biotechnol. Bioeng. 2011;108: 2359–2370. © 2011 Wiley Periodicals, Inc.     

Self‐association of TPR domains: Lessons learned from a designed,consensus‐based TPR oligomer

The tetratricopeptide repeat (TPR) motif is a protein–protein interaction module that acts as an organizing centre for complexes regulating a multitude of biological processes. Despite accumulating evidence for the formation of TPR oligomers as an additional level of regulation there is a lack of structural and solution data explaining TPR self‐association. In the present work we characterize the trimeric TPR‐containing protein YbgF, which is linked to the Tol system in Gram‐negative bacteria. By subtracting previously identified TPR consensus residues required for stability of the fold from residues conserved across YbgF homologs, we identified residues involved in oligomerization of the C‐terminal YbgF TPR domain. Crafting these residues, which are located in loop regions between TPR motifs, onto the monomeric consensus TPR protein CTPR3 induced the formation of oligomers. The crystal structure of this engineered oligomer shows an asymmetric trimer where stacking interactions between the introduced tyrosines and displacement of the C‐terminal hydrophilic capping helix, present in most TPR domains, are key to oligomerization. Asymmetric trimerization of the YbgF TPR domain and CTPR3Y3 leads to the formation of higher order oligomers both in the crystal and in solution. However, such open‐ended self‐association does not occur in full‐length YbgF suggesting that the protein's N‐terminal coiled‐coil domain restricts further oligomerization. This interpretation is borne out in experiments where the coiled‐coil domain of YbgF was engineered onto the N‐terminus of CTPR3Y3 and shown to block self‐association beyond trimerization. Our study lays the foundations for understanding the structural basis for TPR domain self‐association and how such self‐association can be regulated in TPR domain‐containing proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Force‐induced globule–coil transition in laminin binding protein and its role for viral–cell membrane fusion

The specific interactions of the pairs laminin binding protein (LBP)–purified tick‐borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single‐molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves‐based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus–cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus–cell membrane fusion, SMDFS data reveal the existence of a force‐induced (effective already for forces as small as 30–70 pN) sharp globule–coil transition for LBP and LBP–fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first‐order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus–cell membrane fusion process. Copyright © 2014 John Wiley & Sons, Ltd.     

Allosteric HIV‐1 integrase inhibitors promote aberrant protein multimerization by directly mediating inter‐subunit interactions: Structural and thermodynamic modeling studies

Allosteric HIV‐1 integrase (IN) inhibitors (ALLINIs) bind at the dimer interface of the IN catalytic core domain (CCD), and potently inhibit HIV‐1 by promoting aberrant, higher‐order IN multimerization. Little is known about the structural organization of the inhibitor‐induced IN multimers and important questions regarding how ALLINIs promote aberrant IN multimerization remain to be answered. On the basis of physical chemistry principles and from our analysis of experimental information, we propose that inhibitor‐induced multimerization is mediated by ALLINIs directly promoting inter‐subunit interactions between the CCD dimer and a C‐terminal domain (CTD) of another IN dimer. Guided by this hypothesis, we have built atomic models of inter‐subunit interfaces in IN multimers by incorporating information from hydrogen‐deuterium exchange (HDX) measurements to drive protein‐protein docking. We have also developed a novel free energy simulation method to estimate the effects of ALLINI binding on the association of the CCD and CTD. Using this structural and thermodynamic modeling approach, we show that multimer inter‐subunit interface models can account for several experimental observations about ALLINI‐induced multimerization, including large differences in the potencies of various ALLINIs, the mechanisms of resistance mutations, and the crucial role of solvent exposed R‐groups in the high potency of certain ALLINIs. Our study predicts that CTD residues Tyr226, Trp235 and Lys266 are involved in the aberrant multimer interfaces. The key finding of the study is that it suggests the possibility of ALLINIs facilitating inter‐subunit interactions between an external CTD and the CCD‐CCD dimer interface.     

Expression of scFv‐Mel‐Gal4 triple fusion protein as a targeted DNA‐carrier in Escherichia Coli

