Biomedical subjects

Z T Farahbakhsh

Publications and source records attributed to Z T Farahbakhsh.

13 recordsLinked to original sources

PubMed1996

Structure and function in rhodopsin. Cysteines 65 and 316 are in proximity in a rhodopsin mutant as indicated by disulfide formation and interactions between attached spin labels.

To probe proximity relationships between different amino acids in the interhelical loops in the cytoplasmic domain of rhodopsin, we are using a general approach in which two cysteine residues are introduced at different locations. Here we report on the characteristics of one such mutant that contains the naturally occurring cysteine 316 near the cytoplasmic end of helix G and a second cysteine at position 65 (H65C), near the cytoplasmic end of helix A. The mutant protein after expression in COS-1 cells and reconstitution with 11-cis-retinal can be bound to anti-rhodopsin antibody 1D4-Sepharose at pH 6 in a form that contains the two cysteines in the free sulfhydryl form. In this form, the mutant protein reacts as expected with N-ethylmaleimide in the dark at room temperature and can be derivatized with nitroxide spin labels. However, under appropriate conditions, the mutant can be isolated with the cysteines in the disulfide form, which has been characterized by analysis of fragments produced on proteolysis with thermolysin. A study of the interactions between nitroxide spin labels attached to the two cysteine residues in the mutant protein indicates that in the dark state they are within about 10 A of each other. On illumination the distance between the spin labels increases. Collectively, the above results show that, upon folding of the mutant opsin in vivo, cysteines 65 and 316, and by inference, helices A and G, are in proximal locations and move further apart upon photoactivation.

Amino Acid Sequence↗

PubMed1996

Structural features and light-dependent changes in the cytoplasmic interhelical E-F loop region of rhodopsin: a site-directed spin-labeling study.

Thirty consecutive single cysteine substitution mutants in the amino acids Q225-I256 of bovine rhodopsin have been prepared and modified with a sulfhydryl specific nitroxide reagent. This sequence includes the E-F interhelical loop, a transducin interaction site. The accessibilities of the attached nitroxides to collisions with hydrophilic and hydrophobic paramagnetic probes in solution were determined, and the electron paramagnetic resonance spectra analyzed in terms of side chain mobility, both in the dark and after photoactivation. Accessibility cata shows that the rhodopsin polypeptide chain crosses an aqueous/ hydrophobic boundary in the range V227-K231 and again in the range V250-V254. In the hydrophobic segments, both the accessibility and mobility data are consistent with helical structures. In the regions of the sequence located within the aqueous phase, periodic variation in both accessibility and mobility of the spin-labeled side chains indicates that the E-F interhelical loop is largely alpha-helical, being formed by regular extensions of the E and F helices by about 1.5 and 3 turns, respectively. Judging from nitroxide mobilities, the putative extension of helix E in the aqueous phase is more dynamic than that of helix F. Changes in the electron paramagnetic resonance characteristics of the spin-labeled rhodopsin upon photoactivation indicate that chromophore isomerization results in patterns of structural changes that can be interpreted in terms of movements of helices that extend into the aqueous loop regions.

Amino Acid Sequence↗

PubMed1995

Mapping light-dependent structural changes in the cytoplasmic loop connecting helices C and D in rhodopsin: a site-directed spin labeling study.

All 20 single cysteine substitution mutants in the sequence Y136-M155 of bovine rhodopsin have been prepared and modified with a sulfhydryl-specific nitroxide reagent. This sequence contains the C-D interhelical loop, a transducin interaction site. The accessibilities of the attached nitroxides to collisions with paramagnetic probes in solution were determined, and the electron paramagnetic resonance spectra were analyzed, both in the dark and after photoexcitation. Accessibility data show that the rhodopsin polypeptide crosses an aqueous/hydrophobic boundary near V138 and H152. The nitroxide mobilities inferred from the spectra are consistent with a model where the C helix extends to at least residue C140, with much of the helix surface in contact with protein rather than lipid near the cytoplasmic surface of the membrane. Upon photoexcitation, electron paramagnetic resonance spectral changes are observed at sites on the putative C helix surface that are in contact with the protein and at specific sites in the C-D interhelical loop. A simple interpretation of these results is that photoexcitation involves a rigid body movement of the C helix relative to the others in the helix bundle.

