Octameric Structure Research Articles

Open and closed structures of L-arginine oxidase by cryo-electron microscopy and X-ray crystallography.

L-arginine oxidase (AROD, EC 1.4.3.25) is an oxidoreductase that catalyzes the deamination of L-arginine, with flavin adenine dinucleotide (FAD) as a cofactor. Recently identified AROD from Pseudomonas sp. TPU 7192 (PT-AROD) demonstrates high selectivity for L-arginine. This enzyme is useful for accurate assays of L-arginine in biological samples. The structural characteristics of the FAD-dependent AROD, however, remain unknown. Here, we report the structure of PT-AROD at a resolution of 2.3 Å by cryo-electron microscopy. PT-AROD adopts an octameric structure with D4 symmetry, which is consistent with its molecular weight in solution, estimated by mass photometry. Comparative analysis of this structure with that determined using X-ray crystallography reveals open and closed forms of the lid-like loop at the entrance to the substrate pocket. Furthermore, mutation of Glu493, located at the substrate binding site, diminishes substrate selectivity, suggesting that this residue contributes significantly to the high selectivity of PT-AROD.

Guanine biomolecule derived nitrogen-doped carbon for efficient oxidative dehydrogenation performance

Biomolecule self-assembly shows great potential in the preparation of ordered functional materials. Here, we prepare a gauzy NC material via water guiding guanine biomolecule self-assemble, which exhibits the better ethylbenzene conversion (∼50%), styrene selectivity (∼91%) and long-term stability (>40 h) in ODH of ethylbenzene to styrene than that of guanine solid self-assembly produced NC (38% conversion vs. 88% selectivity) sample. The study shows that water not only facilitates guanine biomolecule self-assembly to form more stable octamer structure but restrains vertical growth of guanine, and resultant NC-1000-12 h holds high density active sites and outstanding oxidation resistance, thus enhancing NC catalytic performance. Overall, current work provides a new understanding on preparation of highly efficient NC materials through supramolecular self-assembly strategy.

Structural and biochemical insights into the mechanism of the Gabija bacterial immunity system

The Gabija system is a newly discovered bacterial immune system that consists of GajA and GajB. Here we report the cryo-EM structure of the Gabija complex from Bacillus cereus VD045 at 3.6 Å, which provides the direct evidence of interactions between GajA and GajB. The Gabija complex is an octameric ring structure with four GajA and four GajB. GajA is an OLD nucleases family protein, while GajB belongs to the SF1 helicases. The Gabija complex has sequence-specific DNA nuclease activity and prefers circular rather than linear DNA as substrate, its activity is more sensitive to concentrations change of nucleotides compared to GajA alone. Our data suggest a mechanism of Gabija immunity: the nuclease activity of Gabija complex is inhibited under physiological conditions, while it is activated by depletion of NTP and dNTP upon the replication and transcription of invading phages and cleave the circular DNA to prevent phage DNA replication.

Global Lrp regulator protein from Haloferax mediterranei: Transcriptional analysis and structural characterization

Haloferax mediterranei, an extreme halophilic archaeon thriving in hypersaline environments, has acquired significant attention in biotechnological and biochemical research due to its remarkable ability to flourish in extreme salinity conditions. Transcription factors, essential in regulating diverse cellular processes, have become focal points in understanding its adaptability. This study delves into the role of the Lrp transcription factor, exploring its modulation of glnA, nasABC, and lrp gene promoters in vivo through β-galactosidase assays. Remarkably, our findings propose Lrp as the pioneering transcriptional regulator of nitrogen metabolism identified in a haloarchaeon. This study suggests its potential role in activating or repressing assimilatory pathway enzymes (GlnA and NasA). The interaction between Lrp and these promoters is analyzed using Electrophoretic Mobility Shift Assay and Differential Scanning Fluorimetry, highlighting l-glutamine's indispensable role in stabilizing the Lrp-DNA complex. Our research uncovers that halophilic Lrp forms octameric structures in the presence of l-glutamine. The study reveals the three-dimensional structure of the Lrp as a homodimer using X-ray crystallography, confirming this state in solution by Small-Angle X-ray Scattering. These findings illuminate the complex molecular mechanisms driving Hfx. mediterranei's nitrogen metabolism, offering valuable insights about its gene expression regulation and enriching our comprehension of extremophile biology.

