Designed Artificial Protein Heterodimers With Coupled Functions Constructed Using Bio-Orthogonal Chemistry

The formation of protein complexes is central to biology, with oligomeric proteins more prevalent than monomers. The coupling of functionally and even structurally distinct protein units can lead to new functional properties not accessible by monomeric proteins alone. While such complexes are driven by evolutionally needs in biology, the ability to link normally functionally and structurally disparate proteins can lead to new emergent properties for use in synthetic biology and the nanosciences. Here we demonstrate how two disparate proteins, the haem binding helical bundle protein cytochrome b562 and the β-barrel green fluorescent protein can be combined to form a heterodimer linked together by an unnatural triazole linkage. The complex was designed using computational docking approaches to predict compatible interfaces between the two proteins. Models of the complexes where then used to engineer residue coupling sites in each protein to link them together. Genetic code expansion was used to incorporate azide chemistry in cytochrome b562 and alkyne chemistry in GFP so that a permanent triazole covalent linkage can be made between the two proteins. Two linkage sites with respect to GFP were sampled. Spectral analysis of the new heterodimer revealed that haem binding and fluorescent protein chromophore properties were retained. Functional coupling was confirmed through changes in GFP absorbance and fluorescence, with linkage site determining the extent of communication between the two proteins. We have thus shown here that is possible to design and build heterodimeric proteins that couple structurally and functionally disparate proteins to form a new complex with new functional properties.

Changes in Hydrophobic Interactions among Gluten Proteins during Dough Formation

In this study, changes in hydrophobic interactions among gluten proteins were analyzed during dough mixing. Size-exclusion high-performance chromatography and two-dimensional fluorescence difference gel electrophoresis were performed on proteins extracted with 1-propanol by weakening the hydrophobic interaction. The amount of proteins extracted with 30% 1-propanol increased from the start of mixing to peak consistency, suggesting that the hydrophobic interactions among the strongly aggregated proteins weakened and resulted in disaggregation. The amount of proteins extracted with 10% 1-propanol decreased during hydration, indicating that these proteins aggregated through relatively weak hydrophobic interactions. The proteins that extractability decreased were mainly low molecular weight glutenin, α-gliadin, and γ-gliadin. The amount of monomeric proteins extracted with 30% 1-propanol decreased after peak consistency. The decreased protein was mainly ω-gliadin, indicating that ω-gliadin aggregated with other proteins through hydrophobic interactions. A front-face fluorescence analysis was performed on the dough with the addition of 8-anilino-1-naphthalenesulfonic acid or thioflavin T. The fluorescence intensity increased as a result of exposure to the hydrophobic groups of the gluten proteins and the formation of protein aggregates during dough mixing. These results indicate the importance of hydrophobic interactions in dough formation.

Chlamydia trachomatis Polymorphic Membrane Proteins (Pmps) Form Functional Homomeric and Heteromeric Oligomers

Chlamydiae are Gram-negative, obligate intracellular bacteria, which infect animals and humans. Adhesion to host cells, the first step in the infection process, is mediated by polymorphic membrane proteins (Pmps). Pmps constitute the largest chlamydial protein family, with 9 members (subdivided into six subtypes) in C. trachomatis and 21 in C. pneumoniae, and are characterized by the presence of multiple copies of GGA(I,L,V) and FxxN motifs. Motif-rich fragments of all nine C. trachomatis Pmps act as adhesins and are essential for infection. As autotransporters, most Pmp proteins are secreted through their β-barrel domain and localize on the surface of the chlamydial cell, where most of them are proteolytically processed. Classical autotransporters are monomeric proteins, which can function as toxins, proteases, lipases and monoadhesive adhesins. Here we show that selected recombinant C. trachomatis Pmp fragments form functional adhesion-competent multimers. They assemble into homomeric and heteromeric filaments, as revealed by non-denaturing gel electrophoresis, size-exclusion chromatography and electron microscopy. Heteromeric filaments reach 2 μm in length, significantly longer than homomeric structures. Filament formation was independent of the number of motifs present in the fragment(s) concerned and their relative affinity for host cells. Our functional studies demonstrated that only adhesion-competent oligomers were able to block a subsequent infection. Pre-loading of infectious chlamydial cells with adhesion-competent Pmp oligomers maintained the subsequent infection, while adhesion-incompetent structures reduced infectivity, presumably by blocking the function of endogenous Pmps. The very large number of possible heteromeric and homomeric Pmp complexes represents a novel mechanism to ensure stable adhesion and possibly host cell immune escape.

The multilayered organization of water-soluble proteins

The structural analysis of proteins has focused primarily on secondary structure, three-dimensional fold and active site while whole surface has been analyzed to a lesser extent and interior has not received much attention. Here we present an analysis of both the surfaces and the interiors of a set of water-soluble monomeric proteins in terms of solvent-excluded surface (SES) and atomic partial charge. The analysis shows that the surface of a soluble monomer has a net negative charge and is much smoother than the interior. Most interestingly with regard to both atomic partial charge and SES-defined geometric property there exists a multilayered organization from the exterior to the interior of a soluble monomer. The multilayered organization is closely related to protein-solvent interaction and should be a general feature of a water-soluble protein. Particularly the multilayered organization may set an upper limit for the size of a water-soluble monomer and plays an important role in the determination of its overall shape in solution.

Trypsin Induced Degradation of Amyloid Fibrils

Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.

