Sequence context modulates the stability of a GxxxG-mediated transmembrane helix-helix dimer.

To quantify the relationship between sequence and transmembrane dimer stability, a systematic mutagenesis and thermodynamic study of the protein-protein interaction residues in the glycophorin A transmembrane helix-helix dimer was carried out. The results demonstrate that the glycophorin A transmembrane sequence dimerizes when its GxxxG motif is abolished by mutation to large aliphatic residues, suggesting that the sequence encodes an intrinsic propensity to self-associate independent of a GxxxG motif. In the presence of an intact GxxxG motif, the glycophorin A dimer stability can be modulated over a span of -0.5 kcal mol(-1) to +3.2 kcal mol(-1) by mutating the surrounding sequence context. Thus, these flanking residues play an active role in determining the transmembrane dimer stability. To assess the structural consequences of the thermodynamic effects of mutations, molecular models of mutant transmembrane domains were constructed, and a structure-based parameterization of the free energy change due to mutation was carried out. The changes in association free energy for glycophorin A mutants can be explained primarily by changes in packing interactions at the protein-protein interface. The energy cost of removing favorable van der Waals interactions was found to be 0.039 kcal mol(-1) per A2 of favorable occluded surface area. The value corresponds well with estimates for mutations in bacteriorhodopsin as well as for those mutations in the interiors of soluble proteins that create packing defects.

Mutation screen of the glutamate decarboxylase-67 gene and haplotype association to unipolar depression.

Abnormally low concentrations of brain gamma-aminobutyric acid (GABA) have been reported in unipolar depression. Almost all of the brain GABA is synthesized by glutamate decarboxylase (GAD) enzymes (GAD67 and GAD65). These enzymes, therefore, play a central role in brain GABA homeostasis. We screened all the 17 exons of the GAD67 gene for mutations using single strand conformation polymorphism (SSCP) or denaturing high pressure liquid chromatography (dHPLC) in a sample of 43 individuals diagnosed with major unipolar depression or other disorders with putative GABAergic dysfunction. We identified eight novel variants (five synonymous base substitutions, two insertion/deletions and one tandem repeat). Three relatively common (minor allele frequency >20%) single nucleotide polymorphisms (SNPs), located in the 5' non-coding region (exon 0), intron 8, and the 3' non-coding region (exon 16) of the gene, were genotyped in 103 European-American (EA) subjects with depression and 125 EA psychiatrically screened controls. Linkage disequilibrium (LD) and haplotype frequencies were estimated using the 3LOCUS program. Significant LD was observed between the intron 8 SNP and the exon 16 SNP and between the exon 0 SNP and the exon 16 SNP. Three common GAD67 haplotypes were observed in this population, which accounted for >90% of the possible GAD67 three-locus haplotypes. Comparison of SNP and haplotype frequencies between individuals with depression and controls revealed no differences. These results demonstrate a significant within-gene LD for GAD67 in the EA population and begin to establish a haplotype map for this gene. Furthermore, these results suggest that common genetic variation within the GAD67 gene does not play a major role in the predisposition to unipolar depression.

Mutation of the novel gene Tmie results in sensory cell defects in the inner ear of spinner, a mouse model of human hearing loss DFNB6.

The recessive mutation at the mouse spinner (sr) locus results in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified the mutant locus responsible for this pathology. The affected gene (Tmie) lies within a 40 kb deletion in the original sr allele. In a newly identified allele, Tmie contains a nonsense mutation expected to truncate the C-terminal end of its product. The 153 amino acid protein encoded by the gene shows no similarity to other known proteins, and is predicted to contain a signal peptide and at least one transmembrane domain. Tmie transcripts were identified in several tissues, including the cochlea. Loss of function of Tmie results in postnatal alterations of sensory hair cells in the cochlea, including defects in stereocilia, the apical projections of hair cells that are important in mechanotransduction of sound. These morphological defects are associated with a profound failure to develop normal auditory function. Consistent with a conserved role for this gene in the cochlea, the genetic mapping data presented here support human TMIE as the gene affected at DFNB6, a non-syndromic hearing loss locus. The spinner mutant is thus a valuable model for insight into mechanisms of human deafness and development of sensory cell function.