Deuterium exchange on micrograms of proteins by attenuated total reflection Fourier transform infrared spectroscopy on silver halide fiber.

We illustrate the use of polycrystalline silver halide fibers (2-20 microns transparency range) for attenuated total internal reflection Fourier transform infrared (IR) spectroscopic measurements of microsamples (10 micrograms of protein). A powerful adjunct technique is a simple method for carrying out deuterium for proton exchange. Spectra of trypsin, soybean trypsin inhibitor, and their complex are easily obtained. Two kinds of difference spectra (DS) are revealing: DS1 (changes in protein on combination with ligand), IR of the trypsin-soybean trypsin inhibitor complex (T.SBTI complex)--sigma [IR of trypsin (T) + IR of soybean trypsin inhibitor (SBTI)], the small values at all wavelengths indicating no conformational change of the proteins upon complexation, and DS2 (changes in materials on deuteration), IR of protioprotein--IR of deuterioprotein, which reveals the infrared bands affected by deuteration. The rate and the extent of the exchange are additional valuable parameters readily measured with this technique. In the present instance, the rate and the amount of the exchange for T.SBTI complex after 30 min was substantially less than that expected from the simple sum of the same parameters for the two individual proteins, T and SBTI. The enzymatic activity of trypsin on the fiber survived for more than a day, no autodegradation being detected by SDS-gel electrophoresis.

para-sulfobenzoyloxybromobimane: a new membrane-impermeable reagent useful for the analysis of thiols and their export from cells.

para-Sulfonylbenzoyloxybromobimane (sBBr) was shown to be similar to the fluorescent labeling agent monobromobimane (mBBr) in reacting rapidly and selectively with thiols to produce stable derivatives which are readily separated by HPLC. Chromatography of the sBBr derivative provides a useful means of confirming the identification of an unknown thiol based upon the chromatography of its mBBr derivative and can be useful for quantitative determination of polycationic thiols for which chromatography of the mBBr derivative is unsatisfactory. Unlike mBBr, which readily penetrates cells, sBBr was found not to be taken up by cells. These characteristics allow sBBr to be used, in conjunction with mBBr, to quantify the export of thiols from cells, as illustrated for GSH and the radioprotective drug WR1065, from V79 cells. Simultaneous determination of GSH and glutathione disulfides in cell culture medium could be achieved by labeling of thiols with sBBr followed by reduction of disulfides with dithiothreitol, labeling of the resulting thiols with mBBr, and HPLC analysis for both glutathione derivatives.

Sensory mechanisms on the molecular level.

As sensory perception is the channel by which we learn about the nature of the world around us it has been the subject of philosophical inquiry since classical antiquity. With the emergence of science in its modern sense in the 17th century, experimental investigation began to be possible but it was only with the relatively recent advent of molecular biology that sensory perception can be studied at its basic level.

Holistic approaches to receptor and channel structure and dynamics.

A detailed three-dimensional structure of a molecule is a necessary, but not sufficient prerequisite to understanding its behaviour. Crystal structures are lacking for protein receptors and ion channels. However, the amino acid sequences for representatives of the most important superfamilies of neuroactive proteins (ligand-gated ion channels, voltage-gated ion channels and G-protein receptors) are known. To address the problem of three-dimensional structure and, subsequently, the dynamic properties of these molecules, a holistic approach was applied to the nicotinic acetylcholine receptor (nAChR), the sodium channel of nerves (NaC) and rhodopsin (Rh) to build 'working' structures. For nAChR and Rh, the chains are placed within an envelope derived from image analysis of electron micrographs. Physical organic principles suggest how to juxtapose binding groups as well as structures for ion channels, leading to the identification of the most common feature of channels, the positively charged 'S4' segment and the ever-present amphiphilic segment of nAChR proteins, which has homologous counterparts in the glutamate receptor and glutamate-binding proteins. The cyclic GMP-gated NaC (cGMP-NaC) is related to the eel and rat neuronal NaC, and has a novel positively charged control sequence.