Liver‐directed gene therapy has become a promising treatment for many liver diseases. In this study, we constructed a multi‐functional targeting molecule, which maintains targeting, endosome‐escaping, and DNA‐binding abilities for gene delivery. Two single oligonucleotide chains of Melittin (M) were synthesized. The full‐length cDNA encoding anti‐hepatic asialoglycoprotein receptor scFv C1 (C1) was purified from C1/pIT2. The GAL4 (G) gene was amplified from pSW50‐Gal4 by polymerase chain reaction. M, C1 and G were inserted into plasmid pGC4C26H to product the recombinant plasmid pGC‐C1MG. The fused gene C1MG was subsequently subcloned into plasmid pET32c to product the recombinant plasmid C1MG/pET32c and expressed in Escherichia coli BL21. The scFv‐Mel‐Gal4 triple fusion protein (C1MG) was purified with a Ni²⁺ chelating HiTrap HP column. The fusion protein C1MG of roughly 64 kD was expressed in inclusion bodies; 4.5 mg/ml C1MG was prepared with Ni²⁺ column purification. Western blot and immunohistochemistry showed the antigen‐binding ability of C1MG to the cell surface of the liver‐derived cell line and liver tissue slices. Hemolysis testing showed that C1MG maintained membrane‐disrupting activity. DNA‐binding capacity was substantiated by luciferase assay, suggesting that C1MG could deliver the DNA into cells efficiently on the basis of C1MG. Successful expression of C1MG was achieved in E. coli, and C1MG recombinant protein confers targeting, endosome‐escaping and DNA‐binding capacity, which makes it probable to further study its liver‐specific DNA delivery efficacy in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Defining the metal specificity of a multifunctional biofilm adhesion protein

The accumulation associated protein (Aap) of Staphylococcus epidermidis mediates intercellular adhesion events necessary for biofilm growth. This process depends upon Zn²⁺‐induced self‐assembly of G5 domains within the B‐repeat region of the protein, forming anti‐parallel, intertwined protein “ropes” between cells. Pleomorphism in the Zn²⁺‐coordinating residues was observed in previously solved crystal structures, suggesting that the metal binding site might accommodate other transition metals and thereby support dimerization. By use of carefully selected buffer systems and a specialized approach to analyze sedimentation velocity analytical ultracentrifugation data, we were able to analyze low‐affinity metal binding events in solution. Our data show that both Zn²⁺ and Cu²⁺ support B‐repeat assembly, whereas Mn²⁺, Co²⁺, and Ni²⁺ bind to Aap but do not support self‐association. As the number of G5 domains are increased in longer B‐repeat constructs, the total concentration of metal required for dimerization decreases and the transition between monomer and dimer becomes more abrupt. These characteristics allow Aap to function as an environmental sensor that regulates biofilm formation in response to local concentrations of Zn²⁺ and Cu²⁺, both of which are implicated in immune cell activity.     

Linkers in the structural biology of protein–protein interactions

Linkers or spacers are short amino acid sequences created in nature to separate multiple domains in a single protein. Most of them are rigid and function to prohibit unwanted interactions between the discrete domains. However, Gly‐rich linkers are flexible, connecting various domains in a single protein without interfering with the function of each domain. The advent of recombinant DNA technology made it possible to fuse two interacting partners with the introduction of artificial linkers. Often, independent proteins may not exist as stable or structured proteins until they interact with their binding partner, following which they gain stability and the essential structural elements. Gly‐rich linkers have been proven useful for these types of unstable interactions, particularly where the interaction is weak and transient, by creating a covalent link between the proteins to form a stable protein–protein complex. Gly‐rich linkers are also employed to form stable covalently linked dimers, and to connect two independent domains that create a ligand‐binding site or recognition sequence. The lengths of linkers vary from 2 to 31 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked partners. Various structures of covalently linked protein complexes have been described using X‐ray crystallography, nuclear magnetic resonance and cryo‐electron microscopy techniques. In this review, we evaluate several structural studies where linkers have been used to improve protein quality, to produce stable protein–protein complexes, and to obtain protein dimers.     

Crystal stuctures of MglB‐2 (TP0684), a topologically variant d‐glucose‐binding protein from Treponema pallidum,reveal a ligand‐induced conformational change

Previously, we determined the crystal structure of apo‐TpMglB‐2, a d ‐glucose‐binding component of a putative ABC transporter from the syphilis spirochete Treponema pallidum. The protein had an unusual topology for this class of proteins, raising the question of whether the d ‐glucose‐binding mode would be different in TpMglB‐2. Here, we present the crystal structures of a variant of TpMglB‐2 with and without d ‐glucose bound. The structures demonstrate that, despite its aberrant topology, the protein undergoes conformational changes and binds d ‐glucose similarly to other Mgl‐type proteins, likely facilitating d ‐glucose uptake in T. pallidum.     

