Major venom proteins of the fire ant Solenopsis invicta: insights into possible pheromone-binding function from mass spectrometric analysis.

Proteins in the venom of the fire ant Solenopsis invicta have been suggested to function in pheromone binding. Venom from queens and workers contains different isoforms of these proteins, consistent with the differing pheromones they secrete, but questions remain about the venom protein composition and glandular source. We found that the queen venom contains a previously uncharacterized pheromone-binding protein paralogue known as Sol i 2X1. Using imaging mass spectrometry, we located the main venom proteins in the poison sac, implying that pheromones might have to compete with venom alkaloids for binding. Using the known structure of the worker venom protein Sol i 2w, we generated three-dimensional homology models of the worker venom protein Sol i 4.02, and of the two main venom proteins in queens and female alates, Sol i 2q and Sol i 2X1. Surprisingly, the models show that the proteins have relatively small internal hydrophobic binding pockets that are blocked by about 10 amino acids of the C-terminal region. For these proteins to function as carriers of hydrophobic ligands, a conformational change would be required to displace the C-terminal region, somewhat like the mechanism known to occur in the silk moth pheromone-binding protein.

Quantitative analysis of pheromone-binding protein specificity.

Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins, using ß-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila odorant-binding protein that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in Escherichia coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between ß-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the ß-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ∼100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ∼200 nM for the silk moth pheromone bombykol and ∼90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the Laughlin, Ha, Jones and Smith model of pheromone reception are discussed.

The major antennal chemosensory protein of red imported fire ant workers.

Some chemosensory proteins (CSPs) are expressed in insect sensory appendages and are thought to be involved in chemical signalling by ants. We identified 14 unique CSP sequences in expressed sequence tag (EST) libraries of the red imported fire ant, Solenopsis invicta. One member of this group (Si-CSP1) is highly expressed in worker antennae, suggesting an olfactory function. A shotgun proteomic analysis of antennal proteins confirmed the high level of Si-CSP1 expression, and also showed expression of another CSP and two odorant-binding proteins (OBPs). We cloned and expressed the coding sequence for Si-CSP1. We used cyclodextrins as solubilizers to investigate ligand binding. Fire ant cuticular lipids strongly inhibited Si-CSP1 binding to the fluorescent dye N-phenyl-naphthylamine, suggesting cuticular substances are ligands for Si-CSP1. Analysis of the cuticular lipids showed that the endogenous ligands of Si-CSP1 are not cuticular hydrocarbons.

Water and carboxyl group environments in the dehydration blueshift of bacteriorhodopsin.

The proton channels of the bacteriorhodopsin (BR) proton pump contain bound water molecules. The channels connect the purple membrane surfaces with the protonated retinal Schiff base at the membrane center. Films of purple membrane equilibrated at low relative humidity display a shift of the 570 nm retinal absorbance maximum to 528 nm, with most of the change occurring below 15% relative humidity. Purple membrane films were dehydrated to defined humidities between about 50 and 4.5% and examined by Fourier transform infrared difference spectroscopy. In spectra of dehydrated-minus-hydrated purple membrane, troughs are observed at 3645 and 3550 cm-1, and peaks are observed at 3665 and 3500 cm-1. We attribute these changes to water dissociation from the proton uptake channel and the resulting changes in hydrogen bonding of water that remains bound. Also, in the carboxylic acid spectral region, a trough was observed at 1742 cm-1 and a peak at 1737 cm-1. The magnitude of the trough to peak difference between 1737 and 1742 cm-1 correlates linearly with the extent of the 528 nm pigment. This suggests that a carboxylic acid group or groups is undergoing a change in environment as a result of dehydration, and that this change is linked to the appearance of the 528 nm pigment. Dehydration difference spectra with BR mutants D96N and D115N show that the 1737-1742 cm-1 change is due to Asp 96 and Asp 115. A possible mechanism is suggested that links dissociation of water in the proton uptake channel to the environmental change at the Schiff base site.

Transmembrane and water-soluble helix bundles display reverse patterns of surface roughness.

Amino acid exposure and surface roughness were calculated for 12 helices from three transmembrane alpha-helix bundles and 13 helices from seven water-soluble alpha-helix bundles. Transmembrane helix bundles have relatively rough surfaces exposed to the lipid bilayer hydrocarbon chains and relatively smooth surfaces along helix-helix interfaces. This pattern is the reverse of what occurs in water-soluble helix bundles, where relatively rough surfaces are at the helix-helix interfaces and relatively smooth surfaces are exposed to water. The relatively rough exposed surfaces and buried smooth surfaces of transmembrane helices are likely to contribute to the stability of transmembrane helical bundles in a phospholipid environment.

