Structural dynamics of the heme pocket and intersubunit coupling in the dimeric hemoglobin from Scapharca inaequivalvis.

Cooperativity is essential for the proper functioning of numerous proteins by allosteric interactions. Hemoglobin from Scapharca inaequivalvis (HbI) is a homodimeric protein that can serve as a minimal unit for studying cooperativity. We investigated the structural changes in HbI after carbon monoxide dissociation using time-resolved resonance Raman spectroscopy and observed structural rearrangements in the Fe-proximal histidine bond, the position of the heme in the pocket, and the hydrogen bonds between heme and interfacial water upon ligand dissociation. Some of the spectral changes were different from those observed for human adult hemoglobin due to differences in subunit assembly and quaternary changes. The structural rearrangements were similar for the singly and doubly dissociated species but occurred at different rates. The rates of the observed rearrangements indicated that they occurred synchronously with subunit rotation and are influenced by intersubunit coupling, which underlies the positive cooperativity of HbI.

Change in vibrational entropy with change in protein volume estimated with mode Grüneisen parameters.

For a small adjustment in average volume, due to a change in state of a protein or other macromolecule at constant temperature, the change in vibrational entropy is related to the mode Grüneisen parameters, which relate shifts in frequency to a small volume change. We report here values of mode Grüneisen parameters computed for two hydrated proteins, cytochrome c and myoglobin, which exhibit trends with mode frequency resembling those of glassy systems. We use the mode Grüneisen parameters to relate volumetric thermal expansion to previously computed values of the isothermal compressibility for several proteins. We also estimate changes in vibrational entropy resulting from the change in volume upon ligand bonding of myoglobin and the homodimeric hemoglobin from Scapharca inaequivalvis (HbI). We compare estimates of the change in entropy upon ligation obtained in terms of mode Grüneisen parameters with the results of normal mode analysis for myoglobin and earlier molecular dynamics simulations of HbI. The results illustrate how small changes in average volume can yield changes in entropy that contribute to ligand binding and allostery.

Locating and Navigating Energy Transport Networks in Proteins.

We review computational methods to locate energy transport networks in proteins that are based on the calculation of local energy diffusion in nanoscale systems. As an illustrative example, we discuss energy transport networks computed for the homodimeric hemoglobin from Scapharca inaequivalvis, where channels for facile energy transport, which include the cluster of water molecules at the interface of the globules, have been found to lie along pathways that experiments reveal are important in allosteric processes. We also review recent work on master equation simulations to model energy transport dynamics, including efforts to relate rate constants in the master equation to protein structural dynamics. Results for apomyoglobin involving relations between fluctuations in the length of hydrogen bonds and the energy flux between them are presented.

Water-mediated biomolecular dynamics and allostery.

Dynamic coupling with water contributes to regulating the functional dynamics of a biomolecule. We discuss protein-water dynamics, with emphasis on water that is partially confined, and the role of protein-confined water dynamics in allosteric regulation. These properties are illustrated with two systems, a homodimeric hemoglobin from Scapharca inaequivalvis (HbI) and an A2A adenosine receptor (A2AAR). For HbI, water-protein interactions, long known to contribute to the thermodynamics of cooperativity, are seen to influence the dynamics of the protein not only around the protein-water interface but also into the core of each globule, where dynamic and entropic changes upon ligand binding are coupled to protein-water contact dynamics. Similarly, hydration waters trapped deep inside the core region of A2AAR enable the formation of an allosteric network made of water-mediated inter-residue contacts. Extending from the ligand binding pocket to the G-protein binding site, this allosteric network plays key roles in regulating the activity of the receptor.

Energy Transport across Interfaces in Biomolecular Systems.

Energy transport during chemical reactions or following photoexcitation in systems of biological molecules is mediated by numerous interfaces that separate chemical groups and molecules. Describing and predicting energy transport has been complicated by the inhomogeneous environment through which it occurs, and general rules are still lacking. We discuss recent work on identification of networks for vibrational energy transport in biomolecules and their environment, with focus on the nature of energy transfer across interfaces. Energy transport is influenced both by structure of the biomolecular system as well as by equilibrium fluctuations of nonbonded contacts between chemical groups, biomolecules, and water along the network. We also discuss recent theoretical and computational work on the related topic of thermal transport through molecular interfaces, with focus on systems important in biology as well as relevant experimental studies.

Water-Mediated Energy Dynamics in a Homodimeric Hemoglobin.

