Voltage-dependent gating in K(+) channels results from the mechanical coupling of voltage sensor movements to pore opening. We used single and double mutations in the pore of the Shaker K(+) channel to analyze a late concerted pore opening transition and interpreted the results in the context of known K(+) channel structures. Gating sensitive mutations are located at mechanistically informative regions of the pore and are coupled energetically across distances up to 15 A. We propose that the pore is intrinsically more stable when closed, and that to open the pore the voltage sensors must exert positive work by applying an outward lateral force near the inner helix bundle.

Initial rates of ATP hydrolysis by wild-type GroEL were measured as a function of ATP concentration from 0 to 0.8 mM. Two allosteric transitions are observed: one at relatively low ATP concentrations (< or = 100 microM) and the second at higher concentrations of ATP with respective midpoints of about 16 and 160 microM. Two allosteric transitions were previously observed also in the case of the Arg-196-->Ala GroEL mutant [Yifrach, O., & Horovitz, A. (1994) J. Mol. Biol. 243, 397-401]. On the basis of these observations a mathematical model for nested cooperativity in ATP hydrolysis by GroEL is developed in which there are two levels of allostery: one within each ring and the second between rings. In the first level, each hepatameric ring is in equilibrium between the T and R states, in accordance with the Monod-Wyman-Changeux (MWC) model of cooperativity [Monod et al. (1965) J. Mol. Biol. 12, 88-118]. A second level of allostery is between the rings of the GroEL particle which undergoes sequential Koshland-Némethy-Filmer (KNF)-type transitions from the TT state via the TR state to the RR state [Koshland et al. (1966) Biochemistry 5, 365-385]. Using our model, we estimate the values of the Hill coefficient for the negative cooperativity between rings in wild-type GroEL and the Arg-196-->Ala mutant to be 0.003 (+/- 0.001) and 0.07 (+/- 0.02), respectively. The inter-ring coupling free energies in wild-type GroEL and the Arg-196-->Ala mutant are -7.5 (+/- 0.4) and -3.9 (+/- 0.3) kcal mol-1, respectively.

Vesicular zinc transporters (ZnTs) play a critical role in regulating Zn2؉ homeostasis in various cellular compartments and are linked to major diseases ranging from Alzheimer disease to diabetes. Despite their importance, the intracellular localization of ZnTs poses a major challenge for establishing the mechanisms by which they function and the identity of their ion binding sites. Here, we combine fluorescence-based functional analysis and structural modeling aimed at elucidating these functional aspects. Expression of ZnT5 was followed by both accelerated removal of Zn 2؉ from the cytoplasm and its increased vesicular sequestration. Further, activity of this zinc transport was coupled to alkalinization of the trans-Golgi network. Finally, structural modeling of ZnT5, based on the x-ray structure of the bacterial metal transporter YiiP, identified four residues that can potentially form the zinc binding site on

The information flow between distal elements of a protein may rely on allosteric communication trajectories lying along the protein's tertiary or quaternary structure. To unravel the underlying features of energy parsing along allosteric pathways in voltagegated K ؉ channels, high-order thermodynamic coupling analysis was performed. We report that such allosteric trajectories are functionally conserved and delineated by well defined boundaries. Moreover, allosteric trajectories assume a hierarchical organization whereby increasingly stronger layers of cooperative residue interactions act to ensure efficient and cooperative long-range coupling between distal channel regions. Such long-range communication is brought about by a coupling of local and global conformational changes, suggesting that the allosteric trajectory also corresponds to a pathway of physical deformation. Supported by theoretical analyses and analogy to studies analyzing the contribution of long-range residue coupling to protein stability, we propose that such experimentally derived trajectory features are a general property of allosterically regulated proteins.is a fundamental property of many allosteric proteins. Information transfer between such elements may be achieved by propagation of conformational changes through a protein structure, induced by changes in chemical or electrical potential. However, while the structures of stable conformational states of several allosteric proteins are known, the mechanism(s) by which ligand-induced structural changes propagate through the molecule remains elusive. In the absence of extensive experimental data accurately describing such allosteric networks, computational analyses have attempted to provide mechanistic explanations for long-range coupling in proteins. Structure-based computer simulations suggest that conformational changes may propagate signal transmission by a redistribution of native-state conformational ensembles (1-3). Others suggest that conformational changes may propagate by simple mechanical deformation of the protein structure along pathways of energetic connectivity, comprising adjacent amino acid positions in the tertiary structure (4, 5). Conformational changes are central to the function of voltage-activated potassium channels (Kv), poreforming proteins that open and close in response to changes in membrane potential. Such conformational transitions regulate the flow of potassium ions across the membrane (6-9), a process underlying many fundamental biological processes, in particular the generation of nerve and muscle action potentials (10). These conformational changes, moreover, play a fundamental role in mediating coupling between the voltage-sensor, activation gate and selectivity filter elements of Kv channels (6)(7)(8)(9)(11)(12)(13)(14).Amenable to rapid and highly accurate functional characterization without the need for protein purification, the Kv channel represents an excellent model for studying the features underlying allosteric communication networks in proteins. R...

GroEL with an intrinsic fluorescent probe was generated by introducing the mutation Phe44 --> Trp. Different concentrations of ATP were rapidly mixed with GroEL containing this mutation, and the time-resolved change in fluorescence emission, upon excitation at 280 nm, was followed. Three kinetic phases were observed: a fast phase with a large amplitude and two slower phases with small amplitudes. The phases were assigned by (i) determining their dependence on ATP concentration; (ii) measuring their sensitivity to the mutation Arg197 --> Ala, which decreases cooperativity in ATP binding; and (iii) by carrying out mixing experiments of GroEL also with ADP, ATPgammaS, and ATP without K+. The apparent rate constant corresponding to the fast phase displays a bi-sigmoidal dependence on ATP concentration with Hill coefficients that are strikingly similar to those determined in steady-state experiments. This phase, which reflects ATP-induced conformational changes, is sensitive to the mutation Arg197 --> Ala in a manner that parallels steady-state experiments. The rate of conformational change in the presence of ATP is >100 sec-1, which is fast relative to most protein folding rates, whereas in the absence of ATP it is approximately 0.7 s-1. The second phase reflects the transition from an ATP-bound state of GroEL to an ADP-bound state. The third phase, with the smallest amplitude, reflects release of residual contaminants. The results in this study are found to be consistent with the nested model for cooperativity in ATP binding by GroEL [Yifrach, O., and Horovitz, A. (1995) Biochemistry 34, 5303-5308].

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Ion channels open and close their pore in a process called gating. On the basis of crystal structures of two voltage-independent K(+) channels, KcsA and MthK, a conformational change for gating has been proposed whereby the inner helix bends at a glycine hinge point (gating hinge) to open the pore and straightens to close it. Here we ask if a similar gating hinge conformational change underlies the mechanics of pore opening of two eukaryotic voltage-dependent K(+) channels, Shaker and BK channels. In the Shaker channel, substitution of the gating hinge glycine with alanine and several other amino acids prevents pore opening, but the ability to open is recovered if a secondary glycine is introduced at an adjacent position. A proline at the gating hinge favors the open state of the Shaker channel as if by preventing inner helix straightening. In BK channels, which have two adjacent glycine residues, opening is significantly hindered in a graded manner with single and double mutations to alanine. These results suggest that K(+) channels, whether ligand- or voltage-dependent, open when the inner helix bends at a conserved glycine gating hinge.

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