Amino Acid Sequence↗

PubMed1995

Interaction of alpha-crystallin with spin-labeled peptides.

alpha-Crystallin is a major protein of the vertebrate lens once thought to be highly specialized for conferring transparency. However, recent work has revealed a wide tissue distribution and a sequence homology to small heat shock proteins, suggesting a more general role for the protein. Like other molecular chaperons, alpha-crystallin is known to bind to unfolded proteins and suppress nonspecific aggregation in vitro. In the present work, spin-labeled derivatives of the insulin B chain and melittin were used to investigate the state of these proteins bound to alpha-crystallin. Insulin was selected since unfolding can be triggered by reduction of the interchain disulfide bonds, a treatment that does not affect alpha-crystallin. Upon reduction of insulin, the separated B chains aggregate. In the presence of alpha-crystallin, the B chains bind to alpha-crystallin and aggregation is suppressed. Melittin, a 26 amino acid peptide from bee venom, was selected for study since it is a random coil under physiological conditions, and its interaction with alpha-crystallin can be directly studied. EPR analysis of the spin-labeled peptides shows that the nitroxide side chains are immobilized in a polar environment on alpha-crystallin and that they are separated by 25 A or more in the complex, indicating that the bound proteins are not clustered. The bound B chains of insulin are not in a fully extended conformation, and melittin does not appear to bind to a hydrophobic surface in alpha-crystallin as an amphipathic helix, as it does to membranes and some other proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallins↗

PubMed1995

Tear lipocalins bind a broad array of lipid ligands.

To identify the native ligands of tear lipocalins, tear proteins were separated by size exclusion chromatography and the lipid content in the major protein fractions identified. Lipids extracted from native tears and purified tear lipocalins comigrated with fatty acids, fatty alcohols, phospholipids, glycolipids, and cholesterol on thin layer chromatograms. Abundant stearic and palmitic acids as well as cholesterol, and lesser amounts of lauric acid were specifically identified in extracts of purified lipocalins by gas chromatography-mass spectroscopy. A preliminary study of the ligand-protein interaction was carried out using nitroxide spin-labeled lipids.

Carrier Proteins↗

PubMed1993

Photoactivated conformational changes in rhodopsin: a time-resolved spin label study.

Rhodopsin has been selectively spin-labeled near the cytoplasmic termini of helices C and G. Photoactivation with a light flash induces an electron paramagnetic resonance spectral change in the millisecond time domain, coincident with the appearance of the active metarhodopsin II intermediate. The spectral change is consistent with a small movement near the cytoplasmic termination of the C helix and reverses upon formation of the MIII state. These results provide an important link between the optical changes associated with the retinal chromophore and protein conformational states.

Amino Acid Sequence↗

PubMed1993

Formation of the meta II photointermediate is accompanied by conformational changes in the cytoplasmic surface of rhodopsin.

Five mutations of rhodopsin have been produced, each of which contains a unique cysteine residue at positions 62, 65, 140, 240, or 316 in the cytoplasmic domain. The single reactive cysteines were derivatized with a sulfhydryl-specific nitroxide spin-label, and the electron paramagnetic resonance (EPR) spectra were analyzed in both lauryl maltoside and digitonin in the dark and after photobleaching. The collision rate of the attached nitroxides with polar and nonpolar paramagnetic agents indicated that they were all exposed to the aqueous environment. Photobleaching of the mutants in digitonin, which arrests the protein at the meta I intermediate, produced little change in mobility of the attached nitroxide. On the other hand, photobleaching in lauryl maltoside produced the meta II intermediate and significant changes in the EPR spectra of the nitroxides attached to positions 140 and 316. These data directly reveal a light-induced conformational change in the cytoplasmic loops that accompanies meta II formation.

Amino Acid Sequence↗

PubMed1992

Spin labeled cysteines as sensors for protein-lipid interaction and conformation in rhodopsin.

