Fluorescence studies on the interaction of myoglobin with mitochondria

To determine the nature and characteristic parameters of the myoglobin-mitochondrion interaction during oxymyoglobin (MbO₂) deoxygenation in the cell, we studied the quenching of the intrinsic mitochondrial flavin and tryptophan fluorescence by different liganded myoglobins in the pH range of 6–8, as well as the quenching of the fluorescence of the membrane probes 1,8-ANS and merocyanine 540 (M 540) embedded into the mitochondrial membrane. Physiologically active MbO₂ and oxidized metmyoglobin (metMb), which are unable to bind oxygen, were used as the quenchers. The absence of quenching of flavin and tryptophan fluorescence implies that myoglobin does not form quenching complexes with either electron transport chain proteins of the inner mitochondrial membrane or with outer membrane proteins. We found, however, that MbO₂ and metMb effectively quench 1,8-ANS and M 540 fluorescence in the pH range of 6–8. Characteristic parameters of 1,8-ANS and M 540 fluorescence quenching by the myoglobins (extent of quenching and quencher binding constant, K _m) are very similar, indicating that both probes are localized in phospholipid sites of the mitochondrial membrane, and myoglobin is complexed with these sites. The dependence of K _m on ionic strength proves the important role of coulombic interactions in the formation of the quenching complex. Since the overall charge of myoglobin is shown not to influence the K _m values, the ionic strength dependence must be due to local electrostatic interactions in which polar groups of some part of the myoglobin molecule participate. The most likely candidates to interact with anionic groups of mitochondrial phospholipids are invariant lysine and arginine residues in the environment of the myoglobin heme cavity, which do not change their ionization state in the pH range investigated.     

Palmitate interaction with physiological states of myoglobin

Background

Previous studies have shown that palmitate (PA) can bind specifically and non-specifically to Fe(III) MbCN. The present study has observed PA interaction with physiological states of Fe(II) Mb, and the observations support the hypothesis that Mb may have a potential role in facilitating intracellular fatty acid transport.

Methods

¹H NMR spectra measurements of the Mb signal during PA titration show signal changes consistent with specific and non-specific binding.

Results

Palmitate (PA) interacts differently with physiological states of Mb. Deoxy Mb does not interact specifically or non-specifically with PA, while the carbonmonoxy myoglobin (MbCO) interaction with PA decreases the intensity of selective signals and produces a 0.15 ppm upfield shift of the PA methylene peak. The selective signal change upon PA titration provides a basis to determine an apparent PA binding constant, which serves to create a model comparing the competitive PA binding and facilitated fatty acid transport of Mb and fatty acid binding protein (FABP).

Conclusions

Given contrasting PA interaction of ligated vs. unligated Mb, the cellular fatty acid binding protein (FABP) and Mb concentration in the cell, the reported cellular diffusion coefficients, the PA dissociation constants from ligated Mb and FABP, a fatty acid flux model suggests that Mb can compete with FABP transporting cellular fatty acid.

General significance

Under oxygenated conditions and continuous energy demand, Mb dependent fatty acid transport could influence the cell's preference for carbohydrate or fatty acid as a fuel source and regulate fatty acid metabolism.     

Circular dichroism studies of myoglobin and leghemoglobin.

The circular dichroism spectra of leghemoglobin a from the root nodules of soybean have been compared with those for sperm whale myoglobin in the fat- and near-ultraviolet and the Soret and visible regions of the spectrum. Circular dichroism spectra in the far-ultraviolet show that the leghemoglobins all have a high alpha-helix content (soybean leghemoglobin a, 55%) regardless of the nature of bound ligands and oxidation or spin state of the heme iron. The known sequence homologies with mammalian hemoglobins may therefore be reflected in conformational homologies as suggested by the x-ray studies of Vainshtein et al. ((1975) Nature (London) 254, 163-164) on lupin leghemoglobin. Removal of the heme moiety decreases helicity by only 9% for leghemoglobins, compared with 23% for myoglobin. This, the much smaller heme contribution to the near-ultraviolet circular dichroism than in myoglobin, and the greater accessibility of the heme moiety to aqueous solvent (Nicola et al. (1974), Proc. Aust. Biochem. Soc. 7, 21) suggest that the association between heme and protein is much weaker in leghemoglobins than in myoglobin. The aromatic Soret and visible circular dichroism spectra for all derivatives of leghemoglobin are opposite in sense to those for myoglobin, showing that the patterns of protein side chain contacts with the heme are different in the two classes of heme proteins. There is strong evidence that one of the two tryptophans whose identity and structural role in myoglobin is known, is present also in plant leghemoglobins, hydrogen-bonded and in a similar nonpolar environment whether heme is present or not. The above findings help to explain the remarkably high oxygen affinity and some other ligand-binding properties of leghemoglobins which differ from those of myoglobin.     

