Deciphering Membrane Insertion of the Diphtheria Toxin T Domain by Specular Neutron Reflectometry and Solid-State NMR Spectroscopy

Insertion and translocation of soluble proteins into and across biological membranes are involved in many physiological and pathological processes, but remain poorly understood. Here, we describe the pH-dependent membrane insertion of the diphtheria toxin T domain in lipid bilayers by specular neutron reflectometry and solid-state NMR spectroscopy. We gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. At pH 6, the native tertiary structure of the T domain unfolds, allowing its binding to the membrane. The membrane-bound state is characterized by a localization of the C-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. In contrast, the amphiphilic N-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. At pH 4, repulsive interactions vanish; the N-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. The C-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. These helices do not adopt a transmembrane orientation. Interestingly, the T domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. We propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane.     

The energetics of transmembrane helix insertion into a lipid bilayer

Free energy profiles for insertion of a hydrophobic transmembrane protein α-helix (M2 from CFTR) into a lipid bilayer have been calculated using coarse-grained molecular dynamics simulations and umbrella sampling to yield potentials of mean force along a reaction path corresponding to translation of a helix across a lipid bilayer. The calculated free energy of insertion is smaller when a bilayer with a thinner hydrophobic region is used. The free energies of insertion from the potentials of mean force are compared with those derived from a number of hydrophobicity scales and with those derived from translocon-mediated insertion. This comparison supports recent models of translocon-mediated insertion and in particular suggests that: 1), helices in an about-to-be-inserted state may be located in a hydrophobic region somewhat thinner than the core of a lipid bilayer; and/or 2), helices in a not-to-be-inserted state may experience an environment more akin (e.g., in polarity/hydrophobicity) to the bilayer/water interface than to bulk water.     

Conformation of membrane-associated proapoptotic tBid

The proapoptotic Bcl-2 family protein Bid is cleaved by caspase-8 to release the C-terminal fragment tBid, which translocates to the outer mitochondrial membrane and induces massive cytochrome c release and cell death. In this study, we have characterized the conformation of tBid in lipid membrane environments, using NMR and CD spectroscopy with lipid micelle and lipid bilayer samples. In micelles, tBid adopts a unique helical conformation, and the solution NMR (1)H/(15)N HSQC spectra have a single well resolved resonance for each of the protein amide sites. In lipid bilayers, tBid associates with the membrane with its helices parallel to the membrane surface and without trans-membrane helix insertion, and the solid-state NMR (1)H/(15)N polarization inversion with spin exchange at the magic angle spectrum has all of the amide resonances centered at (15)N chemical shift (70-90 ppm) and (1)H-(15)N dipolar coupling (0-5 kHz) frequencies associated with NH bonds parallel to the bilayer surface, with no intensity at frequencies associated with NH bonds in trans-membrane helices. Thus, the cytotoxic activity of tBid at mitochondria may be similar to that observed for antibiotic polypeptides, which bind to the surface of bacterial membranes as amphipathic helices and destabilize the bilayer structure, promoting the leakage of cell contents.     

31P and 2H relaxation studies of helix VII and the cytoplasmic helix of the human cannabinoid receptors utilizing solid-state NMR techniques

Cannabinoid receptors are G-protein-coupled receptors comprised of seven transmembrane helices. We hypothesized that the extended helix of the receptor interacts differently with POPC bilayers due to the differing distribution of charged amino acid residues. To test this, hCB1(T377-E416) and hCB2(K278-H316) peptides were studied with 31P and 2H solid-state NMR spectroscopy by incorporating them into 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine bilayers. Lipid affinities of the 40- and 39-residue peptides were analyzed on the basis of 31P and 2H spectral line shapes, order parameters, and T1 relaxation measurements of the POPC bilayers. Lipid headgroup perturbations were noticed in the 31P NMR spectra in the lipid/peptide mixtures when compared with the pure lipids. 2H order parameters were calculated from the quadrupolar splitting of the de-Paked 2H NMR spectra. At the top of the acyl chain, pure lipids had an average S(CD) approximately = 0.20, whereas S(CD) approximately = 0.16 and S(CD) approximately = 0.18 were found in the presence of hCB1(T377-E416) and hCB2(K278-H316), respectively. S(CD) values decreased in the central part of the acyl chains when compared to the pure POPC lipids, indicating a change in the dynamic properties of the lipid membrane in the presence of the cannabinoid peptides. R(1Z) vs S2(CD) plots exhibited a linear dependency with and without the peptides, with an increase in slope upon addition of the peptides to the POPC, indicating that the dynamics of the lipid bilayer is dominated by fast axially symmetric motion. This study provides insights into the interaction of cannabinoid peptides with the membrane bilayer by investigating the headgroup and acyl chain dynamics.     

Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1-TH3 as a hydrophilic tether

After low pH-triggered membrane insertion, the T domain of diphtheria toxin helps translocate the catalytic domain of the toxin across membranes. In this study, the hydrophilic N-terminal helices of the T domain (TH1-TH3) were studied. The conformation triggered by exposure to low pH and changes in topography upon membrane insertion were studied. These experiments involved bimane or BODIPY labeling of single Cys introduced at various positions, followed by the measurement of bimane emission wavelength, bimane exposure to fluorescence quenchers, and antibody binding to BODIPY groups. Upon exposure of the T domain in solution to low pH, it was found that the hydrophobic face of TH1, which is buried in the native state at neutral pH, became exposed to solution. When the T domain was added externally to lipid vesicles at low pH, the hydrophobic face of TH1 became buried within the lipid bilayer. Helices TH2 and TH3 also inserted into the bilayer after exposure to low pH. However, in contrast to helices TH5-TH9, overall TH1-TH3 insertion was shallow and there was no significant change in TH1-TH3 insertion depth when the T domain switched from the shallowly inserting (P) to deeply inserting (TM) conformation. Binding of streptavidin to biotinylated Cys residues was used to investigate whether solution-exposed residues of membrane-inserted T domain were exposed on the external or internal surface of the bilayer. These experiments showed that when the T domain is externally added to vesicles, the entire TH1-TH3 segment remains on the cis (outer) side of the bilayer. The results of this study suggest that membrane-inserted TH1-TH3 form autonomous segments that neither deeply penetrate the bilayer nor interact tightly with the translocation-promoting structure formed by the hydrophobic TH5-TH9 subdomain. Instead, TH1-TH3 may aid translocation by acting as an A-chain-attached flexible tether.     

The molecular behavior of a single β‐amyloid inside a dipalmitoylphosphatidylcholine bilayer at three different temperatures: An atomistic simulation study: Aβ interaction with DPPC: Atomistic simulation

The behavior of a single Aβ40 molecule within a dipalmitoylphosphatidylcholine (DPPC) bilayer was studied by all‐atom molecular dynamics simulations. The effect of membrane structure was investigated on Aβ40 behavior, secondary structure, and insertion depth. Simulations were performed at three temperatures (323, 310, and 300 K) to probe three different bilayer fluidities. Results show that at all above temperatures, the peptide contains two short helices, coil, bend, and turn structures. At 300 K, the peptide contains a region with β structure in C‐terminal region. Our results also show that Aβ decreases the bilayer thickness and the order of lipids in its vicinity which leads to water insertion into the bilayer and concomitant increase in the local fluidity. The peptide remains embedded in the bilayer at all temperatures, and become inserted into the bilayer up to several residues at 323 and 310 K. At 310 and 300 K, the dominant interaction energy between Aβ and bilayer changes from electrostatic to van der Waals. It can be proposed that at higher temperatures (e.g., 323 K), Lys28 and the C‐terminal region of the peptide play the role of two anchors that keep Aβ inside the top leaflet. This study demonstrates that Aβ molecule can perturb the integrity of cellular membranes. Proteins 2017; 85:1298–1310. © 2017 Wiley Periodicals, Inc.     