We examine energy dynamics in the unliganded and liganded states of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI), which exhibits cooperativity mediated by the cluster of water molecules at the interface upon ligand binding and dissociation. We construct and analyze a dynamic network in which nodes representing the residues, hemes, and water cluster are connected by edges that represent energy transport times, as well as a nonbonded network (NBN) indicating regions that respond rapidly to local strain within the protein via nonbonded interactions. One of the two largest NBNs includes the Lys30-Asp89 salt bridge critical for stabilizing the dimer. The other includes the hemes and surrounding residues, as well as, in the unliganded state, the cluster of water molecules between the globules. Energy transport in the protein appears to be controlled by the Lys30-Asp89 salt bridge critical for stabilizing the dimer, as well as the interface water cluster in the unliganded state. Possible connections between energy transport dynamics in response to local strain identified here and allosteric transitions in HbI are discussed.

Linking Intertidal and Subtidal Food Webs: Consumer-Mediated Transport of Intertidal Benthic Microalgal Carbon.

We examined stable carbon and nitrogen isotope ratios for a large variety of consumers in intertidal and subtidal habitats, and their potential primary food sources [i.e., microphytobenthos (MPB), phytoplankton, and Phragmites australis] in a coastal bay system, Yeoja Bay of Korea, to test the hypothesis that the transfer of intertidal MPB-derived organic carbon to the subtidal food web can be mediated by motile consumers. Compared to a narrow δ13C range (-18 to -16‰) of offshore consumers, a broad δ13C range (-18 to -12‰) of both intertidal and subtidal consumers indicated that 13C-enriched sources of organic matter are an important trophic source to coastal consumers. In the intertidal areas, δ13C of most consumers overlapped with or was 13C-enriched relative to MPB. Despite the scarcity of MPB in the subtidal, highly motile consumers in subtidal habitat had nearly identical δ13C range with many intertidal foragers (including crustaceans and fish), overlapping with the range of MPB. In contrast, δ13C values of many sedentary benthic invertebrates in the subtidal areas were similar to those of offshore consumers and more 13C-depleted than motile foragers, indicating high dependence on phytoplankton-derived carbon. The isotopic mixing model calculation confirms that the majority of motile consumers and also some of subtidal sedentary ones depend on intertidal MPB for more than a half of their tissue carbon. Finally, although further quantitative estimates are needed, these results suggest that direct foraging by motile consumers on intertidal areas, and thereby biological transport of MPB-derived organic carbon to the subtidal areas, may provide important trophic connection between intertidal production and the nearshore shallow subtidal food webs.

Insight into the allosteric mechanism of Scapharca dimeric hemoglobin.

Allosteric regulation is an essential function of many proteins that control a variety of different processes such as catalysis, signal transduction, and gene regulation. Structural rearrangements have historically been considered the main means of communication between different parts of a protein. Recent studies have highlighted the importance, however, of changes in protein flexibility as an effective way to mediate allosteric communication across a protein. Scapharca dimeric hemoglobin (HbI) is the simplest possible allosteric system, with cooperative ligand binding between two identical subunits. Thermodynamic equilibrium studies of the binding of oxygen to HbI have shown that cooperativity is an entropically driven effect. The change in entropy of the system observed upon ligand binding may arise from changes in the protein, the ligand, or the water of the system. The goal of this study is to determine the contribution of the change in entropy of the protein backbone to HbI cooperative binding. Molecular dynamics simulations and nuclear magnetic resonance relaxation techniques have revealed that the fast internal motions of HbI contribute to the cooperative binding to carbon monoxide in two ways: (1) by contributing favorably to the free energy of the system and (2) by participating in the cooperative mechanism at the HbI subunit interface. The internal dynamics of the weakly cooperative HbI mutant, F97Y, were also investigated with the same methods. The changes in backbone NH dynamics observed for F97Y HbI upon ligand binding are not as large as for the wild type, in agreement with the reduced cooperativity observed for this mutant. The results of this study indicate that interface flexibility and backbone conformational entropy of HbI participate in and are important for the cooperative mechanism of carbon monoxide binding.

Anti-wear properties of the molluscan shell Scapharca subcrenata: influence of surface morphology, structure and organic material on the elementary wear process.

As a typical natural biological mineralisation material, molluscan shells have excellent wear-resistance properties that result from the interactions amongst biological coupling elements such as morphology, structure and material. The in-depth study of the wear-resistance performance of shells and the contribution made by each coupling element may help to promote the development of new bionic wear-resistant devices. The objective of this study was to investigate the influence of surface morphology (rib distribution on the shell), structure (rib coupled with nodules) and material (organic matter) on the anti-wear performance of the molluscan Scapharca subcrenata shell. The effect and contribution of each of these biological coupling elements were systematically investigated using the comparative experiment method. All three were found to exert significant effects on the shell's wear-resistance ability, and their individual contributions to that ability were revealed. Organic material can be classified as the principal coupling element, rib morphology as the secondary coupling element and the combined rib-nodule structure as the general coupling element.