Csx28 is a membrane pore that enhances CRISPR-Cas13b-dependent antiphage defense.

Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. We show that Csx28, of type VI-B2 systems, is a transmembrane protein that assists to slow cellular metabolism upon viral infection, increasing antiviral defense. High-resolution cryo-electron microscopy reveals that Csx28 forms an octameric pore-like structure. These Csx28 pores localize to the inner membrane in vivo. Csx28's antiviral activity in vivo requires sequence-specific cleavage of viral messenger RNAs by Cas13b, which subsequently results in membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Our work suggests a mechanism by which Csx28 acts as a downstream, Cas13b-dependent effector protein that uses membrane perturbation as an antiviral defense strategy.

Atomic force microscopic analysis of the light-harvesting complex 2 from purple photosynthetic bacterium Thermochromatium tepidum.

Structural information on the circular arrangements of repeating pigment-polypeptide subunits in antenna proteins of purple photosynthetic bacteria is a clue to a better understanding of molecular mechanisms for the ring-structure formation and efficient light harvesting of such antennas. Here, we have analyzed the ring structure of light-harvesting complex 2 (LH2) from the thermophilic purple bacterium Thermochromatium tepidum (tepidum-LH2) by atomic force microscopy. The circular arrangement of the tepidum-LH2 subunits was successfully visualized in a lipid bilayer. The average top-to-top distance of the ring structure, which is correlated with the ring size, was 4.8 ± 0.3nm. This value was close to the top-to-top distance of the octameric LH2 from Phaeospirillum molischianum (molischianum-LH2) by the previous analysis. Gaussian distribution of the angles of the segments consisting of neighboring subunits in the ring structures of tepidum-LH2 yielded a median of 44°, which corresponds to the angle for the octameric circular arrangement (45°). These results indicate that tepidum-LH2 has a ring structure consisting of eight repeating subunits. The coincidence of an octameric ring structure of tepidum-LH2 with that of molischianum-LH2 is consistent with the homology of amino acid sequences of the polypeptides between tepidum-LH2 and molischianum-LH2.

High-Resolution Single-Particle Cryo-EM Hydrated Structure of Streptococcus pyogenes Enolase Offers Insights into Its Function as a Plasminogen Receptor.

Cellular plasminogen (Pg) receptors (PgRs) are utilized to recruit Pg; stimulate its activation to the serine protease, plasmin (Pm); and sterically protect the surface Pm from inactivation by host inhibitors. One such PgR is the moonlighting enzyme, enolase, some of which leaves the cytoplasm and resides at the cell surface to potentially function as a PgR. Since microbes employ conscription of host Pg by PgRs as one virulence mechanism, we explored the structural basis of the ability of Streptococcus pyogenes enolase (Sen) to function in this manner. Employing single-particle cryo-electron microscopy (cryo-EM), recombinant Sen from S. pyogenes was modeled at 2.6 Å as a stable symmetrical doughnut-shaped homooctamer with point group 422 (D4) symmetry, with a monomeric subunit molecular weight of ∼49 kDa. Binding sites for hPg were reported in other studies to include an internal K252,255 and the COOH-terminal K434,435 residues of Sen. However, in native Sen, the latter are buried within the minor interfaces of the octamer and do not function as a Pg-binding epitope. Whereas Sen and hPg do not interact in solution, when Sen is bound to a surface, hPg interacts with Sen independently of K252,255,434,435. PgRs devoid of COOH-terminal lysine utilize lysine isosteres comprising a basic residue, "i", and an anionic residue at "i + 3" around one turn of an α-helix. We highlight a number of surface-exposed potential hPg-binding lysine isosteres and further conclude that while the octameric structure of Sen is critical for hPg binding, disruption of this octamer without dissociation exposes hPg-binding epitopes.

A conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1

Sterile alpha (SAM) and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1) is an autoinhibitory NAD-consuming enzyme that is activated by the accumulation of nicotinamide mononucleotide (NMN) during axonal injury. Its activation mechanism is not fully understood. Here, we generate a nanobody, Nb-C6, that specifically recognizes NMN-activated SARM1. Nb-C6 stains only the activated SARM1 in cells stimulated with CZ-48, a permeant mimetic of NMN, and partially activates SARM1 in vitro and in cells. Cryo-EM of NMN/SARM1/Nb-C6 complex shows an octameric structure with ARM domains bending significantly inward and swinging out together with TIR domains. Nb-C6 binds to SAM domain of the activated SARM1 and stabilized its ARM domain. Mass spectrometry analyses indicate that the activated SARM1 in solution is highly dynamic and that the neighboring TIRs form transient dimers via the surface close to one BB loop. We show that Nb-C6 is a valuable tool for studies of SARM1 activation.