A structural and dynamic model for the nicotinic acetylcholine receptor.

Folding of the five polypeptide subunits (alpha 2 beta gamma delta) of the nicotinic acetylcholine receptor (AChR) into a functional structural model is described. The principles used to arrange the sequences into a structure include: (1) Hydrophobicity----membrane crossing segments (2) amphipathic character----ion-carrying segments (ion channel with single group rotations) (3) molecular shape (elongated, pentagonal cylinder)----folding dimensions of exobilayer portion (4) choice of acetylcholine binding sites----specific folding of exobilayer segments (5) location of reducible disulfides (near agonist binding site)----additional specification of exobilayer arrangement (6) genetic homology----consistency of functional group choices (7) noncompetitive antagonist labeling----arrangement of bilayer helices. The AChR model is divided into three parts (a) exobilayer: 11 antiparallel beta-strands from each subunit (b) bilayer: 4 hydrophobic and 1 amphiphilic alpha-helices from each subunit and (c) cytoplasmic: one (folded) loop from each subunit. The exobilayer strands can form a closed "flower" (the "resting state") which is opened ("activated") by agonists bound perpendicular to the strands. Rearrangement of the agonists to a strand-parallel position and partial closing of the "flower" leads to a desensitized receptor. The actions of acetylcholine and succinoyl and suberoyl bis-cholines are clarified by the model. The opening and closing of the exobilayer "flower" controls access to the ion channel which is composed of the 5 amphiphilic bilayer helices. A molecular mechanism for ion flow in the channel is given. The unusual photolabeling of intrabilayer serines in alpha, beta and delta, but not in gamma-subunits near the binding site for non-competitive antagonists (NCAs) is explained. The dynamic behavior of the AChR channel and many experimental results can be interpreted in terms of the model.

A partial structure for the gamma-aminobutyric acid (GABAA) receptor is derived from the model for the nicotinic acetylcholine receptor. The anion-exchange protein of cell membranes is related to the GABAA receptor.

Based on the nicotinic acetylcholine receptor model [(1987) Eur. J. Biochem. 168, 431-449], a partial model is constructed for the exobilayer portion of the GABAA receptor, an approach justified by the superfamily relationship of the two receptors [(1987) Nature 328, 221-227]. The model predicts successfully the excess positive charge on interior strands which constitute the ligand-responsive portion of the receptor. Binding to GABA expands the exobilayer portion of the receptor, opening a pathway to a chloride channel. Separate binding sites for antianxiolytics (benzodiazepines) and hypnotics (barbiturates) are suggested, with prolongation of chloride entry projected as a consequence of stabilization of the open form. The anion-exchange protein (AEP) of membranes (band 3 of red blood cell membranes) is similar in some respects to the gamma-aminobutyric acid (GABAA) receptor. Both proteins are inhibited and labeled by diisocyanatostilbenedisulfonate (DIDS), both transport Cl- and HCO-3, and both are membrane proteins. Starting with the lysines known to be labeled in band 3 protein, searches of the amino acid sequences of the GABAA receptor alpha- and beta-subunits reveal at least 4 reasonably homologous sequences. The relationship between AEP and GABAA receptor leads to the idea that the chloride/bicarbonate channel may be the ancestor of all ligand-gated channels, with ligand gating by gamma-aminobutyric acid and acetylcholine arising later in evolution.

Assignment of groups responsible for the "opsin shift" and light absorptions of rhodopsin and red, green, and blue iodopsins (cone pigments).

