Site-directed solid-state NMR measurement of a ligand-induced conformational change in the serine bacterial chemoreceptor

The challenging nature of studies of membrane proteins has made it difficult to determine the molecular mechanism of transmembrane signaling. For the bacterial chemoreceptor family, there are crystal structures of the internal and external domains, structural models of the transmembrane domain, and evidence for subtle ligand-induced conformational changes, but the signaling mechanism remains controversial. We have used a novel site-directed solid-state NMR distance measurement approach, using (13)C(19)F REDOR, to measure a ligand-induced change of 1.0 +/- 0.3 A in the distance between helices alpha 1 and alpha 4 of the ligand-binding domain in the intact, membrane-bound serine receptor. This distance change is shown not to be due to motion of the side chain and thus is due to motion of either the alpha 1 or the alpha 4 helix. Additional distance measurements can be used to determine the type of backbone motion and to follow it to the cytoplasm, to test and refine current proposals for the mechanism of transmembrane signaling. This is a promising general method for high-resolution measurements of local structure in intact, membrane-bound proteins.     

Site-directed spin labeling of a bacterial chemoreceptor reveals a dynamic, loosely packed transmembrane domain

We used site-directed spin labeling and electron paramagnetic resonance spectroscopy to investigate dynamics and helical packing in the four-helix transmembrane domain of the homodimeric bacterial chemoreceptor Trg. We focused on the first transmembrane helix, TM1, particularly on the nine-residue sequence nearest the periplasm, because patterns of disulfide formation between introduced cysteines had identified that segment as the region of closest approach among neighboring transmembrane helices. Along this sequence, mobility and accessibility of the introduced spin label were characteristic of loosely packed or solvent-exposed side chains. This was also the case for eight additional positions around the circumference and along the length of TM1. For the continuous nine-residue sequence near the periplasm, mobility and accessibility varied only modestly as a function of position. We conclude that side chains of TM1 that face the interior of the four-helix domain interact with neighboring helices but dynamic movement results in loose packing. Compared to transmembrane segments of other membrane proteins reconstituted into lipid bilayers and characterized by site-directed spin labeling, TM1 of chemoreceptor Trg is the most dynamic and loosely packed. A dynamic, loosely packed chemoreceptor domain can account for many experimental observations about the transmembrane domains of chemoreceptors.     

A solid-state NMR study of the phospholamban transmembrane domain: local structure and interactions with Ca(2+)-ATPase

The structure and dynamics of a double (13)C-labelled 24-residue synthetic peptide ([(13)C(2)]CAPLB(29-52)), corresponding to the membrane-spanning sequence of phospholamban (PLB), were examined using (13)C cross-polarisation magic-angle spinning (CP-MAS) NMR spectroscopy. CP-MAS spectra of [(13)C(2)]CAPLB(29-52) reconstituted into unsaturated lipid membranes indicated that the peptide was mobile at temperatures down to -50 degrees C. The NMR spectra showed that peptide motion became constrained in the presence of the SERCA1 isoform of Ca(2+)-ATPase, and chemical cross-linking experiments indicated that [(13)C(2)]CAPLB(29-52) and Ca(2+)-ATPase came into close contact with one another. These results together suggested that the peptide and the 110-kDa calcium pump were interacting in the membrane. Rotational resonance CP-MAS (13)C-(13)C distance measurements on [(13)C(2)]CAPLB(29-52) reconstituted into lipid bilayers confirmed that the sequence spanning Phe-32 and Ala-36 was alpha-helical, and that this structure was not disrupted by interaction with Ca(2+)-ATPase. These results support the finding that the transmembrane domain of PLB is partially responsible for regulation of Ca(2+) transport through interactions with cardiac muscle Ca(2+)-ATPase in the lipid bilayer, and also demonstrate the feasibility of performing structural measurements on PLB peptides when bound to their physiological target.     

Analysis of protein structure in intact cells: crosslinking in vivo between introduced cysteines in the transmembrane domain of a bacterial chemoreceptor.