Pore-forming moss protein bryoporin is structurally and mechanistically related to actinoporins from evolutionarily distant cnidarians

Pore-forming proteins perforate lipid membranes and consequently affect their integrity and cell fitness. Therefore, it is not surprising that many of these proteins from bacteria, fungi, or certain animals act as toxins. While pore-forming proteins have also been found in plants, there is little information about their molecular structure and mode of action. Bryoporin is a protein from the moss Physcomitrium patens, and its corresponding gene was found to be upregulated by various abiotic stresses, especially dehydration, as well as upon fungal infection. Based on the amino acid sequence, it was suggested that bryoporin was related to the actinoporin family of pore-forming proteins, originally discovered in sea anemones. Here, we provide the first detailed structural and functional analysis of this plant cytolysin. The crystal structure of monomeric bryoporin is highly similar to those of actinoporins. Our cryo-EM analysis of its pores showed an actinoporin-like octameric structure, thereby revealing a close kinship of proteins from evolutionarily distant organisms. This was further confirmed by our observation of bryoporin’s preferential binding to and formation of pores in membranes containing animal sphingolipids, such as sphingomyelin and ceramide phosphoethanolamine; however, its binding affinity was weaker than that of actinoporin equinatoxin II. We determined bryoporin did not bind to major sphingolipids found in fungi or plants, and its membrane-binding and pore-forming activity was enhanced by various sterols. Our results suggest that bryoporin could represent a part of the moss defense arsenal, acting as a pore-forming toxin against membranes of potential animal pathogens, parasites, or predators.

The Crystal Structure of L-Leucine Dehydrogenase from Pseudomonas aeruginosa.

Leucine dehydrogenase (LDH, EC 1.4.1.9) catalyzes the reversible deamination of branched-chain L-amino acids to their corresponding keto acids using NAD+ as a cofactor. LDH generally adopts an octameric structure with D4 symmetry, generating a molecular mass of approximately 400 kDa. Here, the crystal structure of the LDH from Pseudomonas aeruginosa (Pa-LDH) was determined at 2.5 Å resolution. Interestingly, the crystal structure shows that the enzyme exists as a dimer with C2 symmetry in a crystal lattice. The dimeric structure was also observed in solution using multiangle light scattering coupled with size-exclusion chromatography. The enzyme assay revealed that the specific activity was maximal at 60°C and pH 8.5. The kinetic parameters for three different amino acid and the cofactor (NAD+) were determined. The crystal structure represents that the subunit has more compact structure than homologs’ structure. In addition, the crystal structure along with sequence alignments indicates a set of non-conserved arginine residues which are important in stability. Subsequent mutation analysis for those residues revealed that the enzyme activity reduced to one third of the wild type. These results provide structural and biochemical insights for its future studies on its application for industrial purposes.

Distinct structural groups of histone H3 and H4 residues have divergent effects on chronological lifespan in Saccharomyces cerevisiae.

We have performed a comprehensive analysis of the involvement of histone H3 and H4 residues in the regulation of chronological lifespan in yeast and identify four structural groups in the nucleosome that influence lifespan. We also identify residues where substitution with an epigenetic mimic extends lifespan, providing evidence that a simple epigenetic switch, without possible additional background modifications, causes longevity. Residues where substitution result in the most pronounced lifespan extension are all on the exposed face of the nucleosome, with the exception of H3E50, which is present on the lateral surface, between two DNA gyres. Other residues that have a more modest effect on lifespan extension are concentrated at the extremities of the H3-H4 dimer, suggesting a role in stabilizing the dimer in its nucleosome frame. Residues that reduce lifespan are buried in the histone handshake motif, suggesting that these mutations destabilize the octamer structure. All residues exposed on the nucleosome disk face and that cause lifespan extension are known to interact with Sir3. We find that substitution of H4K16 and H4H18 cause Sir3 to redistribute from telomeres and silent mating loci to secondary positions, often enriched for Rap1, Abf1 or Reb1 binding sites, whereas H3E50 does not. The redistribution of Sir3 in the genome can be reproduced by an equilibrium model based on primary and secondary binding sites with different affinities for Sir3. The redistributed Sir3 cause transcriptional repression at most of the new loci, including of genes where null mutants were previously shown to extend chronological lifespan. The transcriptomic profiles of H4K16 and H4H18 mutant strains are very similar, and compatible with a DNA replication stress response. This is distinct from the transcriptomic profile of H3E50, which matches strong induction of oxidative phosphorylation. We propose that the different groups of residues are involved in binding to heterochromatin proteins, in destabilizing the association of the nucleosome DNA, disrupting binding of the H3-H4 dimer in the nucleosome, or disrupting the structural stability of the octamer, each category impacting on chronological lifespan by a different mechanism.