Conformational change in bacterio-opsin on binding to retinal.

The detailed mechanism of retinal binding to bacterio-opsin is important to understanding retinal pigment formation as well as to the process of membrane protein folding. We have measured the temperature dependence of bacteriorhodopsin formation from bacterio-opsin and all-trans retinal. An Arrhenius plot of the apparent second-order rate constants gives an activation energy of 11.6 +/- 0.7 kcal/mol and an activation entropy of -4 +/- 2 cal/mol deg. Comparison of the activation entropy to model compound reactions suggests that chromophore formation in bacteriorhodopsin involves a substantial protein conformational change. Cleavage of the polypeptide chain between residues 71 and 72 has little effect on the activation energy or entropy, indicating that the connecting loop between helices B and C is not involved in this conformational change.

Guanidinium restores the chromophore but not rapid proton release in bacteriorhodopsin mutant R82Q.

Replacement of the Arg residue at position 82 in bacteriorhodopsin by Gln or Ala was previously shown to slow the rate of proton release and raise the pK of Asp 85, indicating that R82 is involved both in the proton release reaction and in stabilizing the purple form of the chromophore. We now find that guanidinium chloride lowers the pK of D85, as monitored by the shift of the 587-nm absorbance maximum to 570 nm (blue to purple transition) and increased yield of photointermediate M. The absorbance shift follows a simple binding curve, with an apparent dissociation constant of 20 mM. When membrane surface charge is taken into account, an intrinsic dissociation constant of 0.3 M fits the data over a range of 0.2-1.0 M cation concentration (Na+ plus guanidinium) and pH 5.4-6.7. A chloride counterion is not involved in the observed spectral changes, as chloride up to 0.2 M has little effect on the R82Q chromophore at pH 6, whereas guanidinium sulfate has a similar effect to guanidinium chloride. Furthermore, guanidinium does not affect the chromophore of the double mutant R82Q/D85N. Taken together, these observations suggest that guanidinium binds to a specific site near D85 and restores the purple chromophore. Surprisingly, guanidinium does not restore rapid proton release in the photocycle of R82Q. This result suggests either that guanidinium dissociates during the pump cycle or that it binds with a different hydrogen-bonding geometry than the Arg side chain of the wild type.

Effect of transmembrane helix packing on tryptophan and tyrosine environments in detergent-solubilized bacterio-opsin.

Bacterio-opsin (bO) is folded in a nearly native conformation in mixed micelles of dimyristoyl phosphatidyl choline (DMPC) and 3-[(3-cholamidopropyl)-dimehtylamonio]-1-propane sulfonic acid (CHAPS), but bO is partially unfolded in sodium dodecyl sulfate (SDS). UV difference spectroscopy was used to study the changes in environment of bO aromatic amino acid side chains that occur upon partial unfolding. The UV difference spectra of peptides in CHAPS/DMPC minus peptides in SDS were measured for bO and the following subfragments of bO: C1 (residues 72-248), C2 (1-71), V1 (1-166), V2 (167-248), CB7 (119-145), CB9 (164-209), and CB10 (72-118). The spectra show that, in partially unfolded bO in SDS, the Tyr and Trp absorbance is blue-shifted. The difference spectra were compared to solvent perturbation difference spectra of N-acetyl-L-tyrosine ethyl ester and N-acetyl-L-tryptophanamide. The exposure change calculated from the difference spectra was found to correlate with the change in the number of van der Waals contacting atoms upon partial unfolding, and also with the number of transmembrane helical segments. This result suggests a simple experimental method of testing helix packing arrangements derived from hydropathy plots and model building.

Long-range effects on the retinal chromophore of bacteriorhodopsin caused by surface carboxyl group modification.

