Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles

Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591-598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca2+- and Mg2+-ATPase activities of CF1 deficient in its inhibitory epsilon subunit (CF1-epsilon) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca2+-ATPase activity by CTAB is irreversible. The Ca2+-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg2+-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-epsilon or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-epsilon from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the alpha/beta heterohexamer.     

Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles

Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591-598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca²⁺- and Mg²⁺-ATPase activities of CF1 deficient in its inhibitory ε subunit (CF1-ε) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca²⁺-ATPase activity by CTAB is irreversible. The Ca²⁺-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg²⁺-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-ε or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-ε from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the α/β heterohexamer.     

Electron microscopic observation of the aggregation of membrane proteins in human erythrocyte by melittin

Human erythrocytes and erythrocyte ghost membranes were treated with native and modified melittins, up to 250 nmol/mg membrane protein. Native melittin induced aggregation of intramembranous particles (IMPs, observed by freeze-fracture electron microscopy), and created large, smooth bilayer areas devoid of IMP. The degree of IMP aggregation increased with increasing concentration of melittin, corresponding to hemolysis results. Membrane ghosts were slightly more susceptible to IMP aggregation than membranes on intact cells. The potency of inducing IMP aggregation was ranked in the order of: native melittin greater than acetylated melittin greater than succinylated melittin = 0. The concentration range of melittin which caused IMP aggregation corresponded to that which caused the immobilization of band 3 proteins as detected by measurement of rotational mobility by transient dichroism (Dufton et al. (1984) Eur. J. Biophys. 11, 17-24). Because both IMP aggregation and band 3 protein immobilization decreased with decreasing positive charge of the melittins used, the nature of melittin-protein interaction is likely to be at least in part electrostatic in the case of human erythrocyte membranes. Possible roles of IMP aggregation and the consequent creation of 'exposed' bilayer areas in the cytotoxic reaction of melittins are discussed.     

The molecular chaperone activity of simian virus 40 large T antigen is required to disrupt Rb-E2F family complexes by an ATP-dependent mechanism

The simian virus 40 large T antigen (T antigen) inactivates tumor suppressor proteins and therefore has been used in numerous studies to probe the mechanisms that control cellular growth and to generate immortalized cell lines. Binding of T antigen to the Rb family of growth-regulatory proteins is necessary but not sufficient to cause transformation. The molecular mechanism underlying T-antigen inactivation of Rb function is poorly understood. In this study we show that T antigen associates with pRb and p130-E2F complexes in a stable manner. T antigen dissociates from a p130-E2F-4-DP-1 complex, coincident with the release of p130 from E2F-4-DP-1. The dissociation of this complex requires Hsc70, ATP, and a functional T-antigen J domain. We also report that the "released" E2F-DP-1 complex is competent to bind DNA containing an E2F consensus binding site. We propose that T antigen disrupts Rb-E2F family complexes through the action of its J domain and Hsc70. These findings indicate how Hsc70 supports T-antigen action and help to explain the cis requirement for a J domain and Rb binding motif in T-antigen-induced transformation. Furthermore, this is the first demonstration linking Hsc70 ATP hydrolysis to the release of E2F bound by Rb family members.     

Lytic activity of monomeric and oligomeric melittin

The haemolytic activities of melittin and melittin tetramer as induced by high phosphate counterion concentration, were monitored. Monomeric melittin was found to be fully lytic, whilst tetrameric melittin lacked such activity. Under conditions where melittin was fully tetrameric attempts were made to covalently cross-link the native tetramer using a series of different chain length bifunctional imido esters. The cross-linked oligomers were fully lytic under conditions where melittin was demonstrated to lack such activity. This finding, together with molecular weight determinations and circular dichroism studies, indicated that the cross-linked melittin was quite different to the native tetramer. The haemolytic activity of melittin-containing solutions was related to the concentration of monomeric melittin. The effect of reduced dielectric constant (?) on the aggregation behaviour of melittin and its derivatives was found to favour monomeric melittin.     