Insertion intermediates of pore-forming colicins in membrane two-dimensional space

The formation of integral membrane voltage-gated ion channels by the initially soluble C-terminal channel polypeptide (CP) of the pore-forming colicins is a fruitful area for studies on membrane protein import. The dependence of CP import on specific membrane parameters can be better understood using liposomes and planar membranes of defined lipid composition. The membrane surface and interfacial layer provide special conditions for the transition of a pore-forming colicin from the soluble to the integral membrane state. The colicin E1 CP is arranged in the membrane interfacial layer as a conformationally mobile helical array that is extended far more in the two dimensions parallel to the membrane surface than in the third dimension perpendicular to it. The alpha-helical content of CP(E1) increases by approximately 30% upon binding to the membrane. The sequence of kinetically distinguishable events in the CP(E1)-membrane interaction is binding, unfolding to a subtended area of 4200 A(2), helix extension, and insertion, the last three events overlapping in their time course ( approximately 10 s(-1)). The extension into two dimensions and the interaction with the membrane surface may explain the reversible denaturation and refolding of secondary structure that occurs after boiling of the CP-membrane complex. Although DSC showed the presence of helix-helix interactions in the membrane-bound state, the change in secondary structure and the extended surface area argue against a molten-globule intermediate in the CP-membrane interaction. However, the surface-bound state is mobile, as surface conformational mobility is a necessary prerequisite for insertion of CP trans-membrane helices into the bilayer. The requirement for this surface protein mobility, described by "thermal melting" FRET experiments, may provide the explanation for the precipitous decrease in the voltage-gated CP channel formation at high values of surface potential of planar bilayer membranes. Thus, the membrane interfacial layer, with the CP backbone situated near the acyl chain carbonyls, provides a favorable environment for the structure changes necessary for the transition from the soluble to the membrane-inserted state.     

Analyzing topography of membrane-inserted diphtheria toxin T domain using BODIPY-streptavidin: at low pH, helices 8 and 9 form a transmembrane hairpin but helices 5-7 form stable nonclassical inserted segments on the cis side of the bilayer

Low pH-induced membrane insertion by diphtheria toxin T domain is crucial for A chain translocation into the cytoplasm. To define the membrane topography of the T domain, the exposure of biotinylated Cys residues to the cis and trans bilayer surfaces was examined using model membrane vesicles containing a deeply inserted T domain. To do this, the reactivity of biotin with external and vesicle-entrapped BODIPY-labeled streptavidin was measured. The T domain was found to insert with roughly 70-80% of the molecules in the physiologically relevant orientation. In this orientation, residue 349, located in the loop between hydrophobic helices 8 and 9, was exposed to the trans side of the bilayer, while other solution-exposed residues along the hydrophobic helices 5-9 region of the T domain located near the cis surface. A protocol developed to detect the movement of residues back and forth across the membranes demonstrated that T domain sequences did not rapidly equilibrate between the cis and the trans sides of the bilayer. Binding streptavidin to biotinylated residues prior to membrane insertion only inhibited T domain pore formation for residues in the loop between helices 8 and 9. Pore formation experiments used an approach avoiding interference from transient membrane defects/leakage that may occur upon the initial insertion of protein. Combined, these results indicate that at low pH hydrophobic helices 8 and 9 form a transmembrane hairpin, while hydrophobic helices 5-7 form a nonclassical deeply inserted nontransmembraneous state. We propose that this represents a novel pre-translocation state that is distinct from a previously defined post-translocation state.     

Position 127 amino acid substitutions affect the formation of CRP:cAMP:lacP complexes but not CRP:cAMP:RNA polymerase complexes at lacP.

The lacP DNA binding and activation characteristics of CRP having amino acid substitutions at position 127 were investigated. Wild-type (WT) and T127C CRP footprinted lacP DNA in the presence of DNase I in a cAMP-dependent manner. The T127G, T127I, and T127S forms of CRP failed to footprint lacP both in the absence and in the presence of cAMP. Consistent with these data, WT and T127C CRP:cAMP complexes exhibited high affinity for the lacP CRP site whereas T127G, T127I, or T127S CRP:cAMP complexes exhibited low affinity for the lacP CRP site. CRP:cAMP:RNA polymerase (RNAP) complexes formed at lacP in reactions that contained WT, T127C, T127G, T127I, or T127S CRP. These results demonstrate that allosteric changes important for cAMP-mediated CRP activation are differentially affected by amino acid substitution at position 127. Proper cAMP-mediated reorientation of the DNA binding helices required either threonine or cysteine at position 127. However, cAMP-dependent interaction of CRP with RNAP was accomplished regardless of the amino acid at position 127. RNAP:lacP complexes that supported high-level lac RNA synthesis formed rapidly in reactions that contained WT or T127C CRP whereas RNAP:lacP complexes that supported only low-level lac RNA synthesis formed at slower rates in reactions that contained T127I or T127S CRP. The T127G CRP:cAMP:RNAP:lacP complex failed to activate lacP. The results of this study lead us to conclude that threonine 127 plays an important role in transduction of the signal from the CRP cyclic nucleotide binding pocket that promotes proper orientation of the DNA binding helices and only a minor, if any, role in the functional exposure of the CRP RNAP interaction domain.     