Carboxyl groups of bacteriorhodopsin (bR) that are modified by 1-ethyl-3-[3-(trimethylamino)-propyl]carbodiimide (ETC) have been identified. Reaction of deionized purple membrane with a 400-fold molar excess of ETC or [14C]ETC for 1 h at 0 degree C incorporates about 3.5 mol of ETC/mol of bR. Proteinase K cleavage of ETC-modified bacterioopsin (bO) produced small 14C-labeled peptides. Amino acid sequence analysis showed three major ETC-modified residues: Glu 234, Asp 38, and Glu 74. Proteolysis of purple membrane with papain removes the ETC site at Glu 234. Treatment of ETC-modified, papain-cleaved purple membrane with hydroxylamine removes half of the remaining ETC label. Subsequent cleavage with chymotrypsin, followed by amino acid sequence analysis, revealed that most of the remaining label was at Glu 74. bR modified by ETC primarily at Glu 74 displays two alterations in the retinal chromophore, located in the membrane interior at a distance more than 2 nm away from the modified carboxyl group. (1) The acid-induced purple-to-blue transition undergoes a shift in apparent pK from 3.2 to 2.3. (2) The second-order rate constant for chromophore regeneration from bO and retinal is diminished from 3600 to 1700 M-1 s-1 in membrane sheets. Most of the shift in the pK of the purple-to-blue transition can be explained by the quaternary ammonium ion of ETC attached to Glu 74 overlapping the postulated location of the guanidinium group of Arg 82.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta IV is the major beta-tubulin isotype in bovine cilia.

Four different isotypes of beta-tubulin are known to be expressed in mammalian brain. Monoclonal antibodies against beta II, beta III, and beta IV were used to characterize the beta-tubulin isotypes in two ciliated bovine tissues: non-motile sensory cilia of retinal rod cells and motile cilia of tracheal epithelium. Retinal rod outer segment (ROS) connecting cilia and cytoskeletons were purified by density gradient centrifugation. This preparation contained more than 20 major protein components, as shown by dodecyl sulfate polyacrylamide gel electrophoresis. Electroblots were used to quantitate the relative amounts of beta II, beta III, and beta IV. The connecting cilium and cytoskeleton of the rod outer segment has less type III beta-tubulin than brain and more type IV. The ratio of beta IV to beta II in the ROS is nearly a factor of 8 larger than in brain. Electron microscopic immunocytochemistry showed extensive labeling of cilia by anti-type IV in thin sections of retinas and trachea, and also in purified ROS cilia and cytoskeletons. Labeling of cilia by anti-beta II was also observed, although in the purified ROS cilia and cytoskeleton, the anti-beta II labeling was primarily on amorphous non-ciliary material. The results suggest that both motile and non-motile cilia are enriched in the type IV beta-tubulin subunit.

Regeneration of bacteriorhodopsin in mixed micelles.

Regeneration of bacteriorhodopsin from bacterioopsin and all-trans-retinal was studied in a mixed micelle system consisting of dodecyl sulfate, CHAPS and a water-soluble phospholipid dihexanoylphosphatidylcholine (hex2-PhosChol). Regeneration to approximately 40,000 M-1.cm-1 extinction at 550 nm (epsilon 550) was obtained with either 2.3 mM or 6.5 mM CHAPS along with 6.9 mM dodecyl sulfate and 4.5 mM hex2-PhosChol in 0.16 M NaCl and 40 mM phosphate (pH 6.0). Without CHAPS, the regeneration in 4.5 mM Hex2-PhosChol gave epsilon 555 = 27,800; without PhosChol, the 1:3 CHAPS/dodecyl sulfate mixture gave epsilon 550 approximately 20,000; and without PhosChol the nearly equimolar CHAPS/dodecyl sulfate mixture gave epsilon 550 approximately 10,000. The composition of the mixed micelles was estimated from fluorescence spectroscopy using pyrene butyryl hydrazine. The molecular weight was estimated by molecular seive chromatography to be 87,100 for 2.3 mM CHAPS, 6.9 mM dodecyl sulfate and 0.67 mM hex2-PhosChol; and 83,200 for 7.0 mM CHAPS, 6.9 mM dodecyl sulfate, and 1.1 mM hex2-PhosChol. These results are consistent with the idea that at low concentrations of CHAPS and dodecyl sulfate, CHAPS organizes the dodecyl sulfate into disk shaped bilayer micelles that are favorable for bacterioopsin refolding. However, a high concentration of either detergent inhibits regeneration. Added hex2-PhosChol can overcome the inhibitory effects of high concentrations of either CHAPS or dodecyl sulfate.

Control of bacteriorhodopsin color by chloride at low pH. Significance for the proton pump mechanism.

