Homomeric and heteromeric assembly of KCNQ (Kv7) K+ channels assayed by total internal reflection fluorescence/fluorescence resonance energy transfer and patch clamp analysis

M-type K(+) channels, consisting of KCNQ1-5 (Kv7.1-7.5) subunits, form a variety of homomeric and heteromeric channels. Whereas all the subunits can assemble into homomeric channels, the ability of the subunits to assemble into heteromultimers is highly variable. KCNQ3 is widely thought to co-assemble with several other KCNQ subtypes, whereas KCNQ1 and KCNQ2 do not. However, the existence of other subunit assemblies is not well studied. To systematically explore the heteromeric assembly of KCNQ channels in individual living cells, we performed fluorescence resonance energy transfer (FRET) between cyan fluorescent protein- and yellow fluorescent protein-tagged KCNQ subunits expressed in Chinese hamster ovary cells under total internal reflection fluorescence microscopy in which excitation light only penetrates several hundred nanometers into the cell, thus isolating membrane events. We found significant FRET between homomeric subunits as expected from their functional expression in heterologous expression systems. Also as expected from previous work, robust FRET was observed between KCNQ2 and KCNQ3. KCNQ3 and KCNQ4 also showed substantial FRET as did KCNQ4 and KCNQ5. To determine functional assembly of KCNQ4/KCNQ5 heteromers, we performed two types of experiments. In the first, we constructed a mutant tetraethylammonium ion-sensitive KCNQ4 subunit and tested its assembly with KCNQ5 by patch clamp analysis of the tetraethylammonium ion sensitivity of the resulting current; however, those data were not conclusive. In the second, we co-expressed a KCNQ4 (G285S) pore mutant with KCNQ5 and found the former to act as a dominant negative, suggesting co-assembly of the two types of subunits. These data confirm that among the allowed assembly conformations are KCNQ3/4 and KCNQ4/5 heteromers.     

Structure of the cytosolic part of the subunit b-dimer of Escherichia coli F0F1-ATP synthase

The structure of the external stalk and its function in the catalytic mechanism of the F₀F₁-ATP synthase remains one of the important questions in bioenergetics. The external stalk has been proposed to be either a rigid stator that binds F₁ or an elastic structural element that transmits energy from the small rotational steps of subunits c to the F₁ sector during catalysis. We employed proteomics, sequence-based structure prediction, molecular modeling, and electron spin resonance spectroscopy using site-directed spin labeling to understand the structure and interfacial packing of the Escherichia coli b-subunit homodimer external stalk. Comparisons of bacterial, cyanobacterial, and plant b-subunits demonstrated little sequence similarity. Supersecondary structure predictions, however, show that all compared b-sequences have extensive heptad repeats, suggesting that the proteins all are capable of packing as left-handed coiled-coils. Molecular modeling subsequently indicated that b₂ from the E. coli ATP synthase could pack into stable left-handed coiled-coils. Thirty-eight substitutions to cysteine in soluble b-constructs allowed the introduction of spin labels and the determination of intersubunit distances by ESR. These distances correlated well with molecular modeling results and strongly suggest that the E. coli subunit b-dimer can stably exist as a left-handed coiled-coil.     

Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray

Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5min. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect differences in both the genotypes and phenotypes of the B. cereus group organisms.     

Genetic variation in the enigmatic Altaian Kazakhs of South-Central Russia: insights into Turkic population history

The Altaian Kazakhs, a Turkic speaking group, now reside in the southern part of the Altai Republic in south-central Russia. According to historical accounts, they are one of several ethnic and geographical subdivisions of the Kazakh nomadic group that migrated from China and Western Mongolia into the Altai region during the 19th Century. However, their population history of the Altaian Kazakhs and the genetic relationships with other Kazakh groups and neighboring Turkic-speaking populations is not well understood. To begin elucidating their genetic history, we analyzed the mtDNAs from 237 Altaian Kazakhs through a combination of SNP analysis and HVS1 sequencing. This analysis revealed that their mtDNA gene pool was comprised of roughly equal proportions of East (A-G, M7, M13, Y and Z) and West (H, HV, pre-HV, R, IK, JT, X, U) Eurasian haplogroups, with the haplotypic diversity within haplogroups C, D, H, and U being particularly high. This pattern of diversity likely reflects the complex interactions of the Kazakhs with other Turkic groups, Mongolians, and indigenous Altaians. Overall, these data have important implications for Kazakh population history, the genetic prehistory of the Altai-Sayan region, and the phylogeography of major mitochondrial lineages in Eurasia.     

Sizing the Bacillus anthracis PA63 channel with nonelectrolyte poly(ethylene glycols)

Nonelectrolyte polymers of poly(ethylene glycol) (PEG) were used to estimate the diameter of the ion channel formed by the Bacillus anthracis protective antigen 63 (PA₆₃). Based on the ability of different molecular weight PEGs to partition into the pore and reduce channel conductance, the pore appears to be narrower than the one formed by Staphylococcus aureus α-hemolysin. Numerical integration of the PEG sample mass spectra and the channel conductance data were used to refine the estimate of the pore's PEG molecular mass cutoff (∼1400 g/mol). The results suggest that the limiting diameter of the PA₆₃ pore is <2 nm, which is consistent with an all-atom model of the PA₆₃ channel and previous experiments using large ions.     

Determination of the transition-state entropy for aggregation suggests how the growth of sickle cell hemoglobin polymers can be slowed

Sickle cell anemia is associated with the mutant hemoglobin HbS, which forms polymers in red blood cells of patients. The growth rate of the polymers is several micrometers per second, ensuring that a polymer fiber reaches the walls of an erythrocyte (which has a 7-μm diameter) within a few seconds after its nucleation. To understand the factors that determine this unusually fast rate, we analyze data on the growth rate of the polymer fibers. We show that the fiber growth follows a first-order Kramers-type kinetics model. The entropy of the transition state for incorporation into a fiber is 95 J mol^− 1 K^− 1, very close to the known entropy of polymerization. This agrees with a recent theoretical estimate for the hydrophobic interaction and suggests that the gain of entropy in the transition state is due to the release of the last layer of water molecules structured around contact sites on the surface of the HbS molecules. As a result of this entropy gain, the free-energy barrier for incorporation of HbS molecules into a fiber is negligible and fiber growth is unprecedentedly fast. This finding suggests that fiber growth can be slowed by components of the red cell cytosol, native or intentionally introduced, which restructure the hydration layer around the HbS molecules and thus lower the transition state entropy for incorporation of an incoming molecule into the growing fiber.