Reconstitution of lipoproteins. II. Lipid-protein interaction between dimyristoyl-lecithin and dimyristoyl-lecithin:cholesterol vesicles and purified apolipoprotein C-I and C-III2 studied by isomeric spin-labelled lecithins

The reconstitution of purified apolipoprotein C-I and C-III2 with sn-3-dimyristoyl-lecithin and sn-3-dimyristoyl-lecithin:cholesterol (10:1) vesicles was studied by electron spin resonance spectroscopy using isomeric 5'-, 12'-, and 16'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithin probes. Results obtained from the temperature-induced changes of lipoprotein recombinants showed the hydrophilic nature of the lipid-protein interactions. The temperature-induced phospholipid phase transition, as measured by 5'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithin probe in recombinants containing apoprotein C-1 or apoprotein C-iii2, is very broad and has a small cooperative unit indicative of extensive lipid-protein interactions occurring at the head group region of the phospholipid bilayer. When 12"- and 16'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithins are used as probes in the same system, similar sharper and more cooperative lipid phase changes are detected. These results indicate a surface location for both apoprotein C-I and apoprotein C-III2 with respect to the phospholipid bilayer in lipoprotein recombinants with and without cholesterol.     

Amino acid sequence of the amino-terminal 24 kDa fragment of the heavy chain of chicken gizzard myosin

Chicken gizzard myosin was modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine (IAEDANS) in the presence of ATP and in 0.15 M KCl, where the myosin assumed 10S conformation. From the tryptic digest of the modified myosin, a fluorescent fragment (24 kilodaltons) was isolated by gel filtration on a Sephadex G-100 column followed by chromatography on a CM 52 column. The amino acid sequence of the fragment was analyzed by conventional methods, and was: (S,Z)K-P-L-S-D-D-E-K-F-L-F-V-D-K-N-F-V-N-N-P-L-A-Q-A-D-W-S-A-K-K- L-V-W-V-P-S-E-K-H-G-F-E-A-A-S-I-K-E-E-K-G-D-E-V-T-V-E-L-Q-E-N-G-K-K- V-T-L-S-K-D-D-I-Q-K-M-N-P-P-K-F-S-K-V-E-D-M-A-E-L-T-C-L-N-E-A-S-V-L- H-N-L-R-E-R-Y-F-S-G-L-I-Y-T-Y-S-G-L-F-C-V-V-I-N-P-Y-K-Q-L-P-I-Y-S-E-K-I- I-D-M-Y-K-G-K-K-R-H-E-M-P-P-H-I-Y-A-I-A-D-T-A-Y-R-S-M-L-Q-D-R-E-D-Q- S-I-L-C-T-G-E-S-G-A-G-K-T-E-N-T-K-K-V-I-Q-Y-L-A-V-V-A-S-S-H-K-G-K. The amino-terminus was blocked, and the fragment was assigned as an amino-terminal part of the heavy chain of gizzard myosin. Position 127 was occupied by epsilon-N-trimethyllysine. Trp-130 of rabbit skeletal myosin heavy chain, which was reported to cross-link to an azide derivative of ATP by Okamoto and Yount (Proc. Natl. Acad. Sci. U.S. 82, 1575-1579 (1985], was replaced by glutamine in gizzard myosin. Cys-93 of the fragment is the amino acid residue whose reaction with IAEDANS alters the ATPase activity of gizzard myosin (Onishi, H. (1985) J. Biochem. 98, 81-86).     