Bi‐directional pH dependency of CALHM1 ion channel

Calcium homeostasis modulator (CALHM) membrane proteins are nonselective ion channels drawing attention regarding their roles in modulating neuronal activity and taste sensation. CALHM1‐expressing cells show voltage‐gated slowly activating ionic currents. The voltage‐dependence of CALHM1 is negatively affected by extracellular Ca2+ ([Ca2+]e), while facilitated by temperature. Here we investigated the effect of extracellular and intracellular pH (pHe and pHi) on the electrophysiological properties of CALHM1 overexpressed in CHO cells. When normalized to the control at pHe 7.4, the amplitudes of CALHM1 current (ICALHM1) were suppressed to 20 % at pHe 6.2, while increased to 500 % at pHe 8.6. In the whole‐cell configuration of patch clamp, changing pHi from 7.4 to 6.2 or 8.6 showed the acid‐induced inhibition and alkali‐induced activation of ICALHM1. The voltage‐dependence of CALHM1 was shifted to the negative membrane potential at the alkaline pHe and pHi, while positive membrane potential at the acidic pHe and pHi. The pHi effect was also confirmed in the inside‐out configuration of patch clamp. The open probability (Po) of multiple CALHM1 channel decreased at pH 6.2, while increased at pH 8.6 compared with pH 7.4. To get a clue to identify the pH sensing amino acid residues, we utilized homology model performed with the SWISS‐MODEL workspace, based on the cryo‐EM structure of human CALHM2. Then we conducted site‐directed mutagenesis of the water‐accessible charged amino acids of CALHM1. Among 23 candidates, mutations at E17, K229, E233, D257 and E259 in the intracellular space reduced the alkali‐induced facilitation of ICALHM1, while not altered the acid‐induced inhibition. According to the molecular structure model of killifish CALHM1 with octameric structure, the five residues located at N or C termini gathered closely in the intracellular space. Considering the large pore diameter of CALHM1 (~14 Å), we cautiously propose that the sensitivity to alkaline pHe could be because the protons transfer between hydronium ions in the pore. The study firstly demonstrated the remarkable pH‐sensitivity of CALHM1 and its pH‐sensing residues, which might be responsible for the modulation of neuronal excitability during dynamic changes of the regional pH in brain or sensory organs.

Nanobody-aided crystallization of the transcription regulator PaaR2 from Escherichia coli O157:H7.

paaR2-paaA2-parE2 is a three-component toxin-antitoxin module found in prophage CP-993P of Escherichia coli O157:H7. Transcription regulation of this module occurs via the 123-amino-acid regulator PaaR2, which forms a large oligomeric structure. Despite appearing to be well folded, PaaR2 withstands crystallization, as does its N-terminal DNA-binding domain. Native mass spectrometry was used to screen for nanobodies that form a unique complex and stabilize the octameric structure of PaaR2. One such nanobody, Nb33, allowed crystallization of the protein. The resulting crystals belong to space group F432, with unit-cell parameter a = 317 Å, diffract to 4.0 Å resolution and are likely to contain four PaaR2 monomers and four nanobody monomers in the asymmetric unit. Crystals of two truncates containing the N-terminal helix-turn-helix domain also interact with Nb33, and the corresponding co-crystals diffracted to 1.6 and 1.75 Å resolution.