In stoichiometric amounts, the spin label N-tempoyl-(p-chloromercuribenzamide) reacts rapidly with one cysteine residue in membrane-bound bovine rhodopsin. This residue is distinct from the two reactive cysteines previously used as attachment sites for spectroscopic labels, and is on the external surface of the protein near the cytoplasmic membrane/aqueous interface. The spin-labeled side chain has revealed a light-induced conformational change in membrane-bound rhodopsin that is apparently not associated with protein aggregation. The changes are reversible upon the addition of 11-cis retinal, and the magnitude of the change is dependent on the identity of the phospholipid in the surrounding bilayer. Alteration of lipid composition has a much larger effect on bleached rhodopsin than rhodopsin itself, indicating that the former is more readily deformable in response to changes in bilayer properties. This is consistent with the loss of 11-cis retinal binding energy in opsin compared to rhodopsin. These results provide direct structural evidence that the conformation of a membrane protein can be modulated by the lipid properties.

Animals↗

PubMed1989

The acid-triggered entry pathway of Pseudomonas exotoxin A.

In this study we examined the pH requirements and reversibility of early events in the Pseudomonas toxin entry pathway, namely, membrane binding, insertion, and translocation. At pH 7.4, toxin binding to vesicles and insertion into the bilayer are very inefficient. Decreasing the pH greatly increases the efficiencies of these processes. Acid-treated toxin exhibits pH 7.4 binding and insertion levels. This indicates that hydrophobic regions that become exposed upon toxin acidficiation become buried again when the pH is raised to 7.4. In contrast, the change in toxin conformation that occurs upon membrane binding is irreversible. Returning samples to pH 7.4, incubation with excess toxin, or dilution with buffer up to 1000-fold leads to very little loss of bound toxin. Bound toxin exhibits an extremely high susceptibility to trypsin compared to free toxin (at both pH 4 and pH 7.4). At pH 4, membrane-associated toxin slowly proceeds to a trypsin-protected state; neutralization halts this process. At low pH, toxin was found to bind and insert into DMPC vesicles very efficiently at temperatures both above and below 23 degrees C, the lipid melting point. With fluid targets, the proportion of bound toxin that was photolabeled from within the bilayer peaked rapidly and then decreased with time. With frozen targets, the efficiency of photolabeling peaked but then remained fairly constant.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP Ribose Transferases↗

PubMed1987

Effect of low pH on the conformation of Pseudomonas exotoxin A.

Previously we examined factors involved in the entry mechanism of Pseudomonas exotoxin A (PTx) at the level of lipid-protein interactions (Farahbakhsh, Z. T., Baldwin, R. L., and Wisnieski, B. J. (1986) J. Biol. Chem. 261, 11404-11408). Exposure to a low pH environment appears to be an obligatory trigger of the entry pathway. In this report we describe the effect of pH upon the conformation of PTx. We have found that the intrinsic fluorescence of PTx is strongly dependent on pH, decreasing between pH 7.4 and 4.0 with a red shift in the emission lambda max. The changes are reversible and associated with the acquisition of a binding site for the fluorescent dye 1-anilino-8-naphthalenesulfonic acid (ANS). The fluorescence intensity of ANS in the presence of PTx increases with decreasing pH and is accompanied by a blue shift in emission spectra, indicative of exposure of hydrophobic surfaces. These changes are also reversible. Both the intrinsic fluorescence and ANS binding profiles show a dramatic dependence on pH, with the transitions centered on pH 5.0 and 4.5, respectively. Circular dichroism studies reveal a 9% decrease in alpha-helicity between pH 7.7 and 4. The susceptibility of toxin to trypsin cleavage is also a function of pH, increasing with decreasing pH. The pH 7.4 cleavage profile is regained when the acid-treated samples are brought back to pH 7.4. The conformational changes observed in these pH shift experiments are likely to be physiologically significant because the conditions closely resemble those that the toxin would encounter if entry into the cytoplasm of a cell involves escape from an endosomal compartment.

ADP Ribose Transferases↗

PubMed1986

Pseudomonas exotoxin A. Membrane binding, insertion, and traversal.

