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Intensive study of bacterial chemoreceptors has not yet revealed how receptor methylation and ligand binding alter the interactions between the receptor cytoplasmic domain and the CheA kinase to control kinase activity. Both monomeric and dimeric forms of an Asp receptor cytoplasmic fragment have been shown to be highly dynamic, with a small core of slowly exchanging amide hydrogens (Seeley, S. K., Weis, R. M., and Thompson, L. K. (1996) Biochemistry 35, 5199-5206). Hydrogen exchange studies of the wild-type cytoplasmic fragment and an S461L mutant thought to mimic the kinase-inactivating state are used to investigate the relationship between the stable core and dimer dissociation. Our results establish that (i) decreasing pH stabilizes the dimeric state, (ii) the stable core is present also in the transition state for dissociation, and (iii) this core is expanded significantly by small changes in electrostatic and hydrophobic interactions. These kinase-inactivating changes stabilize both the monomeric and the dimeric states of the protein, which has interesting implications for the mechanism of kinase activation. We conclude that the cytoplasmic domain is a flexible region poised for stabilization by small changes in electrostatic and hydrophobic interactions such as those caused by methylation of glutamate residues and by ligand-induced conformational changes during signaling.

The challenging nature of studies of membrane proteins has made it difficult to determine the molecular mechanism of transmembrane signaling. For the bacterial chemoreceptor family, there are crystal structures of the internal and external domains, structural models of the transmembrane domain, and evidence for subtle ligand-induced conformational changes, but the signaling mechanism remains controversial. We have used a novel site-directed solid-state NMR distance measurement approach, using (13)C(19)F REDOR, to measure a ligand-induced change of 1.0 +/- 0.3 A in the distance between helices alpha 1 and alpha 4 of the ligand-binding domain in the intact, membrane-bound serine receptor. This distance change is shown not to be due to motion of the side chain and thus is due to motion of either the alpha 1 or the alpha 4 helix. Additional distance measurements can be used to determine the type of backbone motion and to follow it to the cytoplasm, to test and refine current proposals for the mechanism of transmembrane signaling. This is a promising general method for high-resolution measurements of local structure in intact, membrane-bound proteins.

We have used a chemiluminescent detection method to improve both the sensitivity and the speed of detection of human genes with oligonucleotide probes. A direct chemiluminescent substrate (AMPPD) was used in combination with an alkaline-phosphatase-labeled oligonucleotide probe to detect the human tissue of plasminogen activator gene by Southern blot analysis. X-ray exposures obtained after 4 h were comparable to those obtained after 7 days with a 32P-labeled oligomer. After 16 h, the signal was 12 times greater than the 32P signal. The detection of the single-copy tissue plasminogen activator gene in 0.25 micrograms of human genomic DNA (76,000 molecules) was achieved. The improved sensitivity obtained by chemiluminescent detection should increase the usefulness of oligonucleotide probes in the direct Southern analysis of human genetic disorders.