Flagellin-based electrochemical sensing layer for arsenic detection in water

Regular monitoring of arsenic concentrations in water sources is essential due to the severe health effects. Our goal was to develop a rapidly responding, sensitive and stable sensing layer for the detection of arsenic. We have designed flagellin-based arsenic binding proteins capable of forming stable filament structures with high surface binding site densities. The D3 domain of Salmonella typhimurium flagellin was replaced with an arsenic-binding peptide motif of different bacterial ArsR transcriptional repressor factors. We have shown that the fusion proteins developed retain their polymerization ability and have thermal stability similar to that of wild-type filament. The strong arsenic binding capacity of the monomeric proteins was confirmed by isothermal titration calorimetry (ITC), and dissociation constants (Kd) of a few hundred nM were obtained for all three variants. As-binding fibers were immobilized on the surface of a gold electrode and used as a working electrode in cyclic voltammetry (CV) experiments to detect inorganic arsenic near the maximum allowable concentration (MAC) level. Based on these results, it can be concluded that the stable arsenic-binding flagellin variant can be used as a rapidly responding, sensitive, but simple sensing layer in a field device for the MAC-level detection of arsenic in natural waters.

Changes in Protein Non-Covalent Bonds and Aggregate Size during Dough Formation

This research investigated changes in the amounts and sizes of monomeric proteins and protein aggregates during dough mixing, with a focus on the contribution of non-covalent bonds in the aggregation of gluten proteins. High protein flour (HF) and low protein flour (LF) were used in this study. As dough mixing progressed from flour to overmixed dough, the total amount of protein aggregates increased while the amount of monomeric protein decreased. Omega-gliadin was the major monomeric protein that decreased in quantity. Interestingly, the amount of larger-sized protein aggregates decreased and that of smaller-sized protein aggregates increased. The amount of gluten protein macro-polymer aggregated through strong non-covalent bonds decreased whereas aggregates formed with weaker non-covalent bonds increased. LF dough behaved similar to HF dough. Large-sized gluten protein aggregates disaggregated due to the weakening of non-covalent bonds and became smaller. Omega-gliadin was incorporated into gluten protein aggregates during dough mixing.

The Human TSHβ Subunit Proteins and Their Binding Sites on the TSH Receptor Using Molecular Dynamics Simulation

To gain further insight into the binding of the normal and variant human TSHβ subunits (TSHβ and TSHβv), we modeled the 2 monomeric proteins and studied their interaction with the TSH receptor ectodomain (TSHR-ECD) using molecular dynamics simulation Furthermore, analyzed their bioactivity in vitro using recombinant proteins to confirm that such binding was physiologically relevant. Examining the interaction of TSHβ and TSHβv with the TSHR-ECD model using molecular dynamic simulation revealed strong binding of these proteins to the receptor ECD. The specificity of TSHβ and TSHβv binding to the TSHR-ECD was examined by analyzing the hydrogen-bonding residues of these subunits to the FSH receptor ECD, indicating the inability of these molecules to bind to the FSH receptors. Furthermore, the modelling suggests that TSHβ and TSHβv proteins clasped the concave surface of the leucine rich region of the TSHR ECD in a similar way to the native TSH using dynamic hydrogen bonding. These mutually exclusive stable interactions between the subunits and ECD residues included some high-affinity contact sites corresponding to binding models of native TSH. Furthermore, we cloned TSHβ and TSHβv proteins using the entire coding ORF and purified the flag-tagged proteins. The expressed TSHβ subunit proteins retained bioactivity both in a coculture system as well as with immune-purified proteins. In summary, we showed that such interactions can result in a functional outcome and may exert physiological or pathophysiological effects in immune cells.

The Botrytis cinerea Crh transglycosylae is a cytoplasmic effector triggering plant cell death and defense response

Crh proteins catalyze crosslinking of chitin and glucan polymers in the fugal cell wall. We revealed a novel and unexpected role of Botrytis cinerea BcCrh1 as a cytoplasmic effector and elicitor of plant defense. During saprophytic growth the BcCrh1 protein is localized in vacuoles and ER. Upon plant infection the protein accumulates to high levels in infection cushions, it is then secreted to the apoplast and translocated into plant cells, where it induces cell death and defense responses. Two regions of 53 and 35 amino acids were found sufficient for protein uptake and cell death induction, respectively. Dimerization of BcCrh proteins was necessary for the transglycosylation activity and proper fungal development, while the monomeric proteins was sufficient for induction of cell death. Arabidopsis lines expressing the bccrh1 gene had reduced sensitivity to B. cinerea, demonstrating the potential use of the protein in plant immunization against necrotrophic pathogens.

Prokaryotic Argonaute from Archaeoglobus fulgidus interacts with DNA as a homodimer

ABSTRACTBackgroundArgonaute (Ago) proteins are found in all three domains of life. The best characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are monomeric proteins, all composed of four major structural/functional domains (N, PAZ, MID and PIWI) and thereby closely resemble eAgos. It is believed that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown.ResultsIn this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal β-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including single-molecule FRET, SAXS and AFM, to test the possible dimerization of AfAgo. SAXS results confirm that WT AfAgo, but not the dimerization surface mutant AfAgoΔ, forms a homodimer both in the apo-form and when bound to a nucleic acid. Single molecule FRET and AFM studies demonstrate that the dimeric WT AfAgo binds two ends of a linear DNA fragment, forming a relatively stable DNA loop.ConclusionOur results show that contrary to other characterized Ago proteins, AfAgo is a stable homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.