Ligand migration and cavities within Scapharca Dimeric HbI: studies by time-resolved crystallo-graphy, Xe binding, and computational analysis.

As in many other hemoglobins, no direct route for migration of ligands between solvent and active site is evident from crystal structures of Scapharca inaequivalvis dimeric HbI. Xenon (Xe) and organic halide binding experiments, along with computational analysis presented here, reveal protein cavities as potential ligand migration routes. Time-resolved crystallographic experiments show that photodissociated carbon monoxide (CO) docks within 5 ns at the distal pocket B site and at more remote Xe4 and Xe2 cavities. CO rebinding is not affected by the presence of dichloroethane within the major Xe4 protein cavity, demonstrating that this cavity is not on the major exit pathway. The crystal lattice has a substantial influence on ligand migration, suggesting that significant conformational rearrangements may be required for ligand exit. Taken together, these results are consistent with a distal histidine gate as one important ligand entry and exit route, despite its participation in the dimeric interface.

Structural characterization and antithrombin activity of dermatan sulfate purified from marine clam Scapharca inaequivalvis.

Glycosaminoglycans from the body of marine clam Scapharca inaequivalvis were extracted at about 0.15- 0.18 mg/g of dry tissue, composed of dermatan sulfate (DS) (approx. 74%) and heparan sulfate (26%). After treatment with nitrous acid, DS was isolated for further complete structural characterization. Agarose-gel electrophoresis in combination with various enzymes, chondroitin ABC lyase, chondroitin B lyase, chondroitin ACII lyase from Arthrobacter aurescens, and chondroitin AC lyase from Flavobacterium heparinum, confirmed the DS nature of this polysaccharide. Furthermore, by evaluating the unsaturated disaccharides produced by the action of the various lyases, this natural polymer was found to be composed of approx. 75% of disaccharides containing iduronic acid (IdoA) mainly found in disaccharides monosulfated in position 4 of N-acetylgalactosamine (GalNAc) and disulfated in position 2 of the IdoA and 4 of GalNAc (disaccharide B typical of DS). In contrast, glucuronic acid was found to be mainly associated with the nonsulfated disaccharide (approx. 92%), while the rest formed low percentages of monosulfated disaccharides in position 4 or 6 of GalNAc preferentially located inside the chains. Generally, this GAG possesses a peculiar structure, due to the presence of significant amounts of nonsulfated disaccharide mainly located close to the nonreducing end, to the elevated percentage of the disaccharide B, and to the presence of not previously reported low amounts of the disaccharide monosulfated in position 2 of the uronic acid. S. inaequivalvis DS was also found to have a mean molecular mass of approx. 27,000 Da and a mean charge density of 1.10 that increases to 1.54 for the carbohydrate backbone composed of IdoA residues. (1)H-NMR and (13)C-NMR analyses confirmed the nature of S. inaequivalvis polymer revealed by the presence of signals related to DS corresponding to the residue of IdoA and GalNAc mainly sulfated at the C4 along with the presence of a signal belonging to the residue of H1 IdoA-2SO(4). S. inaequivalvis DS was further depolymerized by partial controlled digestion with chondroitinase ABC and separated into oligosaccharides by online HPLC/ESI-MS to obtain sequence information. The most prominent generated oligosaccharides comprised the repeating unit Delta Hex-GalNAcSO(4) thus confirming the results obtained by disaccharide analysis and the structures of the major oligosaccharides (from 6- to 10-mer) confirmed, by means of the LC-MS, the presence of approx. 20% of nonsulfated disaccharide. Furthermore, a minor but significant percentage of a monosaccharide having an m/z 300 and corresponding to GalNAcSO(4) belonging to the DS nonreducing end was observed along with saturated hexasaccharide derived from the nonreducing terminus of the intact DS ending with a uronic acid residue. Finally, S. inaequivalvis DS was calculated to possess a high heparin cofactor II activity of 169.2 +/- 10.7% fairly similar to that of several DS samples purified from porcine and bovine tissues.

Ligand migration and binding in the dimeric hemoglobin of Scapharca inaequivalvis.

Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of the dimeric hemoglobin of Scapharca inaequivalvis (HbI) after photodissociation. By combining these studies with X-ray crystallography, three transient ligand docking sites were identified: a primary docking site B in close vicinity to the heme iron, and two secondary docking sites C and D corresponding to the Xe4 and Xe2 cavities of myoglobin. To assess the relevance of these findings for physiological binding, we also performed flash photolysis experiments on HbICO at room temperature and equilibrium binding studies with dioxygen. Our results show that the Xe4 and Xe2 cavities serve as transient docking sites for unbound ligands in the protein, but not as way stations on the entry/exit pathway. For HbI, the so-called histidine gate mechanism proposed for other globins appears as a plausible entry/exit route as well.

Inhibition of mouse macrophages interleukin-12 production: suppression of nuclear factor-kappaB binding activity by a specific factor isolated from Scapharca broughtonii.

Pharmacological inhibition of interleukin-12 (IL-12) production may allow a therapeutic strategy for preventing development and progression of disease in experimental models of autoimmunity. In this study we investigated the effects of an ethanol fraction of the Scapharca broughtonii, on the production of IL-12 by mouse macrophages stimulated with lipopolysaccharides (LPS). The ethanol fraction (S3) prepared from Scapharca broughtonii potently inhibited LPS-induced IL-12 production in the RAW264.7 monocyte cell-line in a dose-dependent manner. The activation effect of the ethanol fraction (S3) on the IL-12 gene promoter was analyzed by transfecting RAW264.7 cells with IL-12 gene promoter/luciferase constructs. The repressive effect mapped to a region in the IL-12 gene promoter that contained a binding site for NF-kappaB. Furthermore, activation of macrophages by LPS resulted in markedly enhanced binding activity to the NF-kappaB site, which significantly decreased upon addition of the ethanol fraction, indicating that the ethanol fraction of the blood shell inhibited IL-12 production in LPS-activated macrophages via inhibition of NF-kappaB binding activity.

Allosteric action in real time: time-resolved crystallographic studies of a cooperative dimeric hemoglobin.

Protein allostery provides mechanisms for regulation of biological function at the molecular level. We present here an investigation of global, ligand-induced allosteric transition in a protein by time-resolved x-ray diffraction. The study provides a view of structural changes in single crystals of Scapharca dimeric hemoglobin as they proceed in real time, from 5 ns to 80 micros after ligand photodissociation. A tertiary intermediate structure forms rapidly (<5 ns) as the protein responds to the presence of an unliganded heme within each R-state protein subunit, with key structural changes observed in the heme groups, neighboring residues, and interface water molecules. This intermediate lays a foundation for the concerted tertiary and quaternary structural changes that occur on a microsecond time scale and are associated with the transition to a low-affinity T-state structure. Reversal of these changes shows a considerable lag as a T-like structure persists well after ligand rebinding, suggesting a slow T-to-R transition.

Residue F4 plays a key role in modulating oxygen affinity and cooperativity in Scapharca dimeric hemoglobin.

Residue F4 (Phe 97) undergoes the most dramatic ligand-linked transition in Scapharca dimeric hemoglobin, with its packing in the heme pocket in the unliganded (T) state suggested to be a primary determinant of its low affinity. Mutation of Phe 97 to Leu (previously reported), Val, and Tyr increases oxygen affinity from 8- to 100-fold over that of the wild type. The crystal structures of F97L and F97V show side chain packing in the heme pocket for both R and T state structures. In contrast, in the highest-affinity mutation, F97Y, the tyrosine side chain remains in the interface (high-affinity conformation) even in the unliganded state. Comparison of these mutations reveals a correlation between side chain packing in the heme pocket and oxygen affinity, indicating that greater mass in the heme pocket lowers oxygen affinity due to impaired movement of the heme iron into the heme plane. The results indicate that a key hydrogen bond, previously hypothesized to have a central role in regulation of oxygen affinity, plays at most only a small role in dictating ligand affinity. Equivalent mutations in sperm whale myoglobin alter ligand affinity by only 5-fold. The dramatically different responses to mutations at the F4 position result from subtle, but functionally critical, stereochemical differences. In myoglobin, an eclipsed orientation of the proximal His relative to the A and C pyrrole nitrogen atoms provides a significant barrier for high-affinity ligand binding. In contrast, the staggered orientation of the proximal histidine found in liganded HbI renders its ligand affinity much more susceptible to packing contacts between F4 and the heme group. These results highlight very different strategies used by cooperative hemoglobins in molluscs and mammals to control ligand affinity by modulation of the stereochemistry on the proximal side of the heme.