Computational studies of adsorption in metal organic frameworks and interaction of nanoparticles in condensed phases

In this review, we describe recent efforts to systematically study nano-structured metal organic frameworks (MOFs), also known as metal organic heat carriers, with particular emphasis on their application in heating and cooling processes. We used both molecular dynamics and grand canonical Monte Carlo simulation techniques to gain a molecular-level understanding of the adsorption mechanism of gases in these porous materials. We investigated the uptake of various gases such as refrigerants R12 and R143a. We also evaluated the effects of temperature and pressure on the uptake mechanism. Our computed results compared reasonably well with available measurements from experiments, thus validating our potential models and approaches. In addition, we investigated the structural, diffusive and adsorption properties of different hydrocarbons in Ni₂(dhtp). Finally, to elucidate the mechanism of nanoparticle dispersion in condensed phases, we studied the interactions among nanoparticles in various liquids, such as n-hexane, water and methanol.     

Analysis of the interaction of gallic acid and myoglobin by UV-vis absorption spectroscopy

UV-vis absorption spectroscopy has been used to analyze the interaction of myoglobin (Мb) and gallic acid (GA). The binding constants (4.38 × 10⁴ M^–1 at 298.15 K and 0.42 × 10⁴ М^–1 at 308.15K), the number of binding sites (h = 1.0), and the thermodynamic parameters of binding (ΔH, ΔS, and ΔG) have been determined. Hydrogen bonds have been shown to play a major role in the stabilization of the GA–Мb complexes. GA binding led to slight changes in the electronic state of the heme ring of the protein.     

Computational approaches to protein-protein interaction

The interactions between proteins allow the cell's life. A number of experimental, genome-wide, high-throughput studies have been devoted to the determination of protein-protein interactions and the consequent interaction networks. Here, the bioinformatics methods dealing with protein-protein interactions and interaction network are overviewed. 1. Interaction databases developed to collect and annotate this immense amount of data; 2. Automated data mining techniques developed to extract information about interactions from the published literature; 3. Computational methods to assess the experimental results developed as a consequence of the finding that the results of high-throughput methods are rather inaccurate; 4. Exploitation of the information provided by protein interaction networks in order to predict functional features of the proteins; and 5. Prediction of protein-protein interactions.     

Computational redesign of protein-protein interaction specificity

We developed a 'computational second-site suppressor' strategy to redesign specificity at a protein-protein interface and applied it to create new specifically interacting DNase-inhibitor protein pairs. We demonstrate that the designed switch in specificity holds in in vitro binding and functional assays. We also show that the designed interfaces are specific in the natural functional context in living cells, and present the first high-resolution X-ray crystallographic analysis of a computer-redesigned functional protein-protein interface with altered specificity. The approach should be applicable to the design of interacting protein pairs with novel specificities for delineating and re-engineering protein interaction networks in living cells.     

Computational prediction and analysis of the DR6-NAPP interaction

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that involves a devastating clinical course and that lacks an effective treatment. A biochemical model for neuronal development, recently proposed by Nikolaev et al., that may also have implications for AD, hinges on a novel protein–protein interaction between the death cell receptor 6 (DR6) ectodomain and an Nterminal fragment of amyloid precursor protein (NAPP), specifically, the growth factor-like domain of NAPP (GFD NAPP). Given all of this, we used a pure computational work-flow to dock a binding competent homology model of the DR6 ectodomain to a binding competent crystal structure of GFD NAPP. The DR6 homology model was built according to a template supplied by the neurotrophin p75 receptor. The best docked model was selected according to an empirical estimate of the binding affinity and represents a high quality model of probable structural accuracy, especially with respect to the residue-level contribution of GFD NAPP. The final model was tested and verified against a variety of biophysical and theoretical data sets. Particularly, worth noting is the excellent observed agreement between the theoretically calculated DR6–GFD NAPP binding free energy and the experimental quantity. The model is used to provide a satisfying structural and energetic interpretation of DR6–GFD NAPP binding and to suggest the possibility of and a mechanism for spontaneous apoptosis. The evidence suggests that the DR6–NAPP model proposed here is of probable accuracy and that it will prove useful in future studies, modeling work, and structure-based AD drug design.     

