Intrinsic helical propensities and stable secondary structure in a membrane-bound fragment (S4) of the shaker potassium channel.

The location and stability of helical secondary structure in a fragment comprising an extended sequence of the S4 transmembrane segment of the Shaker potassium channel was determined in methanol, and when bound to vesicles composed of egg phosphatidylcholine: egg phosphatidylglycerol (4:1; mol:mol) in water. The N-acetylated, C-amidated peptide corresponds to the sequence comprising residues A355-I384 in the Shaker potassium channel. Although NOEs characteristic of helical structure encompass essentially the full peptide sequence in methanol, analysis of amide and CH(alpha) chemical shifts, and amide exchange protection factors establish that stable helical structure comprises only around the first 22 amino acids of the 30 residue peptide. This sequence corresponds to that predicted to have the highest helical stability in water, indicating that while helical structure is considerably stabilised in methanol, the relative helical propensities of amino acids in methanol may be similar to those in water.In the presence of vesicles containing negatively charged lipids, helical structure corresponding to a maximum of around 40 % of the extended S4 peptide is induced; no helical structure is induced in the presence of vesicles composed only of neutral lipids. The location of stable helical structure in the membrane-bound peptide was determined by amide hydrogen-deuterium exchange trapping, and was shown to encompass the sequence between residues near M2 and I18. This sequence is similar to that having high helix propensity in water and methanol, supporting the idea that intrinsic helical propensities are important in defining the location of stable helical structure in polypeptides bound in the interfacial region of lipid bilayers. The study defines an approach to determining the location of, and contributions to, the stability of helical secondary structure in membrane-reconstituted polypeptides.     

Cytological Staining Procedures Applicable to Methacrylate-Embedded Tissues

Experiments indicate that osmic-fixed, plastic-embedded sections are suitable for examination in the light microscope. Nuclei, mitochondia, cellular membranes and cytoplasmic granules are readily demonstrable by phase microscopy. Connective tissue stains permit the identification of elastic and collagenous fibers. Glycogen and other carbohydrate-containing structures are demonstrable by the periodic acid-Schiff and the ammoniacal silver nitrate procedures. It is, therefore, possible to cross-check individual structures by comparing alternate thick and thin sections, examined in the light microscope and electron microscope respectively. Several other advantages pertain to plastic embedded tissues. The sections compare favorably in translucency and in their lack of distortion with material embedded in celloidin, yet the procedure is simpler and much more rapid. Sections of any desired thinness can be prepared, and alternate thick and thin sections are easily forthcoming. When examined in the phase-contrast microscope, mitochondrial preparations become routinely available without the uncertainties of most of the mitochondrial staining methods. It appears, therefore, that plastic embedding should find a useful place among the methods for light microscopy as well as in the armamentarium of the electron microscopist.