The oligomerization of amyloid beta-protein begins intracellularly in cells derived from human brain

The progressive aggregation and deposition of amyloid beta-protein (Abeta) in brain regions subserving memory and cognition is an early and invariant feature of Alzheimer's disease, the most common cause of cognitive failure in aged humans. Inhibiting Abeta aggregation is therapeutically attractive because this process is believed to be an exclusively pathological event. Whereas many studies have examined the aggregation of synthetic Abeta peptides under nonphysiological conditions and concentrations, we have detected and characterized the oligomerization of naturally secreted Abeta at nanomolar levels in cultures of APP-expressing CHO cells [Podlisny, M. B., Ostaszewski, B. L., Squazzo, S. L., Koo, E. H., Rydell, R. E., Teplow, D. B., and Selkoe, D. J. (1995) J. Biol. Chem. 270, 9564-9570 (1); Podlisny, M. B., Walsh, D. M., Amarante, P., Ostaszewski, B. L., Stimson, E. R., Maggio, J. E., Teplow, D. B., and Selkoe, D. J. (1998) Biochemistry 37, 3602-3611 (2)]. To determine whether similar species occur in vivo, we probed samples of human cerebrospinal fluid (CSF) and detected SDS-stable dimers of Abeta in some subjects. Incubation of CSF or of CHO conditioned medium at 37 degrees C did not lead to new oligomer formation. This inability to induce oligomers extracellularly as well as the detection of oligomers in cell medium very early during the course of pulse-chase experiments suggested that natural Abeta oligomers might first form intracellularly. We therefore searched for and detected intracellular Abeta oligomers, principally dimers, in primary human neurons and in neuronal and nonneural cell lines. These dimers arose intracellularly rather than being derived from the medium by reuptake. The dimers were particularly detectable in neural cells: the ratio of intracellular to extracellular oligomers was much higher in brain-derived than nonbrain cells. We conclude that the pathogenically critical process of Abeta oligomerization begins intraneuronally.     

Effect of the state of association of melittin and phospholipids on their reciprocal binding

In solutions of increasing ionic strength, the molecular weight of melittin varies from 2840 (monomeric melittin) to 11 200. This polymerization, concomitant with an important change in conformation (Talbot, J.C., Dufourcq, J., De Bony, J., Faucon, J.F. and Lussan, C. (1979) FEBS Lett. 102, 191–193), is accompanied by a significant alteration in the partial specific volume of the molecule. The binding of melittin to phospholipids (phosphatidylserine, lysolecithin, dihexanoyl-, dioctanoyl- and lysolauroylphosphatidylcholine) depends on the state of association of the toxin and on the critical micelle concentration of lipids. No interaction is observed between monomeric melittin and free lipids, whereas tetrameric melittin can bind free lipids to form mixed micelles. At phospholipid concentrations above the critical micelle concentration, melittin in any state of self-association can bind lipids. The mixed micelles formed at saturation appear to be independent of the initial state of association of melittin.     

A transthyretin monomer intermediate undergoes local unfolding and transient interaction with oligomers in a kinetically concerted aggregation pathway

Transthyretin (TTR) amyloidosis is associated with tissue deposition of TTR aggregates. TTR aggregation is initiated by dissociation of the native tetramer to form a monomeric intermediate, which locally unfolds and assembles into soluble oligomers and higher-order aggregates. However, a detailed mechanistic understanding requires kinetic and structural characterization of the low population intermediates formed. Here, we show that the monomeric intermediate exchanges with an ensemble of oligomers on the millisecond timescale. This transient and reversible exchange causes broadening of the ¹⁹F resonance of a trifluoromethyl probe coupled to the monomeric intermediate at S85C. We show the ¹⁹F linewidth and R₂ relaxation rate increase with increasing concentration of the oligomer. Furthermore, introduction of ¹⁹F probes at additional TTR sites yielded distinct ¹⁹F chemical shifts for the TTR tetramer and monomer when the trifluoromethyl probe was attached at S100C, located near the same subunit interface as S85C, but not with probes attached at S46C or E63C, which are distant from any interfaces. The ¹⁹F probe at E63C shows that part of the DE loop, which is solvent accessible in the tetramer, becomes more buried in the NMR-visible oligomers. Finally, using backbone amides as probes, we show that parts of the EF helix and H-strand become highly flexible in the otherwise structured monomeric intermediate at acidic pH. We further find that TTR aggregation can be reversed by increasing pH. Taken together, this work provides insights into location-dependent conformational changes in the reversible early steps of a kinetically concerted TTR aggregation pathway.     