A modified structural model of rhodopsin is presented. Seven (alpha-helical) segments of 24 largely hydrophobic amino acid residues are assembled with exobilayer connecting strands into an aligned set, using the sequences of human red, green, and blue iodopsins (cone pigments) and human and bovine rod rhodopsins. (Aligned set numbering is used in this article). The inner region of the heptahelical hydrophobic domain includes one His-Glu (Asp) ion pair (red, green rod) near the retinylidene moiety in addition to an iminium ion Asp-99 pair. The negative charges posited in the "point-charge model" to cause the shift of the retinylidene iminium ion light absorption to longer wavelengths in the protein ("opsin shift") are Asp-99 (red, green rod), Glu-102 (red, green), and Glu-138 (rod). Blue iodopsin lacks both an ion pair and a counter charge to the iminium ion in the inner region, a fact that explains its absorption relative to rod rhodopsin. The spectroscopic difference between rod rhodopsin and the red/green iodopsins is due to the influence of Glu-102 in the latter. The red-green difference is due to the net effect of seven OH groups around the chromophore, all such groups being found within one helix turn of the retinylidene location. The tryptophan, which rotates as the retinylidene group isomerizes, may be Trp-142 or Trp-177. The geometric change (the rhodopsin "photoswitch") resulting from cis-trans isomerization in the first excited electronic state (S1), ultimately leads to RX (photoactivated rhodopsin, metarhodopsin II) and changes the activity of exobilayer groups, possibly causing dissociation of Lys-83 and Arg-85 from the carboxylate groups at positions 263 and 265.

A structural and dynamic model for the nicotinic acetylcholine receptor.

Folding of the five polypeptide subunits (alpha 2 beta gamma delta) of the nicotinic acetylcholine receptor (AChR) into a functional structural model is described. The principles used to arrange the sequences into a structure include: (1) hydrophobicity----membrane-crossing segments; (2) amphipathic character----ion-carrying segments (ion channel with single group rotations); (3) molecular shape (elongated, pentagonal cylinder)----folding dimensions of exobilayer portion; (4) choice of acetylcholine binding sites----specific folding of exobilayer segments; (5) location of reducible disulfides (near agonist binding site)----additional specification of exobilayer arrangement; (6) genetic homology----consistency of functional group choices; (7) noncompetitive antagonist labeling----arrangement of bilayer helices. The AChR model is divided into three parts: (a) exobilayer consisting of 11 antiparallel beta-strands from each subunit; (b) bilayer consisting of four hydrophobic and one amphiphilic alpha-helix from each subunit; (c) cytoplasmic consisting of one (folded) loop from each subunit. The exobilayer strands can form a closed 'flower' (the 'resting state') which is opened ('activated') by agonists bound perpendicular to the strands. Rearrangement of the agonists to a strand-parallel position and partial closing of the 'flower' leads to a desensitized receptor. The actions of acetylcholine and succinoyl and suberoyl bis-cholines are clarified by the model. The opening and closing of the exobilayer 'flower' controls access to the ion channel which is composed of the five amphiphilic bilayer helices. A molecular mechanism for ion flow in the channel is given. Openings interrupted by short duration closings (50 microseconds) depend upon channel group motions. The unusual photolabeling of intrabilayer serines in alpha, beta and delta subunits but not in gamma subunits near the binding site for non-competitive antagonists is explained along with a mechanism for the action of these antagonists such as phencyclidine. The unusual alpha 192Cys-193Cys disulfide may have a special peptide arrangement, such as a cis-peptide bond to a following proline (G.A. Petsko and E.M. Kosower, unpublished results). The position of phosphorylatable sites and proline-rich segments are noted for the cytoplasmic loops. The dynamic behavior of the AChR channel and many different experimental results can be interpreted in terms of the model. An example is the lowering of ionic conductivity on substitution of bovine for Torpedo delta M2 segment. The model represents a useful construct for the design of experiments on AChR.

Mapping a region associated with Na channel inactivation using antibodies to a synthetic peptide corresponding to a part of the channel.

Antibodies to the synthetic peptide (carrier-coupled) corresponding to amino acids 210-223 of the primary sequence of eel Na channel (C1+ peptide) were generated. The antipeptide antibodies were used to identify functional roles as well as the accessibility from the external membrane surface of the C1+ domains. Rabbit antipeptide antibodies bound specifically to the C1+ synthetic peptide and to an eel membrane fraction bearing a high density of Na channels. When applied to the external surface of cultured dorsal root ganglion cells obtained from newborn rats, the antibodies modify Na channel inactivation by shifting the steady-state Na current-inactivation parameter, h infinity, curve to more negative potentials in fast and slow Na currents. The rate of inactivation of the slow channel is shown to be increased. The antibodies do not have a significant effect on activation of the channels. Part of the amino acid sequence corresponding to C1+ peptide is therefore accessible, in the mammalian Na channel, from the external membrane surface and is associated with the inactivation gate.