Spectroscopic and electrochemical studies of horse myoglobin in dimethyl sulfoxide

This paper reports the first report of rapid, reversible direct electron transfer between a redox protein, specifically, horse myoglobin, and a solid electrode substrate in nonaqueous media and the spectroscopic (UV-vis, fluorescence, and resonance Raman) characterization of the relevant redox forms of myoglobin (Mb) in dimethyl sulfoxide (DMSO). In DMSO, the heme active site of metmyoglobin (metMb) appears to remain six-coordinate high-spin, binding water weakly. Changes in the UV-fluorescence spectra for metMb in DMSO indicate that the protein secondary structure has been perturbed and suggest that helix A has moved away from the heme. UV-vis and RR spectra for deoxyMb in DMSO suggest that the heme iron is six-coordinate low-spin, most likely coordinating DMSO. Addition of CO to deoxyMb in DMSO produces a single, photostable six-coordinate CO adduct. UV-vis and RR for Mb-CO in DMSO are consistent with a six-coordinate low-spin heme iron binding His93 weakly, if at all. The polarity of the distal heme pocket is comparable to that of the closed form of horse Mb-CO in aqueous solution, pH 7. Direct electron transfer between horse Mb and Au in DMSO solution was investigated by cyclic voltammetry. Mb exhibits stable and well-defined electrochemical responses that do not appear to be affected by the water content (1.3-7.5%). The electrochemical characteristics are consistent with a one-electron, quasi-reversible, diffusion-controlled charge transfer process at Au. E degrees for horse Mb in DMSO at Au is -0.241+/-0.005 V vs. NHE. The formal heterogeneous electron transfer rate constant, calculated from delta E(p) at 20 mV/s, is 1.7+/-0.5 x 10(-4) cm/s. The rate, which is unaffected by the presence of 1.3-7.5% water, is competitive with that previously reported for horse Mb in aqueous solution.     

Computational studies of membrane channels

The determination of the structure of several members of the K+ channel and aquaporin family represents a unique opportunity to explain the mechanism of these biomolecular systems. With their ability to go beyond static structures, molecular dynamics simulations offer a unique route for relating functional properties to membrane channel structure. The recent progress in this area is reviewed.     

Fluctuation domains in myoglobin. Fluorescence quenching studies

The dynamics of two domains in the myoglobin molecule, close to the heme and inside the protein medium including the surface, are investigated through the study of the fluorescence oxygen quenching of two probes imbedded in the heme pocket: zinc protoporphyrin IX (with a fluorescence lifetime of 2.1 ns) and metal-free protoporphyrin IX (with a fluorescence lifetime of 17.8 ns).     

Computational analysis of human protein interaction networks

Large amounts of human protein interaction data have been produced by experiments and prediction methods. However, the experimental coverage of the human interactome is still low in contrast to predicted data. To gain insight into the value of publicly available human protein network data, we compared predicted datasets, high-throughput results from yeast two-hybrid screens, and literature-curated protein-protein interactions. This evaluation is not only important for further methodological improvements, but also for increasing the confidence in functional hypotheses derived from predictions. Therefore, we assessed the quality and the potential bias of the different datasets using functional similarity based on the Gene Ontology, structural iPfam domain-domain interactions, likelihood ratios, and topological network parameters. This analysis revealed major differences between predicted datasets, but some of them also scored at least as high as the experimental ones regarding multiple quality measures. Therefore, since only small pair wise overlap between most datasets is observed, they may be combined to enlarge the available human interactome data. For this purpose, we additionally studied the influence of protein length on data quality and the number of disease proteins covered by each dataset. We could further demonstrate that protein interactions predicted by more than one method achieve an elevated reliability.     

Effect of fatty acid interaction on myoglobin oxygen affinity and triglyceride metabolism

Recent studies have suggested myoglobin (Mb) may have other cellular functions in addition to storing and transporting O₂. Indeed, NMR experiments have shown that the saturated fatty acid (FA) palmitate (PA) can interact with myoglobin (Mb) in its ligated state (MbCO and MbCN) but does not interact with Mb in its deoxygenated state. The observation has led to the hypothesis that Mb can also serve as a fatty acid transporter. The present study further investigates fatty acid interaction with the physiological states of Mb using the more soluble but unsaturated fatty acid, oleic acid (OA). OA binds to MbCO but does not bind to deoxy Mb. OA binding to Mb, however, does not alter its O₂ affinity. Without any Mb, muscle has a significantly lower level of triglyceride (TG). In Mb knock-out (MbKO) mice, both heart and skeletal muscles have lower level of TG relative to the control mice. Training further decreases the relative TG in the MbKO skeletal muscle. Nevertheless, the absence of Mb and lower TG level in muscle does not impair the MbKO mouse performance as evidenced by voluntary wheel running measurements. The results support the hypothesis of a complex physiological role for Mb, especially with respect to fatty acid metabolism.