Melittin induces both time-dependent aggregation and inhibition of Na,K-ATPase from duck salt glands however these two processes appear to occur independently

Using cupric phenanthroline as a cross-linking agent, we have shown that melittin induced time-dependent aggregations of Na,K-ATPase in microsomal fractions and in preparations of purified Na,K-ATPase from duck salt glands. Incubation of melittin with these preparations also led to the progressive loss of Na,K-ATPase activity. At melittin/protein molar ratio of 5:1, we did not observe inhibition of Na,K-ATPase in the microsomal fraction but the process of enzyme aggregation occurred. At higher melittin/protein molar ratios (10:1 and 30:1), the inhibition of the enzyme and its aggregation proceeded simultaneously but the rates of these processes and maximal values achieved were different. At a melittin/protein ratio of 30:1, Na,K-ATPase inhibition may be described as a biexponential curve with the values for pseudo-first order rate constants being 2.7 and 0.15 min(-1). However, the aggregation may be presented by a monoexponential curve with a pseudo-first order rate constant of 0.15 min(-1). In purified preparations of Na,K-ATPase, the maximal aggregation (about 90%) was achieved at a melittin/protein molar ratio of 2:1, and a further increase in the melittin/protein ratio increased the rate of aggregation but did not affect the value of maximal aggregation. The results show that melittin induced both aggregation and inhibition of Na,K-ATPase but these two processes proceeded independently.     

Monoclonal antibodies selective for α‐synuclein oligomers/protofibrils recognize brain pathology in Lewy body disorders and α‐synuclein transgenic mice with the disease‐causing A30P mutation

Inclusions of intraneuronal alpha‐synuclein (α‐synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of α‐synuclein is a central feature of the disease pathogenesis. Among the different α‐synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large α‐synuclein oligomers were generated. These antibodies, which do not bind amyloid‐beta or tau, recognize Lewy body pathology in brains from patients with Parkinson's disease and dementia with Lewy bodies and detect pathology earlier in α‐synuclein transgenic mice than linear epitope antibodies. An oligomer‐selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of α‐synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of α‐synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer‐selective α‐synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.     

Melittin induces both time-dependent aggregation and inhibition of Na,K-ATPase from duck salt glands however these two processes appear to occur independently

Using cupric phenanthroline as a cross-linking agent, we have shown that melittin induced time-dependent aggregations of Na,K-ATPase in microsomal fractions and in preparations of purified Na,K-ATPase from duck salt glands. Incubation of melittin with these preparations also led to the progressive loss of Na,K-ATPase activity. At melittin/protein molar ratio of 5:1, we did not observe inhibition of Na,K-ATPase in the microsomal fraction but the process of enzyme aggregation occurred. At higher melittin/protein molar ratios (10:1 and 30:1), the inhibition of the enzyme and its aggregation proceeded simultaneously but the rates of these processes and maximal values achieved were different. At a melittin/protein ratio of 30:1, Na,K-ATPase inhibition may be described as a biexponential curve with the values for pseudo-first order rate constants being 2.7 and 0.15 min⁻¹. However, the aggregation may be presented by a monoexponential curve with a pseudo-first order rate constant of 0.15 min⁻¹. In purified preparations of Na,K-ATPase, the maximal aggregation (about 90%) was achieved at a melittin/protein molar ratio of 2:1, and a further increase in the melittin/protein ratio increased the rate of aggregation but did not affect the value of maximal aggregation. The results show that melittin induced both aggregation and inhibition of Na,K-ATPase but these two processes proceeded independently.     