Using vesicle targets composed of phosphatidylcholine and cholesterol (1:1 molar ratio), we found that Pseudomonas aeruginosa exotoxin A (PTx) binding and insertion are not only dependent on pH (Zalman, L.S., and Wisnieski, B.J. (1985) Infect. Immun. 50, 630-635) but also on ionic strength, reaching a maximum in pH 4 buffer that contains 150-200 mM NaCl. Insertion was monitored by photolabeling with an intramembranous probe. Higher levels of binding and insertion were attained with vesicles that contained 2.5 mol% dicetylphosphate than with neutral vesicles. Positively charged vesicles (2.7 mol% stearylamine) were the least effective targets. At pH 7.4, all binding levels were depressed. While PTx binding increased with increasing temperature, the relative proportion of the vesicle-associated toxin that was photolabeled decreased. The most likely explanation for the decrease is that the bilayer translocation rates increased with increasing temperature, and hence fewer PTx molecules were accessible at the time of photolabeling. At 37 degrees C, binding and insertion both plateaued within 10 min of lowering the pH to 4. After 10 min, the amount of bound toxin decreased slightly with time but there was a dramatic decrease in photolabeling, indicating that inserted PTx had begun to cross the bilayer. This was verified by the finding that when PTx was incubated with vesicles that contained trypsin, cleavage occurred only in those samples in which the pH was shifted down to pH 4. Entry is triggered by an acid-induced conformational change that promotes productive binding and insertion. After insertion, the kinetics of membrane traversal appear to be regulated by the physical properties of the bilayer.

ADP Ribose Transferases↗

PubMed1986

Enzymatic reconstitution of brain membrane and membrane opiate receptors.

A new method using lysophosphatide and acyl-CoA as detergents has been used to solubilize the rat brain opiate receptor. After solubilization, lysophosphatide and acyl-CoA can be almost completely removed by an enzymatic reaction that uses an acyltransferase from rat liver microsomes and reconstitutes the solubilized receptor in membranous vesicles. Morphological studies performed with negative staining and freeze-fracture electron microscopy revealed that the general appearance and intramembrane particle distribution of fracture faces in the reconstituted membrane are similar to those of the native membrane; this indicates that hydrophobic protein components of the original membrane were incorporated during reconstitution. Reconstituted membrane, however, contained higher levels of phosphatidylcholine and lower levels of cholesterol. The activities of the membrane-bound enzymes Na+, K+-ATPase and Ca2+, Mg2+-ATPase in the reconstituted system were 24 and 3%, respectively, those of the native membrane. Although binding of opiate ligands to the reconstituted membrane was stereospecific and saturable, higher concentrations of some of the unlabeled ligands were required to inhibit binding of the radiolabeled ligands. These changes in receptor characteristics are likely due to changes in lipid composition, physical state, and/or distribution of the lipids in the reconstituted membrane bilayer. This conclusion is supported by an increase in the affinity of opiate ligands for reconstituted membrane after adjustment of the latter's lipid composition to match more closely that of the original membrane. This was accomplished by treatment with phospholipid exchange protein to remove the excess phosphatidylcholine and by incorporation of cholesterol into the reconstituted membrane.

Acyltransferases↗

PubMed1984

Age-related decrement in hormone-stimulated lipolysis.

The ability of hormones to activate responses in a variety of tissues decreases with age. The mechanism(s) responsible for these alterations are unclear. We have confirmed that the ability of a beta-adrenergic receptor agonist to activate lipolysis in isolated rat adipocytes decreases with age. Maximum response to isoproterenol was greater in 2-mo-old rats (600 +/- 30 nmol of glycerol released/10(5) cells per h) than 12-mo-old rats (250 +/- 25 nmol/10(5) cells per h), P less than 0.001. Similarly, ACTH is less effective in activating lipolysis in the adipocytes from the older rats. However, the cAMP analogue 8-(4-chlorophenothio)adenosine 3',5'-monophosphate cyclic activated lipolysis equally in the two groups, suggesting that the deficit in adipocytes from the older rats was proximal to cAMP-dependent protein kinase activation. Both isoproterenol and ACTH were significantly less effective in promoting cAMP accumulation in adipocytes isolated from 12-mo-old rats. There was no difference in phosphodiesterase activity of the adipocytes between the two groups. beta-Adrenergic receptors were measured using the antagonist radioligand [125I]cyanopindolol. The number of beta-adrenergic receptors was actually increased in the adipocytes from 12-mo-old rats (26,000 +/- 2,600 receptors/cell) compared with cells from 2-mo-old rats (7,200 +/- 1,300 receptors/cell). The results suggest that diminished cAMP production is responsible for the diminished lipolytic response in the adipocytes of older rats. The mechanism responsible for this change is uncertain but cannot be explained by a loss in beta-adrenergic receptors.

Adipose Tissue↗