A multifunctional chemical cue drives opposing demographic processes and structures ecological communities.

Foundation species provide critical resources to ecological community members and are key determinants of biodiversity. The barnacle Balanus glandula is one such species and dominates space among the higher reaches of wave-swept shores (Northeastern Pacific Ocean). This animal produces a cuticular glycoprotein (named "MULTIFUNCin") of 199.6 kDa, and following secretion, a 390 kDa homodimer in native form. From field and lab experiments, we found that MULTIFUNCin significantly induces habitat selection by conspecific larvae, while simultaneously acting as a potent feeding stimulant to a major barnacle predator (whelk, Acanthinucella spirata). Promoting immigration via settlement on the one hand, and death via predation on the other, MULTIFUNCin drives opposing demographic processes toward structuring predator and prey populations. As shown here, a single compound is not restricted to a lone species interaction or sole ecological function. Complex biotic interactions therefore can be shaped by simple chemosensory systems and depend on the multifunctional properties of select bioactive proteins.

MULTIFUNCin: A Multifunctional Protein Cue Induces Habitat Selection by, and Predation on, Barnacles.

Foundation species provide critical resources to ecological community members and are major determinants of biodiversity. The barnacle Balanus glandula is one such species and dominates space among the higher reaches on wave-swept shores. Here, we show that B. glandula produces a 199.6-kDa glycoprotein (named "MULTIFUNCin"), and following secretion, a 390-kDa homodimer in its native state. MULTIFUNCin expression is localized in the epidermis, cuticle, and new shell material. Consequently, this molecule can specify upon contact the immediate presence of a live barnacle. Shared, conserved domains place MULTIFUNCin in the α2-macroglobulin (A2M) subgroup of the thioester-containing protein family. Although previously undescribed, MULTIFUNCin shares 78% nucleotide sequence homology with a settlement-inducing pheromone (SIP) of the barnacle, Amphibalanus amphitrite Based on this and further evidence, we propose that the two proteins are orthologues and evolved ancestrally in structural and immunological roles. More recently, they became exploited as chemical cues for con- and heterospecific organisms, alike. MULTIFUNCin and SIP both induce habitat selection (settlement) by conspecific barnacle larvae. In addition, MULTIFUNCin acts as a potent feeding stimulant to major barnacle predators (sea stars and several whelk species). Promoting immigration via settlement on the one hand, and death via predation on the other, MULTIFUNCin simultaneously mediates opposing demographic processes toward structuring both predator and prey populations. As a multifunctional protein cue, MULTIFUNCin provides valuable sensory information, conveys different messages to different species, and drives complex biotic interactions.

Synthetic approach to homodimeric protein-polymer conjugates.

Synthesis of well-defined homodimeric protein-polymer conjugates using RAFT polymerization is described.

Synthesis of Maleimide-End Functionalized Star Polymers and Multimeric Protein-Polymer Conjugates.

Protein-polymer conjugates exhibit superior properties to unmodified proteins, generating a high demand for these materials in the fields of medicine, biotechnology, and nanotechnology. Multimeric conjugates are predicted to surpass the activity of monomeric conjugates. Herein, we report a straightforward method to synthesize multimeric polymer-conjugates. Four armed poly(N-isopropylacrylamide) (pNIPAAm) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization in the presence of a tetra-functionalized trithiocarbonate chain transfer agent (CTA). The polymer molecular weight, architecture and polydispersity index (PDI) were verified by gel permeation chromatography (GPC), dynamic light scattering gel permeation chromatography (DLS-GPC), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This approach afforded well-defined polymers (PDI's < 1.06) and the ability to target various molecular weights. Maleimide functional groups were introduced at the chain ends by heating the polymers in the presence of a furan-protected azo-initiator. This allowed for site-specific conjugation of V131C T4 lysozyme to the polymers to generate multimeric protein-polymer conjugates. MALDI-TOF mass spectrometry, electrospray ionization gas-phase electrophoretic-mobility macromolecule analysis (ESI-GEMMA), gel electrophoresis, and liquid chromatography tandem mass spectrometry (LC-MS/MS) of the trypsin digests demonstrated that multimeric protein-polymer conjugates had formed. This simple strategy provides ready access to star protein-polymer conjugates for application in the fields of drug discovery, drug delivery, and nanotechnology.

Similar enzymes, different structures: phthalate dioxygenase is an alpha3alpha3 stacked hexamer, not an alpha3beta3 trimer like "normal" Rieske oxygenases.