Oxidative crosslinking of cysteines introduced by site-specific mutagenesis is a powerful tool for structural analysis of proteins, but the approach has been limited to studies in vitro. We recently reported that intact cells of Escherichia coli could be treated with Cu(II)-(o-phenanthroline)3 or molecular iodine in a way that left unperturbed flagellar function or general chemotactic response, yet crosslinks were quantitatively formed between select cysteines in adjoining transmembrane helices of chemoreceptor Trg. This suggested that oxidative crosslinking might be utilized for structural analysis in vivo. Thus, we used our comprehensive collection of Trg derivatives, each containing a single cysteine at one of the 54 positions in the two transmembrane segments of the receptor monomer to characterize patterns of crosslinking in vivo and in vitro for this homodimeric protein. We found that in vivo crosslinking compared favorably as a technique for structural analysis with the more conventional in vitro approach. Patterns of crosslinking generated by oxidation treatments of intact cells indicated extensive interaction of transmembrane segment 1 (TM1) with its homologous partner (TM1') in the other subunit and a more distant placement of TM2 and TM2', the same relationships identified by crosslinking in isolated membranes. In addition, the same helical faces for TM1-TM1' interaction and TM2-TM2' orientation were identified in vivo and in vitro. The correspondence of the patterns also indicates that structural features identified by analysis of in vitro crosslinking are relevant to the organization of the chemoreceptor in its native environment, the intact, functional cell. It appears that the different features of the two functionally benign treatments used for in vivo oxidations can provide insights into protein dynamics.     

Evidence from solid-state NMR for nonhelical conformations in the transmembrane domain of the amyloid precursor protein

The amyloid precursor protein (APP) is subject to proteolytic processing by γ-secretase within neuronal membranes, leading to Alzheimer's disease-associated β-amyloid peptide production by cleavage near the midpoint of the single transmembrane (TM) segment of APP. Conformational properties of the TM segment may affect its susceptibility to γ-secretase cleavage, but these properties have not been established definitively, especially in bilayer membranes with physiologically relevant lipid compositions. In this article, we report an investigation of the APP-TM conformation, using ¹³C chemical shifts obtained with two-dimensional solid-state NMR spectroscopy as site-specific conformational probes. We find that the APP-TM conformation is not a simple α-helix, particularly at 37°C in multilamellar vesicles with compositions that mimic the composition of neuronal cell membranes. Instead, we observe a mixture of helical and nonhelical conformations at the N- and C-termini and in the vicinity of the γ-cleavage site. Conformational plasticity of the TM segment of APP may be an important factor in the γ-secretase cleavage mechanism.     

Retinylidene ligand structure in bovine rhodopsin, metarhodopsin-I, and 10-methylrhodopsin from internuclear distance measurements using 13C-labeling and 1-D rotational resonance MAS NMR.

Rhodopsin is the G-protein coupled photoreceptor that initiates the rod phototransduction cascade in the vertebrate retina. Using specific isotope enrichment and magic angle spinning (MAS) NMR, we examine the spatial structure of the C10-C11=C12-C13-C20 motif in the native retinylidene chromophore, its 10-methyl analogue, and the predischarge photoproduct metarhodopsin-I. For the rhodopsin study 11-Z-[10,20-(13)C(2)]- and 11-Z-[11,20-(13)C(2)]-retinal were synthesized and incorporated into bovine opsin while maintaining a natural lipid environment. The ligand is covalently bound to Lys(296) in the photoreceptor. The C10-C20 and C11-C20 distances were measured using a novel 1-D CP/MAS NMR rotational resonance experimental procedure that was specifically developed for the purpose of these measurements [Verdegem, P. J. E., Helmle, M., Lugtenburg, J., and de Groot, H. J. M. (1997) J. Am. Chem. Soc. 119, 169]. We obtain r(10,20) = 0.304 +/- 0.015 nm and r(11,20) = 0.293 +/- 0.015 nm, which confirms that the retinylidene is 11-Z and shows that the C10-C13 unit is conformationally twisted. The corresponding torsional angle is about 44 degrees as indicated by Car-Parrinello modeling studies. To increase the nonplanarity in the chromophore, 11-Z-[10,20-(13)C(2)]-10-methylretinal and 11-Z-[(10-CH(3)), 13-(13)C(2)]-10-methylretinal were prepared and incorporated in opsin. For the resulting analogue pigment r(10,20) = 0.347 +/- 0.015 nm and r((10)(-)(CH)()3())(,)(13) = 0.314 +/- 0.015 nm were obtained, consistent with a more distorted chromophore. The analogue data are in agreement with the induced fit principle for the interaction of opsin with modified retinal chromophores. Finally, we determined the intraligand distances r(10,20) and r(11,20) also for the photoproduct metarhodopsin-I, which has a relaxed all-E structure. The results (r(10,20) >/= 0.435 nm and r(11,20) = 0.283 +/- 0.015 nm) fully agree with such a relaxed all-E structure, which further validates the 1-D rotational resonance technique for measuring intraligand distances and probing ligand structure. As far as we are aware, these results represent the first highly precise distance determinations in a ligand at the active site of a membrane protein. Overall, the MAS NMR data indicate a tight binding pocket, well defined to bind specifically only one enantiomer out of four possibilities and providing a steric complement to the chromophore in an ultrafast ( approximately 200 fs) isomerization process.     