Calcium ions promote formation of amyloid β-peptide (1-40) oligomers causally implicated in neuronal toxicity of Alzheimer's disease

Amyloid β-peptide (Aβ) is directly linked to Alzheimer's disease (AD). In its monomeric form, Aβ aggregates to produce fibrils and a range of oligomers, the latter being the most neurotoxic. Dysregulation of Ca(2+) homeostasis in aging brains and in neurodegenerative disorders plays a crucial role in numerous processes and contributes to cell dysfunction and death. Here we postulated that calcium may enable or accelerate the aggregation of Aβ. We compared the aggregation pattern of Aβ(1-40) and that of Aβ(1-40)E22G, an amyloid peptide carrying the Arctic mutation that causes early onset of the disease. We found that in the presence of Ca(2+), Aβ(1-40) preferentially formed oligomers similar to those formed by Aβ(1-40)E22G with or without added Ca(2+), whereas in the absence of added Ca(2+) the Aβ(1-40) aggregated to form fibrils. Morphological similarities of the oligomers were confirmed by contact mode atomic force microscopy imaging. The distribution of oligomeric and fibrillar species in different samples was detected by gel electrophoresis and Western blot analysis, the results of which were further supported by thioflavin T fluorescence experiments. In the samples without Ca(2+), Fourier transform infrared spectroscopy revealed conversion of oligomers from an anti-parallel β-sheet to the parallel β-sheet conformation characteristic of fibrils. Overall, these results led us to conclude that calcium ions stimulate the formation of oligomers of Aβ(1-40), that have been implicated in the pathogenesis of AD.     

Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogues

Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2). MLN4924 is an adenosine sulfamate analogue that was identified as a selective, mechanism-based inhibitor of NEDD8-activating enzyme (NAE), another E1 enzyme, by forming a NEDD8-MLN4924 adduct that tightly binds at the active site of NAE, a novel mechanism termed substrate-assisted inhibition (Brownell, J. E., Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump, N. J., Soucy, T. A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke, H. K., Lingaraj, T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis, C. A., Langston, S. P., Vyskocil, S., Sells, T. B., Mallender, W. D., Visiers, I., Li, P., Claiborne, C. F., Rolfe, M., Bolen, J. B., and Dick, L. R. (2010) Mol. Cell 37, 102-111). In the present study, substrate-assisted inhibition of human UAE (Ube1) by another adenosine sulfamate analogue, 5'-O-sulfamoyl-N(6)-[(1S)-2,3-dihydro-1H-inden-1-yl]-adenosine (Compound I), a nonselective E1 inhibitor, was characterized. Compound I inhibited UAE-dependent ATP-PP(i) exchange activity, caused loss of UAE thioester, and inhibited E1-E2 transthiolation in a dose-dependent manner. Mechanistic studies on Compound I and its purified ubiquitin adduct demonstrate that the proposed substrate-assisted inhibition via covalent adduct formation is entirely consistent with the three-step ubiquitin activation process and that the adduct is formed via nucleophilic attack of UAE thioester by the sulfamate group of Compound I after completion of step 2. Kinetic and affinity analysis of Compound I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of adduct formation and the affinity between the adduct and E1 contribute to the overall potency. Because all E1s are thought to use a similar mechanism to activate their cognate ubiquitin-like proteins, the substrate-assisted inhibition by adenosine sulfamate analogues represents a promising strategy to develop potent and selective E1 inhibitors that can modulate diverse biological pathways.     

Hsc70 contacts helix III of the J domain from polyomavirus T antigens: addressing a dilemma in the chaperone hypothesis of how they release E2F from pRb