Phthalate dioxygenase (PDO) is a member of a class of bacterial oxygenases that contain both Rieske [2Fe-2S] and Fe(II) mononuclear centers. Recent crystal structures of several Rieske dioxygenases showed that they exist as alpha(3)beta(3) multimers with subunits arranged head-to-tail in alpha and beta stacked planar rings. The structure of PDO, which consists of only alpha-subunits, remains to be solved. Although similar to other Rieske dioxygenases in many aspects, PDO was shown to differ in the mechanism of catalysis. Gel filtration and analytical centrifugation experiments, supplemented with mass spectrometric analysis (both ESI-MS and ESI-GEMMA), in this work showed a hexameric arrangement of subunits in the PDO multimer. Our proposed model for the subunit arrangement in PDO postulates two alpha(3) planar rings one on top the other, similar to the alpha(3)beta(3) arrangement in other Rieske dioxygenases. Unlike other Rieske dioxygenases, this arrangement brings two Rieske and two mononuclear centers, all on separate subunits, into proximity, allowing their cooperation for catalysis. Potential reasons necessitating this unusual structural arrangement are discussed.

Sizing large proteins and protein complexes by electrospray ionization mass spectrometry and ion mobility.

Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method of gas-phase electrophoretic mobility molecular analysis (GEMMA), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions, which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared with other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and X-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm(3). Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water.

The vault exterior shell is a dynamic structure that allows incorporation of vault-associated proteins into its interior.

Vaults are 13 million Da ribonucleoprotein particles with a highly conserved structure. Expression and assembly by multimerization of an estimated 96 copies of a single protein, termed the major vault protein (MVP), is sufficient to form the minimal structure and entire exterior shell of the barrel-shaped vault particle. Multiple copies of two additional proteins, VPARP and TEP1, and a small untranslated vault RNA are also associated with vaults. We used the Sf9 insect cell expression system to form MVP-only recombinant vaults and performed a series of protein-mixing experiments to test whether this particle shell is able to exclude exogenous proteins from interacting with the vault interior. Surprisingly, we found that VPARP and TEP1 are able to incorporate into vaults even after the formation of the MVP vault particle shell is complete. Electrospray molecular mobility analysis and spectroscopic studies of vault-interacting proteins were used to confirm this result. Our results demonstrate that the protein shell of the recombinant vault particle is a dynamic structure and suggest a possible mechanism for in vivo assembly of vault-interacting proteins into preformed vaults. Finally, this study suggests that the vault interior may functionally interact with the cellular milieu.

Electrospray ionization mass spectrometry and ion mobility analysis of the 20S proteasome complex.

Mass spectrometry and gas phase ion mobility [gas phase electrophoretic macromolecule analyzer (GEMMA)] with electrospray ionization were used to characterize the structure of the noncovalent 28-subunit 20S proteasome from Methanosarcina thermophila and rabbit. ESI-MS measurements with a quadrupole time-of-flight analyzer of the 192 kDa alpha7-ring and the intact 690 kDa alpha7beta7beta7alpha7 are consistent with their expected stoichiometries. Collisionally activated dissociation of the 20S gas phase complex yields loss of individual alpha-subunits only, and it is generally consistent with the known alpha7beta7beta7alpha7 architecture. The analysis of the binding of a reversible inhibitor to the 20S proteasome shows the expected stoichiometry of one inhibitor for each beta-subunit. Ion mobility measurements of the alpha7-ring and the alpha7beta7beta7alpha7 complex yield electrophoretic diameters of 10.9 and 15.1 nm, respectively; these dimensions are similar to those measured by crystallographic methods. Sequestration of multiple apo-myoglobin substrates by a lactacystin-inhibited 20S proteasome is demonstrated by GEMMA experiments. This study suggests that many elements of the gas phase structure of large protein complexes are preserved upon desolvation, and that methods such as mass spectrometry and ion mobility analysis can reveal structural details of the solution protein complex.

Oligomers of ERBB3 have two distinct interfaces that differ in their sensitivity to disruption by heregulin.

ErbB receptors associate in a ligand-dependent or -independent manner, and overexpression of epidermal growth factor receptor (ErbB1) or ErbB2 results in ligand-independent activation. Ligand-independent activation is poorly understood, and dimerization alone is not sufficient for activation. ErbB receptors also form higher order oligomers, but the mechanism of oligomer formation and their contribution to signaling are not known. The kinase-deficient ErbB3 as well as its extracellular domains are particularly prone to ligand-independent oligomerization, and oligomers are destabilized by binding of the ligand heregulin. In contrast, ligand binding facilitates heterodimerization with ErbB2 and is expected to stabilize an extended conformation of the ErbB3 extracellular domain (ECD) in which the dimerization interface is exposed. In the absence of ligand, ErbB3 can adopt a closed conformation that is held together by an intramolecular tether. We used a constitutively extended form of the ErbB3-ECD to analyze the conformation of the ECD in oligomers and the mechanism of oligomer disruption by heregulin. The extended conformation of the ECD forms oligomers more readily, suggesting the crystallographically defined dimer interface is one of the interfaces involved in oligomerization. Heregulin destabilizes oligomeric complexes but not dimers, which are neither stabilized nor disrupted by ligand binding, indicating a distinct second interface in oligomers of ErbB3. Cross-linking and activation studies on membrane-embedded ErbB3/ErbB2 chimeras confirm this dual effect of heregulin. Most of the ErbB3-ECD on the cell surface is apparently kept in an open conformation through oligomerization, and the resulting oligomers adopt a conformation representing a state of reduced activity.