Design of novel ligands of CDP‐methylerythritol kinase by mimicking direct protein‐protein and solvent‐mediated interactions

The methylerythritol 4‐phosphate (MEP) pathway for the biosynthesis of the isoprenoid universal building blocks (isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)) is present in most of human pathogens and is absent in animals, turning it into a promising therapeutic druggable pathway. Two different strategies, a pharmacophore‐directed virtual screening and a protein‐protein interaction (PPI)‐mimicking cyclic peptide were used to search for compounds that bind to the PPI surface of the 4‐(cytidine 5‐diphospho)‐2C‐methyl‐D‐erythritol kinase (CMK), which catalyzes the fourth step of the MEP pathway. A significant part of the pharmacophore hypothesis used in this study was designed by mimicking water‐mediated PPI relevant in the CMK homodimer complex stabilization. After database search and with the aid of docking and molecular dynamics (MD) simulations, a 7H‐furo[3,2‐g]chromen‐7‐one derivative and a cyclic peptide were chosen as candidates to be ligands of CMK. Their binding affinities were measured using surface plasmon resonance (SPR) technology. Copyright © 2010 John Wiley & Sons, Ltd.     

Kinetics of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle: mechanism of protective action of alpha-crystallin

The kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscle have been studied by dynamic light scattering (0.08M Hepes, pH 6.8, containing 0.1M NaCl; 48 degrees C). The hydrodynamic radius of the start aggregates determined from the initial linear parts of the dependences of the hydrodynamic radius (R(h)) on time was found to be 16.7 +/- 1.0 nm. At rather high values of time, the R(h) value for the protein aggregates becomes proportional to t(1/1.8) = t(0.56) suggesting that the aggregation process proceeds in the regime of diffusion-limited cluster-cluster aggregation. In the presence of alpha-crystallin, a protein possessing the chaperone-like activity, the process of protein aggregation switches to the regime of reaction-limited cluster-cluster aggregation as indicated by the exponential dependence of the R(h) value on time. It was shown that the addition of alpha-crystallin raises the rate of thermal inactivation of Phb. These data in combination with the results of the study of interaction of Phb with alpha-crystallin by analytical ultracentrifugation suggest that alpha-crystallin interacts with the intermediates of unfolding of the Phb molecule.     

Trichloroacetic acid‐induced protein precipitation involves the reversible association of a stable partially structured intermediate

Sample preparation for proteomic analysis involves precipitation of protein using 2,2,2‐trichloroacetic acid (TCA). In this study, we examine the mechanism of the TCA‐induced protein precipitation reaction. TCA‐induced protein precipitation curves are U‐shaped and the shape of the curve is observed to be independent of the physicochemical properties of proteins. TCA is significantly less effective in precipitating unfolded states of proteins. Results of the 1‐anilino‐8‐napthalene sulfonate (ANS) and size‐exclusion chromatography, obtained using acidic fibroblast growth factor (aFGF), show that a stable “molten globule‐like” partially structured intermediate accumulates maximally in 5% (w/v) of trichloroacetate. Urea‐induced unfolding and limited proteolytic digestion data reveal that the partially structured intermediate is significantly less stable than the native conformation. ¹H‐¹⁵N chemical shift perturbation data obtained using NMR spectroscopy indicate that interactions stabilizing the β‐strands at the N‐ and C‐ terminal ends (of aFGF) are disrupted in the trichloroacetate‐induced “MG‐like” state. The results of the study clearly demonstrate that TCA‐induced protein precipitation occurs due to the reversible association of the “MG‐like” partially structured intermediate state(s). In our opinion, the findings of this study provide useful clues toward development of efficient protocols for the isolation and analysis of the entire proteome.     

Defining the protein complexome of translation termination factor eRF1: Identification of four novel eRF1‐containing complexes that range from 20S to 57S in size

The eukaryotic eRF1 translation termination factor plays an important role in recognizing stop codons and initiating the end to translation. However, which exact complexes contain eRF1 and at what abundance is not clear. We have used analytical ultracentrifugation with fluorescent detection system to identify the protein complexome of eRF1 in the yeast Saccharomyces cerevisiae. In addition to eRF1 presence in translating polysomes, we found that eRF1 associated with five other macromolecular complexes: 77S, 57S, 39S, 28S, and 20S in size. Generally equal abundances of each of these complexes were found. The 77S complex primarily contained the free 80S ribosome consistent with in vitro studies and did not appear to contain significant levels of the monosomal translating complex that co‐migrates with the free 80S ribosome. The 57S and 39S complexes represented, respectively, free 60S and 40S ribosomal subunits bound to eRF1, associations not previously reported. The novel 28S and 20S complexes (containing minimal masses of 830 KDa and 500 KDa, respectively) lacked significant RNA components and appeared to be oligomeric, as eRF1 has a mass of 49 KDa. The majority of polysomal complexes containing eRF1 were both substantially deadenylated and lacking in closed‐loop factors eIF4E and eIF4G. The thirteen percent of such translating polysomes that contained poly(A) tails had equivalent levels of eIF4E and eIF4G, suggesting these complexes were in a closed‐loop structure. The identification of eRF1 in these unique and previously unrecognized complexes suggests a variety of new roles for eRF1 in the regulation of cellular processes.