Hsc70's expected binding site on helix II of the J domain of T antigens appears to be blocked in its structure bound to tumor suppressor pRb. We used NMR to map where mammalian Hsc70 binds the J domain of murine polyomavirus T antigens (PyJ). The ATPase domain of Hsc70 unexpectedly has its biggest effects on the NMR peak positions of the C-terminal end of helix III of PyJ. The Hsc70 ATPase domain protects the C-terminal end of helix III of PyJ from an uncharged paramagnetic probe of chelated Gd(III), clearly suggesting the interface. Effects on the conserved HPD loop and helix II of PyJ are smaller. The NMR results are supported by a novel assay of Hsc70's ATP hydrolysis showing that mutations of surface residues in PyJ helix III impair PyJ-dependent stimulation of Hsc70 activity. Evolutionary trace analysis of J domains suggests that helix III usually may join helix II in contributing specificities for cognate hsp70s. Our novel evidence implicating helix III differs from evidence that Escherichia coli DnaK primarily affects helix II and the HPD loop of DnaJ. We find the pRb-binding fragment of E2F1 to be intrinsically unfolded and a good substrate for Hsc70 in vitro. This suggests that E2F1 could be a substrate for Hsc70 recruited by T antigen to an Rb family member. Importantly, our results strengthen the chaperone hypothesis for E2F release from an Rb family member by Hsc70 recruited by large T antigen. That is, it now appears that Hsc70 can freely access helix III and the HPD motif of large T antigen bound to an Rb family member.     

Formation of non-beta 6.3-helical gramicidin channels between sequence-substituted gramicidin analogues.

Using the linear gramicidins as an example, we have previously shown how the statistical properties of heterodimeric (hybrid) channels (formed between the parent [Val1]gramicidin A (gA) and a sequence-altered analogue) can be used to assess whether the analogue forms channels that are structurally equivalent to the parent channels (Durkin, J. T., R. E. Koeppe II, and O. S. Andersen. 1990. J. Mol. Biol. 211:221-234). Generally, the gramicidins are tolerant of amino acid sequence alterations. We report here an exception. The optically reversed analogue, gramicidin M- (gM-) (Heitz, F., G. Spach, and Y. Trudelle. 1982. Biophys. J. 40:87-89), forms channels that are the mirror-image of [Val1]gA channels; gM- should thus form no hybrid channels with analogues having the same helix sense as [Val1]gA. Surprisingly, however, gM- forms hybrid channels with the shortened analogues des-Val1-[Ala2]gA and des-Val1-gC, but these channels differ fundamentally from the parent channels: (a) the appearance rate of these heterodimers is only approximately 1/10 of that predicted from the random assortment of monomers into conducting dimers, indicating the existence of an energy barrier to their formation (e.g., monomer refolding into a new channel-forming conformation); and (b), once formed, the hybrid channels are stabilized approximately 1,000-fold relative to the parent channels. The increased stability suggests a structure that is joined by many hydrogen bonds, such as one of the double-stranded helical dimers shown to be adopted by gramicidins in organic solvents (Veatch, W. R., E. T. Fossel, and E. R. Blout. 1974. Biochemistry. 13:5249-5256).     

A role for ADP ribosylation factor in the control of cargo uptake during COPI-coated vesicle biogenesis

ARF-mediated hydrolysis of GTP has been demonstrated to regulate coat disassembly of Golgi-derived COPI transport vesicles (Tanigawa, G., Orci, L., Amherdt, M., Ravazzola, M., Helms, J.B. and Rothman, J.E. (1993) J. Cell Biol. 123, 1365-1371). In addition, a requirement for GTP hydrolysis at an early stage of COPI vesicle biogenesis has been established since cargo uptake is impaired in the presence of GTPgammaS (Nickel, W., Malsam, J., Gorgas, K., Ravazzola, M., Jenne, N., Helms, J.B. and Wieland, F.T. (1998) J. Cell Sci. 111, 3081-3090), a non-hydrolyzable analogue of GTP. We now demonstrate that the GTPase involved in the regulation of cargo uptake is ARF, revealing a multi-functional role of this GTPase in COPI-mediated vesicular transport. The molecular mechanism of cargo uptake as well as the functional implications of these findings on the overall process of COPI vesicle biogenesis are discussed.     

