Template-directed interference footprinting for RNA based on inosine-specific cleavage.

We report here the development of a Template-directed Interference (TDI) footprinting assay for RNA. The TDI nucleotide analogue inosine (I) lacks the exocyclic amine of G and is a suitable probe for the role of this group in RNA structure and function. Using an I-specific cleavage protocol we identified three functionally significant G residues in the Tetrahymena ribozyme. These residues are proximal to the active site of the folded intron and likely contribute to the positioning of substrates at the catalytic core.

Structure-function relationships in helix-bundle channels probed via total chemical synthesis of alamethicin dimers: effects of a Gln7 to Asn7 mutation.

Alamethicin channels are prototypical helix bundles that may serve as tractable models for more complex protein ion channels. Solid-phase peptide synthesis of alamethicin analogues using FMOC-amino acid fluorides followed by chemical dimerization of these peptides facilitates structure-function studies of particular channel states in bilayer membranes. State 3 in particular, tentatively assigned to a hexameric helix bundle, is sufficiently long-lived that current-voltage measurements can be made during the lifetime of an individual channel opening. Molecular models of hexameric helix bundles, generated using restrained molecular dynamics with simulated annealing, indicate that a Gln7-->Asn7 (Q7-->N7) mutation will increase channel diameter locally. Experimentally, the conductance of state 3 of the N7-alm channel is found to be larger than that of the Q7-alm channel when ion flow is in the usual direction (cations entering the C-terminal end of the channel). When ion flow is in the opposite direction, no difference in the conductances of state 3 of Q7 and state 3 of N7 channels is observed. These results indicate that the effect of a change in pore diameter at position 7 is dependent on the magnitude of other barriers to permeation and that these barriers are voltage-dependent.

Intrinsic rectification of ion flux in alamethicin channels: studies with an alamethicin dimer.

Covalent dimers of alamethicin form conducting structures with gating properties that permit measurement of current-voltage (I-V) relationships during the lifetime of a single channel. These I-V curves demonstrate that the alamethicin channel is a rectifier that passes current preferentially, with voltages of the same sign as that of the voltage that induced opening of the channel. The degree of rectification depends on the salt concentration; single-channel I-V relationships become almost linear in 3 M potassium chloride. These properties may be qualitatively understood by using Poisson-Nernst-Planck theory and a modeled structure of the alamethicin pore.

Development of a novel thiol reagent for probing ion channel structure: studies in a model system.

We have synthesized a novel thiol reagent, 2-[(methylsulfonyl)thio]ethyl [N-(N,N-dimethylamino)ethyl]carbamate (MTSAC), that contains a carbamate functional group as well as a (positively charged) terminal amino group. The carbamate C-N bond isomerizes on a millisecond time scale and significantly alters the three-dimensional shape of the reagent. The behavior of this reagent was contrasted with that of the commonly used thiol reagent, [(methylsulfonyl)thio]ethylamine MTSEA [Akabas, M. H., & Karlin, A. (1995) Biochemistry 34, 12496-12500], with respect to its effect on single-channel currents passing through modified gramicidin channels. While both reagents decreased single-channel currents, the MTSAC-treated channels also showed a pattern of steps in the current recordings on the time scale of the carbamate bond isomerization. Moreover, the pattern and size of these steps were sensitive to the location of the thiol-reactive site in relation to the channel entrance. Thus, MTSAC may prove useful as a reagent for establishing the proximity to the pore in studies of ion channel proteins of unknown structure.

C-terminal amino groups facilitate membrane incorporation of gramicidin derivatives.

Gramicidin derivatives with (positively-charged) C-terminal amino groups are found to incorporate readily and refold quickly when added to dioleoylphosphatidylcholine lipid vesicles from concentrated methanol solutions. Neutral and negatively-charged derivatives do not.