In vitro folding of KvAP, a voltage-gated K+ channel

In this contribution, we report in vitro folding of the archaebacterial voltage-gated K(+) channel, K(v)AP. We show that in vitro folding of the K(v)AP channel from the extensively unfolded state requires lipid vesicles and that the refolded channel is biochemically and functionally similar to the native channel. The in vitro folding process is slow at room temperature, and the folding yield depends on the composition of the lipid bilayer. The major factor influencing refolding is temperature, and almost quantitative refolding of the K(v)AP channel is observed at 80 °C. To differentiate between insertion into the bilayer and folding within the bilayer, we developed a cysteine protection assay. Using this assay, we demonstrate that insertion of the unfolded protein into the bilayer is relatively fast at room temperature and independent of lipid composition, suggesting that temperature and bilayer composition influence folding within the bilayer. Further, we demonstrate that in vitro folding provides an effective method for obtaining high yields of the native channel. Our studies suggest that the K(v)AP channel provides a good model system for investigating the folding of a multidomain integral membrane protein.     

The membrane-bound conformation of alpha-lactalbumin studied by NMR-monitored 1H exchange

The interaction of bovine alpha-lactalbumin (BLA) with negatively charged phospholipid bilayers was studied by NMR monitored 1H exchange to characterize the conformational transition that enables a water-soluble protein to associate with and partially insert into a membrane. BLA was allowed to exchange in deuterated buffer in the absence (reference) and the presence (membrane-bound) of acidic liposomes at pH 4.5, experimental conditions that allow efficient protein-membrane interaction. After adjusting the pH to 6.0, to dissociate the protein from the membrane, reference and membrane-released samples of BLA were analysed by (F1) band-selective homonuclear decoupled total correlation spectroscopy in the alphaH-NH region. The overall exchange behaviour of the membrane-bound state is molten globule-like, suggesting an overall destabilization of the polypeptide. Nevertheless, the backbone amide protons of residues R10, L12, C77, K94, K98, V99 and W104 show significant protection against solvent exchange in the membrane-bound protein. We propose a mechanism for the association of BLA with negatively charged membranes that includes initial protonation of acidic side-chains at the membrane interface, and formation of an interacting site with the membrane which involves helixes A and C. In the next step these helices would slide away from each other, adopting a parallel orientation to the membrane, and would rotate to maximize the interaction between their hydrophobic residues and the lipid bilayer.     

Arrangement and mobility of the voltage sensor domain in prokaryotic voltage-gated sodium channels

Prokaryotic voltage-gated sodium channels (Na(V)s) form homotetramers with each subunit contributing six transmembrane α-helices (S1-S6). Helices S5 and S6 form the ion-conducting pore, and helices S1-S4 function as the voltage sensor with helix S4 thought to be the essential element for voltage-dependent activation. Although the crystal structures have provided insight into voltage-gated K channels (K(V)s), revealing a characteristic domain arrangement in which the voltage sensor domain of one subunit is close to the pore domain of an adjacent subunit in the tetramer, the structural and functional information on Na(V)s remains limited. Here, we show that the domain arrangement in NaChBac, a firstly cloned prokaryotic Na(V), is similar to that in K(V)s. Cysteine substitutions of three residues in helix S4, Q107C, T110C, and R113C, effectively induced intersubunit disulfide bond formation with a cysteine introduced in helix S5, M164C, of the adjacent subunit. In addition, substituting two acidic residues with lysine, E43K and D60K, shifted the activation of the channel to more positive membrane potentials and consistently shifted the preferentially formed disulfide bond from T110C/M164C to Q107C/M164C. Because Gln-107 is located closer to the extracellular side of helix S4 than Thr-110, this finding suggests that the functional shift in the voltage dependence of activation is related to a restriction of the position of helix S4 in the lipid bilayer. The domain arrangement and vertical mobility of helix S4 in NaChBac indicate that the structure and the mechanism of voltage-dependent activation in prokaryotic Na(V)s are similar to those in canonical K(V)s.     

Cryo-electron microscopy of coagulation Factor VIII bound to lipid nanotubes

Factor VIII (FVIII) is a key protein in blood coagulation, deficiency or malfunction of which causes Haemophilia A. The sole cure for this condition is intravenous administration of FVIII, whose membrane-bound structure we have studied by Cryo-electron microscopy and image analysis. Self-assembled lipid nanotubes were optimised to bind FVIII at close to native conditions. The tubes diameter was constant at 30 nm and the lipid bilayer resolved. The FVIII molecules were well defined, forming an 8.5 nm thick outer layer, and appeared to reach the hydrophobic core of the bilayer. The two known FVIII atomic models were superimposed with the averaged 2D protein densities. The insertion of the FVIII within the membrane was evaluated, reaffirming that the membrane-binding C2 or C1-C2 domain(s) fully penetrate the outer leaflet of the lipid layer. The presented results lay the basis for new models of the FVIII overall orientation and membrane-binding mechanism.     

