30 research outputs found
Intramolecular remote functionalisation of steroids by benzophenone- increased specificity by solvent-induced hydrophobic interactions
Proximity of reactant sites is one of the major factors that contributes to specificity and high reaction rates observed in enzyme catalysis. Enzymes achieve this proximity between the reactant sites by having high affinity for the substrate. Structural studies on enzyme-substrate complexes provide sufficient evidence in this context and indicate that weak bonding interaction are involved in formation of such complexes. We have exploited the hydrophobic interaction between cholesterol and benzophenone to carry out photoinduced remote functionalisation of cholesterol at specific sites. Thus, using polar solvents intramolecular hydrophobic interaction between cholesterol and benzophenone permitted exclusive functionalisation of ring D in cholesterol. The current study indicates that weak interactions between the reactants can be used to bring them in proximity and photochemical reactions can provide the method for functionalising even inert sites like C-H bonds
Design and synthesis of new water soluble forskolin derivatives for positive inotropic activity
979-98
Cholesterol and phospholipids in membranes: the hydrogen bonding problem
Cholesterol and phospholipids are major components of biological membranes. The role of cholesterol in membranes is not metabolic and is known to be a regulator of membrane fluidity which in turn regulates various biological phenomena. We have studied the nature of cholesterol and phospholipid interaction in artificial membranes using 13C nmr spectroscopy. This involved preparation of phospholipids specifically labeled with 13C in the ester carbonyl group. Though the chemical shift data did not provide very useful information the T1 and T2 measurements indicated that previously proposed H bonding between the ester carbonyl group and hydroxyl group of cholesterol seems unprobable
Transverse location of new fluorene based depth dependent fluorescent probes in membranes- quenching studies with 9,10-dibromostearic acid
Fluorescence quenching technique has been used to determine the transverse location of the fluorescent fluorenyl fatty acids in single bilayer vesicles prepared from phosphatidylcholine. The fluorenyl fatty acids used here are 2-fluorenyl acetic, butyric, hexanoic and octanoic acid. In addition a new type of fluorescent probe, 7-n-butyl-fluorene-2-butyric acid, wherein a hydrophobic tail is attached to 2-fluorenyl-butyric acid has also been used to study its effect on alignment of these probes in the membrane. The association properties of the quencher 9,10-dibromostearic acid have been analysed. It is observed that the quencher association involves partitioning into the vesicles and does not involve any binding to the vesicles. The absolute partition coefficient of the 9,10-dibromostearic acid which partitions between the aqueous and the lipid phases of the phospholipid dispersion has been evaluated. Using this information the corrected Stern-Volmer plots were drawn and the bimolecular quenching constant evaluated
Unfolding of diphtheria toxin: identification of hydrophobic sites exposed on lowering of pH by photolabeling
We report here the use of a hydrophobic photoactivable reagent, 2-[3H]diazofluorene (DAF), to map the hydrophobic sites exposed when the pH is lowered in diphtheria toxin (DT). The reagent binds to DT, and on photolysis with light of wavelength >350 nm, it covalently attaches itself to DT. The labeling was observed to increase considerably when the pH was lowered from 7.4 to 5.2. Although both A- and B-chains were labeled to a similar degree at pH 7.4, at lower pH (5.2), B-chain was labeled to a much higher extent. Subsequent chemical and enzymatic fragmentation of DT followed by separation indicated that the putative transmembrane domain was labeled to its maximum extent at pH 5.2, with the bulk of labeling associated with residues 340-459. Protein sequencing analysis indicated that the two buried hydrophobic helices, identified in the crystal structure and suggested to insert and span the membrane bilayer, corresponding to residues 326–347 and 358–376, are strongly labeled. The Pro-345 residue was observed to be labeled maximally at lower pH values. Finally, the DAF labeling pattern indicated that the parent structural motifs are retained at low pH, suggesting that the low pH conformation of DT corresponds to an equilibrium molten globule state
Fluorescence studies on erythrocyte membrane isolated from Plasmodium berghei infected mice