Interaction of amphipathic peptides with an immobilised model membrane

Summary The interaction of melittin and a truncated analogue of melittin with an immobilised phosphatidylcholine monolayer has been studied using dynamic elution techniques. The melittin analogue (21Q analogue) had five amino acids omitted from the C-terminal region of melittin. The influence of temperature and methanol concentration on the binding affinity of the two peptides was determined and compared to the binding behaviour of two control moleculesN-acetyltryptophanamide and diphenylalanine. Both peptides exhibited non-linear dependence of affinity on % methanol at different temperatures, whileN-acetyltryptophanamide and diphenylalanine exhibited linear behaviour. In addition, both melittin and the 21Q analogue exhibited significant band broadening under a range of experimental conditions, which was not evident forN-acetyltryptophanamide and diphenylalanine. As melittin is known to adopt a significant degree of α-helical conformation in the presence of lipids, the results suggest that melittin and the 21Q analogue adopt different conformations and orientations upon binding to the immobilised phosphatidylcholine surface. Overall, the results of this study demonstrate that the immobilised lipid monolayer provides a powerful system to rapidly assess the affinity of peptides for different lipid surfaces.     

Fluorescence and 13C NMR determination of side-chain and backbone dynamics of synthetic melittin and melittin analogues in isotropic solvents

The dynamics in isotopic solvents of selectively 13C labeled synthetic melittin and three analogues have been investigated by using NMR and fluorescence techniques both separately and in combination. In conjunction with the "model-free" approach to interpretation of NMR relaxation data [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4570], the availability of steady-state fluorescence anisotropy and lifetime data augment T1, T2, and NOE data to provide quantitative information about fluorophore dynamics in these peptides. A method is presented for using combined fluorescence and NMR data to obtain technique- and model-independent values for parameters describing local motion of 13C-labeled fluorophores in peptides and proteins. The dynamics of melittin and melittin analogues are found to be consistent with structural characteristics inferred from CD, fluorescence, and NMR spectral information presented in the preceding paper (Weaver et al., 1989). In particular, the mobility of the random coil peptide monomers is shown to be quite similar, while side-chain as well as peptide backbone motion in the aggregated or oligomeric species differs markedly among the analogues. For melittin itself, experimentally determined overall rotational correlation times for the monomer and tetramer agree very well with values predicted on the basis of solvent-accessible protein surface area. The local dynamics of selectively 13C-labeled Trp-19 and Gly-12 residues of melittin are also found to be consistent with peptide structure. In random coil melittin monomer, a specific model for the motion indicates that the Trp side chain moves through an approximate angle of +/- 71 degrees about the beta-gamma bond with a correlation time of 159 +/- 24 ps. In melittin tetramer, the indole moiety is spatially more confined with a flip angle of +/- 37 degrees, yet demonstrates an increased rate of motion with a correlation time of 56 +/- 8 ps. The constrained mobility of the Trp-19 side chain is consistent with motional constraints inferred from the X-ray structure of melittin tetramer. These results show that protein side-chain motion, even of moieties as large as indole, can occur on the picosecond time scale and that these motions are reasonably similar to those inferred from molecular dynamics simulations.     

Fluctuation methods to study protein aggregation in live cells: concanavalin A oligomers formation

Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging.     

Melittin induces HII phase formation in cardiolipin model membranes

The interaction of melittin with bovine heart cardiolipin model membranes was investigated via binding assays, 31P-NMR, freeze-fracture electron microscopy, small angle X-ray diffraction and fluorescence based fusion assays. A strong binding (Kd less than 10(-7) M) appeared to be accompanied by the formation of large structures, resulting from a fusion process of extremely fast initial rate. As the melittin content is increased, bilayer structure is gradually lost and from a cardiolipin to melittin ratio of about 6 the lipid starts to organize itself in an hexagonal HII phase. At lower temperatures (T less than 40 degrees C) the coexistence of another structure is observed, characterized by a broad isotropic 31P-NMR signal and giving rise to sharp X-ray reflections, most probably a cubic phase, as suggested also be freeze-fracture images, showing orderly stacked particles. The results are discussed in relation to contrasting observations on the structural changes induced by melittin in the zwitterionic phospholipid system of dipalmitoylphosphatidylcholine (Dufourcq. J. et al. (1986) Biochim. Biophys. Acta 859, 33-48). The biological relevance of the observations with respect to the process of protein insertion into membranes is indicated.