One-Pot Synthesis of a Cyclic Pyrene Octamer from Two Bifunctionalized Pyrene Monomers

AbstractA 2,2′-tert-butyl-5,9-6′,8′-cyclooctameric pyrenylene ([8]CP) was synthesized by a one-pot Suzuki–Miyaura cross-coupling reaction from two kinds of bifunctionalized monomers, as a rare example of a cyclic octamer. The octameric molecular structure of [8]CP was revealed by single-crystal X-ray diffraction analysis.

A\u03b2(1-42) tetramer and octamer structures reveal edge conductivity pores as a mechanism for membrane damage

Formation of amyloid-beta (Aβ) oligomer pores in the membrane of neurons has been proposed to explain neurotoxicity in Alzheimerʼs disease (AD). Here, we present the three-dimensional structure of an Aβ oligomer formed in a membrane mimicking environment, namely an Aβ(1-42) tetramer, which comprises a six stranded β-sheet core. The two faces of the β-sheet core are hydrophobic and surrounded by the membrane-mimicking environment while the edges are hydrophilic and solvent-exposed. By increasing the concentration of Aβ(1-42) in the sample, Aβ(1-42) octamers are also formed, made by two Aβ(1-42) tetramers facing each other forming a β-sandwich structure. Notably, Aβ(1-42) tetramers and octamers inserted into lipid bilayers as well-defined pores. To establish oligomer structure-membrane activity relationships, molecular dynamics simulations were carried out. These studies revealed a mechanism of membrane disruption in which water permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core β-sheets edges of the oligomers.

Structures and Properties of Higher-Degree Aggregates of Methylammonium Iodide toward Halide Perovskite Solar Cells

Organic–inorganic halide perovskite solar cells have been in the limelight in recent years due to their outstanding power conversion efficiency (PCE) and facile synthesis. Despite of the ever increasing PCE that astonishes the solar cell community, many fundamental structural and theoretical aspects remain elusive, which inhibit our understanding on this fascinating material. Methylammonium iodide (MAI) is one important precursor to fabricate the halide perovskite materials based on the prototypical CH3NH3PbI3. Some MAI might remain in the CH3NH3PbI3 layer, forming a MAI-rich layer that is considered strongly affecting the perovskite solar cell performance. Therefore it is helpful to take one step back to re-evaluate the structures of MAI that is overlooked in many perovskite solar cell studies. In this manuscript, we identify the alternative structures of MAI aggregates via the ab-initio calculations, including the monomeric, dimeric, trimeric, tetrameric, pentameric, hexameric, heptameric, octameric and decameric structures of MAI. These aggregate structures are proposed to act as a simplified, yet efficient model to represent the MAI-rich region in perovskite solar cells that are usually simulated using the computationally expensive slab model. We find that the aggregates of MAI are universally stabilized by the hydrogen bonds. We further evaluate these MAI aggregate structures via studying the interactions between the MAI aggregates and an experimentally-proven additive iodopentafluorobenzene (IPFB) as an example, and show that the iodine atoms in the MAI aggregates acting as potential charge traps could be passivated by the additive molecule. The present study provides an efficient modeling platform for the MAI-rich halide perovskite materials without high computational cost.

Histone Octamer Structure Is Altered Early in ISW2 ATP-Dependent Nucleosome Remodeling

Nucleosomes are the fundamental building blocks ofchromatin that regulate DNA access and are composed of histone octamers. ATP-dependent chromatin remodelers like ISW2 regulate chromatin access by translationally moving nucleosomes to different DNA regions. We find that histone octamers are more pliable than previously assumed and distorted by ISW2 early in remodeling before DNA enters nucleosomes and the ATPase motor moves processively on nucleosomal DNA. Uncoupling the ATPase activity of ISW2 from nucleosome movement with deletion of the SANT domain from the C terminus of the Isw2 catalytic subunit traps remodeling intermediates in which the histone octamer structure is changed. We find restricting histone movement by chemical crosslinking also traps remodeling intermediates resembling those seen early in ISW2 remodeling with loss of the SANT domain. Other evidence shows histone octamers are intrinsically prone to changing their conformation and can be distorted merely by H3-H4 tetramer disulfide crosslinking.

Human keratin 1/10-1B tetramer structures reveal a knob-pocket mechanism in intermediate filament assembly.