Chemical modification and fluorescence labeling study of Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum using iodoacetamide and its N-substituted derivatives

Sarcoplasmic reticulum membrane vesicles from rabbit skeletal muscle were treated with iodoacetamide (IAA) at pH 7.0 and 30 degrees C. At 1.0 mM IAA, 1 mol of IAA per mol of ATPase peptide was bound in 1 h. Under these conditions, IAA was attached specifically to the B-tryptic fragment portion of the peptide. The binding of IAA did not affect the Ca2+-transporting activity of ATPase. Three fluorescent derivatives of iodoacetamide, 5-(2-acetamidoethyl)aminonaphthalene-1-sulfonate (IAEDANS), 5-iodoacetamido fluorescein (IAF), and 5-iodoacetamido eosin (IAE), were also tested for reactivity toward sarcoplasmic reticulum ATPase at 30 degrees C and pH 7.0. In 1 h at 50 microM concentration, each of these fluorescent labels modified ATPase to a labeling density of 1 mol per mol of ATPase. Neither IAEDANS nor IAF at this labeling density affected Ca2+-transporting activity, but IAE reduced it to 20% of the untreated control. The target site of IAEDANS at this labeling density was located exclusively on the B-fragment portion, as was the case with IAA, but IAF label was found on both A1 and B fragments after limited tryptic digestion. IAEDANS was used as a B-fragment portion-directed conformational probe of Ca2+-transport ATPase, and an increase in fluorescence intensity accompanying E1Ca-P formation was detected. The fluorescence enhancement was abolished when E1Ca-P X ADP beta S was formed by adding ADP beta S to preformed E1Ca-P. This suggests that the conformation of ATPase in the neighborhood of the IAEDANS binding site may be altered in response to the dissociation of ADP from the phosphorylated intermediate.     

Filter flexibility in a mammalian K channel: models and simulations of Kir6.2 mutants

The single-channel conductance varies significantly between different members of the inward rectifier (Kir) family of potassium channels. Mutations at three sites in Kir6.2 have been shown to produce channels with reduced single-channel conductance, the largest reduction (to 40% of wild-type) being for V127T. We have used homology modeling (based on a KcsA template) combined with molecular dynamics simulations in a phosphatidycholine bilayer to explore whether changes in structural dynamics of the filter were induced by three such mutations: V127T, M137C, and G135F. Overall, 12 simulations of Kir6.2 models, corresponding to a total simulation time of 27 ns, have been performed. In these simulations we focused on distortions of the selectivity filter, and on the presence/absence of water molecules lying behind the filter, which form interactions with the filter and the remainder of the protein. Relative to the wild-type simulation, the V127T mutant showed significant distortion of the filter such that approximately 50% of the simulation time was spent in a closed conformation. While in this conformation, translocation of K(+) ions between sites S1 and S2 was blocked. The distorted filter conformation resembles that of the bacterial channel KcsA when crystallized in the presence of a low [K(+)]. This suggests filter distortion may be a possible general model for determining the conductance of K channels.     

Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements

Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T. utilis 5S RNA, its RNase T1 and RNase T2 digested fragments, and a second yeast (Saccharomyces cerevisiae) 5S RNA whose nucleotide sequence differs at only six residues from that of T. utilis 5S RNA. The near-identical chemical shifts and NOE behavior of most of the common peaks from these four RNAs strongly suggest that helices I, IV, and V retain the same conformation after RNase digestion and that both T. utilis and S. cerevisiae 5S RNAs share a common secondary and tertiary structure. Of the four G X U base pairs identified in the intact 5S RNA, two are assigned to the terminal stem (helix I) and the other two to helices IV and V. Seven of the nine base pairs of the terminal stem have been assigned. Our experimental demonstration of a G X U base pair in helix V supports the 5S RNA secondary structural model of Luehrsen and Fox [Luehrsen, K. R., & Fox, G.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2150-2154]. Finally, the base-pair proton peak assigned to the terminal G X U in helix V of the RNase T2 cleaved fragment is shifted downfield from that in the intact 5S RNA, suggesting that helices I and V may be coaxial in intact T. utilis 5S RNA.