The chromophore of bacteriorhodopsin undergoes a transition from purple (570 nm absorbance maximum) to blue (605 nm absorbance maximum) at low pH or when the membrane is deionized. The blue form was stable down to pH 0 in sulfuric acid, while 1 M NaCl at pH 0 completely converted the pigment to a purple form absorbing maximally at 565 Other acids were not as effective as sulfuric in maintaining the blue form, and chloride was the best anion for converting blue membrane to purple membrane at low pH. The apparent dissociation constant for Cl- was 35 mM at pH 0, 0.7 M at pH 1 and 1.5 M at pH 2. The pH dependence of apparent Cl- binding could be modeled by assuming two different types of chromophore-linked Cl- binding site, one pH-dependent. Chemical modification of bacteriorhodopsin carboxyl groups (probably Asp-96, -102 and/or -104) by 1-ethyl-3-dimethlyaminopropyl carbodiimide, Lys-41 by dansyl chloride, or surface arginines by cyclohexanedione had no effect on the conversion of blue to purple membrane at pH 1. Fourier transform infrared difference spectroscopy of chloride purple membrane minus acid blue membrane showed the protonation of a carboxyl group (trough at 1392 cm -1 and peak at 1731 cm -1). The latter peak shifted to 1723 cm -1 in D2O. Ultraviolet difference spectroscopy of chloride purple membrane minus acid blue membrane showed ionization of a phenolic group (peak at 243 nm and evidence for a 295 nm peak superimposed on a tryptophan perturbation trough). This suggests the possibility of chloride-induced proton transfer from a tyrosine phenolic group to a carboxylate side-chain. We propose a mechanism for the purple to acid blue to chloride purple transition based on these results and the proton pump model of Braiman et al. (Biochemistry 27 (1988) 8516-8520).

Resolution of ion translocating proteolipid subclasses active in bacterial calcification.

Formation of calcium hydroxyapatite occurs on membrane surfaces via interaction of calcium, inorganic phosphate, phospholipids, calcifiable proteolipids, and ion flux to and from the nucleating site. Recently, this laboratory reported that proteolipids from the calcifying bacterium, Bacterionema matruchotti, act as an ionophore when reconstituted into bacteriorhodopsin-proteoliposomes. This ionophoric activity is blocked by [14C]dicyclohexylcarbodiimide ([14C]DCCD). SDS-PAGE shows that [14C]DCCD binds to a single band of Mr 8500. To determine whether proteins other than the [14C]DCCD-binding protein are involved, we examined the function of proteolipid species extracted by solvents of differing polarity. Proteolipids were isolated independently from chloroform:methanol (2:1) and chloroform:methanol:HCl (200:100:1) extracts of the bacteria by Sephadex LH-20 chromatography and were electrophoresed on 12.5% acrylamide gels. The chloroform:methanol extract contained a major hand at Mr 10,000 that was not present in gels of proteolipid isolated by acidified solvent. Proteolipids extracted in chloroform:methanol:HCl included a broad band at Mr 8500, which co-migrated with the [14C] DCCD-binding protein. The rate and extent of proton translocation were not altered when either proteolipid extract was added individually to bacteriorhodopsin proteoliposomes. However, when proteolipids isolated from the chloroform:methanol and chloroform:methanol:HCl extracts were combined, the rate and extent of translocation were increased. These data demonstrate that at least two proteolipid proteins are necessary for ionophoric activity, the Mr 10,000 protein isolated by chloroform:methanol 2:1 and the [14C]DCCD-binding protein requiring acidified solvent for extraction.

Altered protein-chromophore interaction in dicyclohexylcarbodiimide-modified purple membrane sheets.

Previous studies of N,N'-dicyclohexylcarbodiimide (DCCD)-modified bacteriorhodopsin (Renthal, R. et al. (1985) Biochemistry 24, 4275-4279) used reaction conditions (detergent micelles) that are not optimal for subsequent physical studies. The present work describes new conditions for reaction of bacteriorhodopsin with DCCD in intact purple membrane sheets in the presence of 4.5% (v/v) diethylether and light. Like the detergent reaction system, the reaction is light induced, incorporates approximately 1 mol [14C]DCCD per mol bacteriorhodospin, and results in a bleached chromophore. Peptide mapping indicates that the likely site of modification in intact membranes is identical to the site in the detergent reaction system: Asp 115. The retinal chromophore of DCCD-modified purple membrane has an absorbance maximum at 390 nm and very little induced circular dichroism. The retinal is easily extracted in hexane, yielding a 3:1 ratio of all-trans to 13-cis retinal. Borohydride reduces the retinal onto the protein within the 1-71 region of the amino acid sequence. These results suggest that Asp-115 is near the retinal binding cavity of bacteriorhodopsin. When DCCD reacts with Asp 115, retinal is displaced from its binding site.

