Anil K. Lala

Probing the Structure of the Nicotinic Acetylcholine Receptor Ion Channel with the Uncharged Photoactivable Compound [3H]Diazofluorene

The uncharged photoactivable probe 2-[3H]diazofluorene ([3H]DAF) was used to examine structural changes in the Torpedo californica nicotinic acetylcholine receptor (AChR) ion channel induced by agonists. Photoincorporation of [3H]DAF into the AChR consisted of the following two components: a nonspecific component consistent with incorporation into residues situated at the lipid-protein interface, and a specific component, inhibitable by noncompetitive antagonists and localized to the M2 hydrophobic segments of AChR subunits. The nonspecific [3H]DAF incorporation was characterized in the M4 segment of each AChR subunit. The observed distribution and periodicity of labeled residues reinforce the conclusion that the M4 segments are organized as transmembrane α-helices with a common “face” of each helix in contact with lipid. Within the M2 segments, in the absence of agonist [3H]DAF specifically labeled homologous residues βVal-261 and δVal-269, with incorporation into δVal-269 at a 5-fold greater efficiency than into βVal-261. This observation, coupled with the lack of detectable incorporation into α-M2 including the homologous αVal-255, indicates that within the resting channel [3H]DAF is bound with its photoreactive diazo group oriented toward δVal-269. In the presence of agonist, there is an ∼90% reduction in the labeling of βVal-261 and δVal-269 accompanied by specific incorporation into residues (βLeu-257, βAla-258, δSer-262, and δLeu-265) situated 1 or 2 turns of an α-helix closer to the cytoplasmic end of the M2 segments. The results provide a further characterization of agonist-induced rearrangements of the M2 (ion channel) domain of the AChR.

Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus.

The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membrane-bound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of alpha-toxin.

Membrane-protein interaction and the molten globule state: Interaction of α-lactalbumin with membranes

The insertion of soluble proteins into membranes has been a topic of considerable interest. We have studied the insertion of bovineα-lactalbumin into single-bilayer vesicles prepared from egg phosphatidylcholine (PC). Fluoresence studies indicated rapid and tight binding of apo-α-lactalbumin (apo-α-LA) to PC vesicles as a function of pH. The binding was maximal at pH values which favor the formation of the molten globule state. As an increase of hydrophobic surface is observed in the molten globule state, this conformational state can provide a molecular basis for insertion of soluble proteins into membranes. The membrane-bound complex formed at low pH (3.0) could be isolated and was found to be stable at neutral pH. The structural characterization of the apo-α-LA-PC complex was studied by fluorescence quenching using iodide, acrylamide, and 9,10-dibromostearic acid. The results obtained indicated that some of the tryptophans of apo-α-LA were buried in the membrane interior and some were exposed on the outer side. Fluorescence quenching and CD studies indicated the membrane-bound conformation of apo-α-LA was some conformational state that is between the soluble, fully folded conformation and the molten globule state.

BTK‐2, a new inhibitor of the Kv1.1 potassium channel purified from Indian scorpion Buthus tamulus

A novel inhibitor of voltage‐gated potassium channel was isolated and purified to homogeneity from the venom of the red scorpion Buthus tamulus. The primary sequence of this toxin, named BTK‐2, as determined by peptide sequencing shows that it has 32 amino acid residues with six conserved cysteines. The molecular weight of the toxin was found to be 3452 Da. It was found to block the human potassium channel hKv1.1 (IC50=4.6 μM). BTK‐2 shows 40–70% sequence similarity to the family of the short‐chain toxins that specifically block potassium channels. Multiple sequence alignment helps to categorize the toxin in the ninth subfamily of the K+ channel blockers. The modeled structure of BTK‐2 shows an α/β scaffold similar to those of the other short scorpion toxins. Comparative analysis of the structure with those of the other toxins helps to identify the possible structure–function relationship that leads to the difference in the specificity of BTK‐2 from that of the other scorpion toxins. The toxin can also be used to study the assembly of the hKv1.1 channel.

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.

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 [(3)H]diazofluorene ([(3)H]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 alpha-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 [(3)H]DAF and therefore likely to be in contact with lipid were identified in the transmembrane segment M3 of the alpha-subunit of the (Na(+)+K(+))-ATPase and in a putative transmembrane beta-strand in VDAC. Collectively these results demonstrate that [(3)H]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 (3)H release.

Purification and characterization of a short insect toxin from the venom of the scorpion Buthus tamulus

A short chain peptide has been isolated from the venom of a red scorpion of Indian origin, Buthus tamulus. This peptide was purified using ion exchange and reverse phase chromatography and was characterized by molecular weight determination and amino acid sequence. The primary structure analysis shows that BtITx3 is a short peptide of 35 amino acid residues having a molecular weight of 3796 Da. The toxin shows toxicity towards the Lepidopteran species of insect Helicoverpa armigera causing flaccid paralysis and even death within 24 h. It shows more than 50% homology with the short insectotoxins having four disulfide bridges, which suggests that the toxin belongs to the class of short chain toxins blocking the chloride ion channels. This sequence homology study has also helped to bring out the structure–function relationship between the various short toxins. Homology modeling done by using template structure of a known toxin indicated that this toxin consists of a similar α/β scaffold, as present in other scorpion toxins.

Fluorescent and photoactivable probes in depth-dependent analysis of membranes

This report summarizes our efforts towards depth-dependent analysis of membranes by design of suitable fluorescent and photoactivable lipid probes, which can be incorporated into membranes. The objective of depth-dependent analysis has been two fold, one to obtain information on lipid domains and other on transmembrane domains of membrane-bound proteins. In view of increasing importance of lipid rafts and other localized domain and limited success in case of structure determination of membrane-bound proteins vis-à-vis their soluble counterparts, it is tempting to rapidly attach fluorescent or photoactivable probes to lipids to get a probes where relatively little attention is paid to design of such probes. We have shown here how careful design of such probes is required to immobilize such probes in membranes for effective depth-dependent analysis of membranes. An effective design has become important when identification of putative transmembrane domains predicted primarily from the genome data based on hydropathy plots, often needs confirmation by contemporary methodology.

Depth-dependent photolabelling of membrane hydrophobic core with 9-diazofluorene-2-butyric acid

Hydrophobic photoactivable reagents, which readily partition into membranes, have proved very useful for studying membrane hydrophobic core. These reagents have been linked to fatty acids in order to obtain amphipathic photoactivable reagents which label membranes more effectively. By varying the length of these amphipathic reagents, an attempt to label membrane hydrophobic core at different depths can be made. We report here 9-diazofluorene-2-butyric acid as a new photoactivable reagent which labels the single bilayer vesicles prepared from egg phosphatidylcholine. The labelling site on the fatty acyl chains could be traced to be between the carbon atom 4 and 6. The new probe thus labels the membrane at a site which is proximal to what can be predicted from its length and transverse location in membranes.

TRANSVERSE LOCATION OF NEW FLUORENE BASED DEPTH DEPENDENT FLUORESCENT PROBES IN MEMBRANES ‐ QUENCHING STUDIES WITH 9,10‐DIBROMOSTEARIC ACID*

Abstract— 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.