Glycine Zipper Motif Search. We started with Swiss-Prot release 41.15 containing 129,996 proteins (6). All sequences residues in length were removed, leaving 125,887 proteins. Helical membrane proteins were identified by using the Eisenberg hydrophobicity scale and a window length of 21 residues (7). A protein was flagged as a membrane protein if the most hydrophobic segment had an average hydrophobicity of >0.68 or if two segments had a summed average hydrophobicity of >1.1. By these criteria, 29,416 proteins (
23.4%) were identified as helical membrane proteins. TM regions within the membrane proteins were identified by using an average hydrophobicity of 0.42. A nonredundant membrane protein data set was made by eliminating sequences with >90% sequence identity. The final data set contained 23,102 proteins. We then searched for proteins containing the GXXXGXXXG motif in the TM segments and found 1,772 proteins and 2,250 motifs. After removing proteins with low complexity G repeats (more than four G residues in a row), 1,726 proteins with the glycine zipper motif remained. The same method was applied to identify proteins with the other strong glycine zipper motifs.
Relative Conservation Score (RCS). The program CONSEQ (http://conseq.bioinfo.tau.ac.il) (8) was used to determine the sequence conservation reflected in multiple sequence alignments of glycine zipper proteins. This server compares the sequence of a reference protein with proteins deposited in Swiss-Prot (6) to find homologs. The number of PSI-BLAST iterations and the E value cutoff used were 1 and 0.001, respectively. All of the sequences that were found to be evolutionarily related with the glycine zipper proteins in the data set were used in conservation scoring. Briefly, the CONSEQ server assigns a conservation score to each residue, taking into account the evolutionary relationships among the family of homologs. The scores are normalized such that the average score is zero, and negative and positive deviations represent the degrees of conservation and variation, respectively. RCS values were calculated by taking normalized ratio between glycines in glycine zippers and other glycines in TMs. RCS = |CSGly_GZ - CSTM|/|CSGly_Ran - CSTM|, where CSGly_GZ, CSGly_Ran, and CSTM are the conservation scores for Gly in glycine zippers, random glycines, and all TM residues, respectively.
Ion Channel Current Measurements. The methods for measuring channel currents were reported in ref. 9. Briefly, planar bilayers were formed by spreading a 50:50 (weight:weight) mixture of 1-palmitoyl-2-oleoyl phosphatidylethanolamine and phosphatidyl serine (Avanti Polar Lipids) over a hole in a Teflon barrier between two chambers. The chamber compartments were filled with symmetrical KCl solution (140 mM KCl/1 mM CaCl/10 mM Hepes, pH7.4). Measurements of mutant channel activity were facilitated by the addition 0.15 nM SDS, which had no effect in the absence of the peptides (10). Ion channel currents were recorded with an Axopatch amplifier (Axon Instruments, Union City, CA) with a 1-kHz low-pass filter during data acquisition. After the formation of the membrane, we verified that there was no channel-like activity before the addition of the amyloid-
(A) 1-42 peptides to the chamber. The peptides were dissolved in 10 mM NaOH at a concentration of 0.42 mM. A1-42 peptide incorporation into acidic phospholipid planar bilayer membranes was achieved by adding peptide solutions into the chamber to make a final peptide concentration of 3 µg/ml. The A1-42 peptides were synthesized and purified to >95% homogeneity by CS Bio (Menlo Park, CA).
Neuronal Cell Viability Assay. Mouse Neuro-2a (N2A) neuroblastoma cells (American Type Culture Collection) were grown on cell culture plates by using standard methods in DMEM. The medium contained high glucose, glutamine, pyridoxine, 110 mg/liter sodium pyruvate, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FBS (Invitrogen). A42 peptides were pre-dissolved in 10 mM sodium hydroxide solution at a concentration of 480 µM. The solutions, along with a control containing no peptide, were diluted 4-fold with Dulbecco's PBS (D-PBS) buffer and immediately added to the cells at final concentrations up to 5 µM. Cell viability was assayed by using the live/dead assay kit from Molecular Probes according to the supplied instructions. Cells were washed with a 1,000x volume of D-PBS, and 200 µl of a solution containing 2 µM ethidium homodimer and 1 µM CalceinAM was added. After incubation for 30-45 min at room temperature, the cells were viewed and counted by using an inverted fluorescence microscope at 580 nm for CalceinAM and 617 nm for ethidium bromide.
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