To characterize keratin intermediate filament assembly mechanisms at atomic resolution, we determined the crystal structure of wild-type human keratin-1/keratin-10 helix 1B heterotetramer at 3.0Å resolution. It revealed biochemical determinants for the A11 mode of axial alignment in keratin filaments. Four regions on a hydrophobic face of the K1/K10-1B heterodimer dictated tetramer assembly: the N-terminal hydrophobic pocket (defined by L227K1, Y230K1, F231K1, and F234K1), the K10 hydrophobic stripe, K1 interaction residues, and the C-terminal anchoring knob (formed by F314K1 and L318K1). Mutation of both knob residues to alanine disrupted keratin 1B tetramer and full-length filament assembly. Individual knob residue mutant F314AK1, but not L318AK1, abolished 1B tetramer formation. The K1-1B knob/pocket mechanism is conserved across keratins and many non-keratin intermediate filaments. To demonstrate how pathogenic mutations cause skin disease by altering filament assembly, we additionally determined the 2.39Å structure of K1/10-1B containing a S233LK1 mutation linked to epidermolytic palmoplantar keratoderma. Light scattering and circular dichroism measurements demonstrated enhanced aggregation of K1S233L/K10-1B in solution without affecting secondary structure. The K1S233L/K10-1B octamer structure revealed S233LK1 causes aberrant hydrophobic interactions between 1B tetramers.

The Oligomerization Landscape of Histones

In eukaryotes, DNA is packaged within nucleosomes. The DNA of each nucleosome is typically centered around an octameric histone protein core: one central tetramer plus two separate dimers. Studying the assembly mechanisms of histones is essential for understanding the dynamics of entire nucleosomes and higher-order DNA packaging. Here, we investigate canonical histone assembly and that of the centromere-specific histone variant, centromere protein A (CENP-A), using molecular dynamics simulations. We quantitatively characterize their thermodynamical and dynamical features, showing that two H3/H4 dimers form a structurally floppy, weakly bound complex, the latter exhibiting large instability around the central interface manifested via a swiveling motion of two halves. This finding is consistent with the recently observed DNA handedness flipping of the tetrasome. In contrast, the variant CENP-A encodes distinctive stability to its tetramer with a rigid but twisted interface compared to the crystal structure, implying diverse structural possibilities of the histone variant. Interestingly, the observed tetramer dynamics alter significantly and appear to reach a new balance when H2A/H2B dimers are present. Furthermore, we found that the preferred structure for the (CENP-A/H4)2 tetramer is incongruent with the octameric structure, explaining many of the unusual dynamical behaviors of the CENP-A nucleosome. In all, these data reveal key mechanistic insights and structural details for the assembly of canonical and variant histone tetramers and octamers, providing theoretical quantifications and physical interpretations for longstanding and recent experimental observations. Based on these findings, we propose different chaperone-assisted binding and nucleosome assembly mechanisms for the canonical and CENP-A histone oligomers.

The influence of truncating the carboxy-terminal amino acid residues of streptococcal enolase on its ability to interact with canine plasminogen.

The native octameric structure of streptococcal enolase from Streptococcus pyogenes increasingly dissociates as amino acid residues are removed one by one from the carboxy-terminus. These truncations gradually convert native octameric enolase into monomers and oligomers. In this work, we investigated how these truncations influence the interaction between Streptococcal enolase and canine plasminogen. We used dual polarization interferometry (DPI), localized surface plasmon resonance (LSPR), and sedimentation velocity analytical ultracentrifugation (AUC) to study the interaction. The DPI was our first technique, was performed on all the truncations and used one exclusive kind of chip. The LSRP was used to show that the DPI results were not dependent on the type of chip used. The AUC was required to show that our surface results were not the result of selecting a minority population in any given sample; the majority of the protein was responsible for the binding phenomenon we observed. By comparing results from these techniques we identified one detail that is essential for streptococcal enolase to bind plasminogen: In our hands the individual monomers bind plasminogen; dimers, trimers, tetramers may or may not bind, the fully intact, native, octamer does not bind plasminogen. We also evaluated the contribution to the equilibrium constant made by surface binding as well as in solution. On a surface, the association coefficient is about twice that in solution. The difference is probably not significant. Finally, the fully octameric form of the protein that does not contain a hexa-his N-terminal peptide does not bind to a silicon oxynitride surface, does not bind to an Au-nanoparticle surface, does not bind to a surface coated with Ni-NTA nor does it bind to a surface coated with DPgn. The likelihood is great that the enolase species on the surface of Streptococcus pyogenes is an x-mer of the native octamer.