Fourier transform infrared spectra of aqueous protein mixtures using a novel attenuated total internal reflectance cell with infrared fibers.

A specially designed cell containing a silver halide (AgBr:AgCl) infrared fiber allows convenient and reproducible loading of viscous protein solutions and suspensions. Attenuated total internal reflectance measurements using an FTIR spectrometer were made for bovine serum albumin-water past. Dynamic changes in the protein films are readily followed, a technique which should be generally useful. A band assigned to a secondary structural feature, the alpha-helix, is similar in intensity to that reported (T.M. Fong, M.G. McNamee, submitted for publication).

A structural and dynamic molecular model for the sodium channel of Electrophorus electricus.

Chemical logic and single group rotation (SGR) theory are applied to the primary structure determined by Noda et al. [(1984) Nature 312, 121-127] to construct a molecular model of the sodium channel of Electrophorus electricus. Both structural and dynamic aspects of the channel are accounted for, including gating current, sensitivity to changes in membrane potential, channel opening, a binding site for sodium, selectivity for sodium over potassium, capacity for rapid sodium flow, sensitivity to batrachotoxin (or other toxins) and inactivation.

Revised assignments for the beta-, gamma- and delta-subunits of the acetylcholine receptor structural model.

Revised assignments for the bilayer helices of the beta-, gamma- and delta-subunits of the acetylcholine receptor are presented. A new feature of the model is extensive charge matching between the polar groups of the ion channel elements of different subunits.

Membrane-mobility agent-promoted fusion of erythrocytes: fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins.

Rat, but not human, erythrocytes undergo fusion promoted by the membrane-mobility agent 2-(2-methoxyethoxy)-ethyl cis-8-(2-octylcyclopropyl)octanoate (A2C). The difference in behavior is correlated with rat erythrocyte membrane protein degradation caused by Ca2+-activated proteases. The human erythrocyte is deficient in such protease activity. Membrane protein degradation is a necessary, but not sufficient, requirement for membrane fusion. Membrane protein degradation probably releases membrane components from certain constraints. In addition, the motion of membrane components precedes fusion and must be promoted by reagents such as A2C, leading to the creation of fusion-potent lipid areas. This sequence of chemical and physical events occurs in other fusion processes.

A hypothesis for the mechanism of sodium channel opening by batrachotoxin and related toxins.

The mechanism of action of one class of sodium channel opening agents (batrachotoxin, veratridine, aconitine and grayanotoxin) is proposed to involve complexation of a triad of agent oxygen atoms with the epsilon-ammonium ion of a channel lysing side chain, holding open the mouth or exit of the ion channel. This idea complements the oxygen triad model derived by structural considerations (Masutani, T., Seyama, I., Narahashi, T. and Iwasa, J. (1981) J. Pharm. Exp. Therap. 217, 812) and extended by crystal structure comparisons (Codding, P.W. (1983) J. Am. Chem. Soc. 105, 3172). The mechanism is based on results for acetylcholine receptor ion channel gating, structure and function, using single group rotation (SGR) theory (cf. Kosower, E.M. (1983) Biochem. Biophys. Res. Commun. 111, 1022 and in press (1983); FEBS Lett. (1983) 155, 245; ibid. 157, 144; Biophys. J. (1983) 45, in press).

A molecular model for the exobilayer portion of the alpha-subunit of the acetylcholine receptor with binding sites for acetylcholine and non-competitive antagonists.

A molecular model for the exobilayer portion of the alpha-subunit of the acetylcholine receptor is presented. Binding sites for an acetylcholine and non-competitive antagonists are indicated.