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Understanding structural dynamics of biomolecules at the single-molecule level is vital to advancing our knowledge of molecular mechanisms. Currently, there are few techniques that can capture dynamics at the sub-nanometre scale and in physiologically relevant conditions. Atomic force microscopy (AFM).sup.1 has the advantage of analysing unlabelled single molecules in physiological buffer and at ambient temperature and pressure, but its resolution limits the assessment of conformational details of biomolecules.sup.2. Here we present localization AFM (LAFM), a technique developed to overcome current resolution limitations. By applying localization image reconstruction algorithms.sup.3 to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions. LAFM enables the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, facilitating single-molecule structural analysis. LAFM is a post-acquisition image reconstruction method that can be applied to any biomolecular AFM dataset. A localization algorithm is applied to datasets obtained with conventional and high-speed atomic force microscopy to increase image resolution beyond the limits set by the radius of the tip used., Author(s): George R. Heath [sup.1] [sup.4] , Ekaterina Kots [sup.2] , Janice L. Robertson [sup.3] , Shifra Lansky [sup.1] , George Khelashvili [sup.2] , Harel Weinstein [sup.2] , Simon Scheuring [...]

Channels and transporters of the ClC family cause the transmembrane movement of inorganic anions in service of a variety of biological tasks, from the unusual--the generation of the kilowatt pulses with which electric fish stun their prey--to the quotidian--the acidification of endosomes, vacuoles and lysosomes (1). The homodimeric architecture of ClC proteins, initially inferred from single-molecule studies of an elasmobranch [Cl.sup.-] channel (2) and later confirmed by crystal structures of bacterial [Cl.sup.-]/[H.sup.+] antiporters (3,4), is apparently universal. Moreover, the basic machinery that enables ion movement through these proteins--the aqueous pores for anion diffusion in the channels and the ion-coupling chambers that coordinate [Cl.sup.-] and [H.sup.+] antiport in the transporters--are contained wholly within each subunit of the homodimer. The near-normal function of a bacterial ClC transporter straitjacketed by covalent crosslinks across the dimer interface and the behaviour of a concatemeric human homologue argue that the transport cycle resides within each subunit and does not require rigid-body rearrangements between subunits (5,6). However, this evidence is only inferential, and because examples are known in which quaternary rearrangements of extramembrane ClC domains that contribute to dimerization modulate transport activity (7), we cannot declare as definitive a 'parallel-pathways' picture in which the homodimer consists of two single-subunit transporters operating independently. A strong prediction of such a view is that it should in principle be possible to obtain a monomeric ClC. Here we exploit the known structure of a ClC [Cl.sup.-]/ [H.sup.+] exchanger, ClC-ec1 from Escherichia coli, to design mutants that destabilize the dimer interface while preserving both the structure and the transport function of individual subunits. The results demonstrate that the ClC subunit alone is the basic functional unit for transport and that cross-subunit interaction is not required for [Cl.sup.-]/[H.sup.+] exchange in ClC transporters., To develop a strategy for generating a monomeric ClC protein, we examined the structure of ClC-ec1 (Fig. 1) for candidate residues mediating dimerization. This homologue is well suited to our [...]

Inward-rectifier potassium (Kir) channels differ from the canonical [K.sup.+] channel structure in that they possess a long extended pore (~85 [Angstrom]) for ion conduction that reaches deeply into the cytoplasm. This unique structural feature is presumably involved in regulating functional properties specific to Kir channels, such as conductance, rectification block, and ligand-dependent gating. To elucidate the underpinnings of these functional roles, we examine the electrostatics of an ion along this extended pore. Homology models are constructed based on the open-state model of KirBac1.1 for four mammalian Kir channels: Kir1.1/ROMK, Kir2.1/IRK, Kir3.1/GIRK, and Kir6.2/KATP. By solving the Poisson-Boltzmann equation, the electrostatic free energy of a [K.sup.+] ion is determined along each pore, revealing that mammalian Kir channels provide a favorable environment for cations and suggesting the existence of high-density regions in the cytoplasmic domain and cavity. The contribution from the reaction field (the self-energy arising from the dielectric polarization induced by the ion's charge in the complex geometry of the pore) is unfavorable inside the long pore. However, this is well compensated by the electrostatic interaction with the static field arising from the protein charges and shielded by the dielectric surrounding. Decomposition of the static field provides a list of residues that display remarkable correspondence with existing mutagenesis data identifying amino acids that affect conduction and rectification. Many of these residues demonstrate interactions with the ion over long distances, up to 40 [Angstrom], suggesting that mutations potentially affect ion or blocker energetics over the entire pore. These results provide a foundation for understanding ion interactions in Kit channels and extend to the study of ion permeation, block, and gating in long, cation-specific pores.

Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at [V.sub.m] = -100 mV from 34 to 21 pS. The slope conductance measured between -60 and -140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by [Ba.sup.2+], suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for [T1.sup.+]:[K.sup.+], accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for [K.sup.+] transport. KEY WORDS: ROMK2 * IRK1 * GIRK1 * cytoplasmic pore * single-channel conductance