The erythrocyte host cell plays a key role in the well defined developmental stages of the malarial parasite growth and propagation in the erythrocyte cycle of malaria. The host cell serves the parasites by supplying metabolites and removing the catabolites produced by the obligatory parasites. It has been observed that the plasma membrane of the infected cells show a substantially higher fluidity probably due to the depletion of cholesterol content from the host cell. The protein component of the membrane is also modulated due to the insertion of new polypeptides of the parasitic origin, which confers upon it new antigenic properties. We have studied the membrane fraction isolated from mice erythrocytes infected with Plasmodium berghei using fluorescent probes like DPH, ANS and series of fluorenyl fatty acids, which permit depth dependent analysis of membrane. We have observed that there is a marked difference in the fluorescence emission wavelength maximum, the dissociation constant Kd of ANS when bound to normal and infected erythrocytes, though relatively small differences are observed in the fluorescence polarisation values of the two cell types. The fluorenyl fatty acids also show the differences when bound to normal and infected erythrocytes, indicating that either they are in a different environment or they have differing binding properties to the two cell types
Glycyl methylene chemical shift non-equivalence in small peptides. Diasteriomers of Leu-Gly-Phe
Main possibilities for the origin of glycyl methylene chemical shift non-equivalence in Gly-containing small peptides are reviewed together with new data obtained at 300 and 360 MHz 1H-nmr spectroscopy. An unexpected behaviour, both in glycyl methylene chemical shift non-equivalence as in the similarity of mean shift values of these protons in the diasteriomeric tripeptides Leu-Gly-Phe rules out the electric field gradient contributions being of major importance. Changes in conformational features, both of the back-bone as of the side chain rotation in function of pH are revealed, as follows from a study of coupling constants
Growth of a yeast mutant on Ring A modified cholesterol derivatives
The ability of cholesterol derivatives without a hydroxyl group or a side-chain, to support the growth of heme and cyclase deficient Saccharomyces cerevisiae mutant GL 7 was tried and found to be in conformity with the results obtained using liposomes. On the other hand, results with other Ring A modified steroids involving saturation or movement of C5-C6 double bond, or isomeric 3-hydroxy-3-methyl cholestane derivatives, indicated that even minor structural variations can cause considerable changes in their growth supporting potential. The consequence of such structural variations need not be obviated by studies using liposomes or vesicles
Design and synthesis of new fluorescent photoaffinity labels to study membrane structure
Photoaffinity labelling has been used as a technique to study membrane structure. This technique necessitates design and synthesis of suitable carbene and nitrene precursors referred to as photoaffinity (PA) labels. The PA labels should preferably be hydrophobic in nature, photolyse with light of wavelength greater than 300 nm to give reactive intermediates i.e., caroenes which should undergo intermolecular insertion exclusively. The latter reaction, on incorporation of the PA labels in membranes, gives rise to crosslinked products, the analyses of which give useful information on the nature of bio-molecular interaction in membranes. We have prepared diazofluorene and quantitatively studied the products formed on photolysis in polar and nonpolar organic solvents. Products from both singlet and triplet carbenes were observed. Various other analogues like iodo, carboxy and alkyl substituted diazofluorenes have been prepared to get greater extinction coefficients and absorptions above 300 nm. These extrinsic PA probes have been incorporated in artificial membranes and photolysed. The covalently-linked fluorescent products so formed have been analysed. Fatty acids containing the diazofluorene unit have also been synthesized and linked to phospholipids. Studies with these intrinsic photolabelled phospholipids as well as erythrocytes are in progress
Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling
To identify membrane-associated polypeptides present in Torpedo nicotinic acetylcholine receptor (AChR)-rich membranes, we used hydrophobic photolabeling with [3H]diazofluorene ([3H]DAF) and 1-azidopyrene (1-AP) to tag the membrane proteins which were then identified by amino-terminal sequence analysis of labeled fragments isolated from proteolytic digests by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reverse-phase high-performance liquid chromatography. In addition to AChR subunits, identified polypeptides include the 95 kDa α-subunit of the (Na++K+)-ATPase, the 89 kDa voltage-gated chloride channel (CLC-0), the 105 kDa SITS-binding protein, and 32 and 34 kDa polypeptides identified as Torpedo homologues of the mitochondrial membrane ATP/ADP carrier protein and the voltage-dependent anion channel (VDAC), respectively. Further, individual amino acids that reacted with [3H]DAF and therefore likely to be in contact with lipid were identified in the transmembrane segment M3 of the α-subunit of the (Na++K+)-ATPase and in a putative transmembrane β-strand in VDAC. Collectively these results demonstrate that [3H]DAF/1-AP photolabeling provides an effective method for tagging the membrane-associated segments of polypeptides in a way that makes it easy to isolate the labeled polypeptide or polypeptide fragments by fluorescence and then to identify amino acids at the lipid-protein interface by 3H release