Fluorescent labeling of bacteriorhodopsin: implications for helix connections.

Purple membrane from Halobacterium halobium was reacted with dansyl (5-dimethylamino-1-naphthalenyl fluorescent labels that have specificity for different protein side chains of bacteriorhodopsin. Dansyl chloride was found to react primarily with Lys-41. Dansyl hydrazine was coupled, with water-soluble carbodiimide, to Glu-74 and/or Asp-85, which was the major modified site after papain-cleavage of the carboxyl-terminal 17 amino acids. Fluorescence energy transfer was used to probe the proximity of the modified sites to the retinal chromophore of bacteriorhodopsin. The dansyl group on Lys-41 was greater than 2.99 nm from retinal, while the dansyl group on Glu-74/Asp-85 was greater than 2.10 nm from retinal. Information available on the location of retinal in the transmembrane profile and probable surface locations of the fluorescent labels was combined with the energy transfer results to calculate distances projected in the plane of the membrane. The projected distances to retinal were 1.64 nm (Lys-41) and 1.65 nm (Gly-74). These measurements, combined with many other labeling experiments that have been reported, restrict the number of likely helix-connection models to only three: EDCABGF, FEDCBAG and FGEABDC (in the nomenclature of Engelman et al. (1980) Proc. Natl. Acad. Sci. USA 77, 2023-2027).

Kinetic comparisons of amniotic fluid inactive renin and renal renin using synthetic and human renin substrates.

Inactive renin has been isolated from pooled amniotic fluid and purified approximately 642-fold. Prior to activation the isolates had approximately 4% of the activity found after activation. The observation is similar to that reported for inactive renin from chorionic cell culture and suggests a placental origin of amniotic fluid inactive renin. Using plasma from an estrogen-treated woman, renin substrate was recovered free of renin and inactive renin and a portion was separated into NMW and HMW components. The NMW form constituted approximately 93% and the HMW form approximately 7% of the renin substrate. Amniotic fluid inactive renin was used for determinations of enzyme-substrate kinetics with the pooled, NMW, and HMW plasma substrate and tetradecapeptide synthetic substrate, and the results were compared to similar determinations using standard renal renin. Using synthetic substrate, the kinetics of renal renin and amniotic fluid inactive renin before and after activation were similar. The kinetics of renal renin with pooled, NMW, and HMW plasma substrate were also similar. Amniotic fluid inactive renin had a lower Km with pooled than with NMW substrate, however, which resulted from a significantly smaller Km with HMW component. Although the affinity constants with pooled substrate were not different for renin and inactive renin, the Km of inactive renin was significantly less with the HMW component of plasma renin substrate. The observations are compatible with a role for placental inactive renin in normal pregnancy and suggest the possibility of a further role in hypertensive pregnancy.

Light activates the reaction of bacteriorhodopsin aspartic acid-115 with dicyclohexylcarbodiimide.

Conditions for a light-induced reaction between the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) and bacteriorhodopsin in Triton X-100 micelles were previously reported [Renthal, R., Dawson, N., & Villarreal, L. (1981) Biochem. Biophys. Res. Commun. 101, 653-657]. We have now located the DCCD site in the bacteriorhodopsin amino acid sequence. [14C]DCCD-bacteriorhodopsin (0.67 mol/mol of bacteriorhodopsin) was cleaved with CNBr. The resulting peptides were purified by gel filtration and reverse-phase high-performance liquid chromatography (HPLC). One major 14C peptide (50%) and two minor fractions were obtained. The modified peptides were completely absent in the absence of DCCD, and 10 times less was obtained when the reaction was run in the dark. Amino acid analysis and sequence analysis showed that the major fraction contained residues 69-118. This region includes six carboxyl side chains. Quantitative sequence analysis ruled out significant amounts of DCCD at Glu-74, Asp-85, Asp-96, Asp-102, and Asp-104. The major 14C peptide was also subjected to pepsin hydrolysis. HPLC analysis of the product gave only a single major radioactive subfragment. Amino acid analysis of the peptic peptide showed that it contained residues 110-118. The only carboxyl side chain in this region is Asp-115. Thus, we conclude that Asp-115 is the major DCCD site. The light sensitivity of this reaction suggests that Asp-115 becomes more exposed or that its environment becomes more acidic during proton pumping. The DCCD reaction blue-shifts the retinal chromophore. Such a result would be expected if Asp-115 is the negative point charge predicted to be near the cyclohexene ring of retinal.