Solution structure and orientation of the transmembrane anchor domain of the HIV-1-encoded virus protein U by high-resolution and solid-state NMR spectroscopy

The structure of the membrane anchor domain (VpuMA) of the HIV-1-specific accessory protein Vpu has been investigated in solution and in lipid bilayers by homonuclear two-dimensional and solid-state nuclear magnetic resonance spectroscopy, respectively. Simulated annealing calculations, using the nuclear Overhauser enhancement data for the soluble synthetic peptide Vpu1-39 (positions Met-1-Asp-39) in an aqueous 2,2,2-trifluoroethanol (TFE) solution, afford a compact well-defined U-shaped structure comprised of an initial turn (residues 1-6) followed by a linker (7-9) and a short helix on the N-terminal side (10-16) and a further longer helix on the C-terminal side (22-36). The side chains of the two aromatic residues (Trp-22 and Tyr-29) in the longer helix are directed toward the center of the molecule around which the hydrophobic core of the folded VpuMA is positioned. As the observed solution structure is inconsistent with the formation of ion-conductive membrane pores defined previously for VpuMA in planar lipid bilayers, the isolated VpuMA domain as peptide Vpu1-27 was investigated in oriented phospholipid bilayers by proton-decoupled 15N cross polarization solid-state NMR spectroscopy. The line widths and chemical shift data of three selectively 15N-labeled peptides are consistent with a transmembrane alignment of a helical polypeptide. Chemical shift tensor calculations imply that the data sets are compatible with a model in which the nascent helices of the folded solution structure reassemble to form a more regular linear alpha-helix that lies parallel to the bilayer normal with a tilt angle of 相似文献   

Structure and dynamics of the HIV-1 Vpu transmembrane domain revealed by solid-state NMR with magic-angle spinning

We report solid-state nuclear magnetic resonance (NMR) measurements on the peptide Vpu(1-40), comprising residues 1-40 of the 81-residue type 1 integral membrane protein Vpu encoded by the HIV-1 genome. On the basis of a combination of 13C and 15N NMR chemical shifts under magic-angle spinning (MAS), effects of local mobility on NMR signal intensities, site-specific MAS NMR line widths, and NMR-detected hydrogen-deuterium exchange, we develop a model for the structure and dynamics of the Vpu(1-40) monomer in phospholipid bilayer membranes. Our data are largely consistent with earlier structural studies of Vpu peptides by Opella and co-workers, in which solution NMR and solid-state NMR without MAS were used, but our data provide new information about local variations in the degree of mobility and structural order. In addition, our data indicate that the transmembrane alpha-helix of Vpu(1-40) extends beyond the hydrophobic core of the bilayer. We find no evidence for heterogeneity in the conformation and intermolecular contacts of the transmembrane alpha-helix, with the exception of two distinct chemical shifts observed for the C alpha and C beta atoms of A18 that may reflect distinct modes of helix-helix interaction. These results have possible implications for the supramolecular structure of Vpu oligomers that form cation-selective ion channels.     

Modeling the transmembrane domain of bacterial chemoreceptors

Bacterial chemoreceptors signal across the membrane by conformational changes that traverse a four-helix transmembrane domain. High-resolution structures are available for the chemoreceptor periplasmic domain and part of the cytoplasmic domain but not for the transmembrane domain. Thus, we constructed molecular models of the transmembrane domains of chemoreceptors Trg and Tar, using coordinates of an unrelated four-helix coiled coil as a template and the X-ray structure of a chemoreceptor periplasmic domain to establish register and positioning. We tested the models using the extensive data for cross-linking propensities between cysteines introduced into adjacent transmembrane helices, and we found that many aspects of the models corresponded with experimental observations. The one striking disparity, the register of transmembrane helix 2 (TM2) relative to its partner transmembrane helix 1, could be corrected by sliding TM2 along its long axis toward the periplasm. The correction implied that axial sliding of TM2, the signaling movement indicated by a large body of data, was of greater magnitude than previously thought. The refined models were used to assess effects of inter-helical disulfides on the two ligand-induced conformational changes observed in alternative crystal structures of periplasmic domains: axial sliding within a subunit and subunit rotation. Analyses using a measure of disulfide potential energy provided strong support for the helical sliding model of transmembrane signaling but indicated that subunit rotation could be involved in other ligand-induced effects. Those analyses plus modeled distances between diagnostic cysteine pairs indicated a magnitude for TM2 sliding in transmembrane signaling of several angstroms.     

Probing the oligomeric state of phospholamban variants in phospholipid bilayers from solid-state NMR measurements of rotational diffusion rates

Phospholamban (PLB) is a small transmembrane protein that regulates calcium transport across the sarcoplasmic reticulum (SR) of cardiac cells. PLB self-associates into pentamers within sodium dodecyl sulfate (SDS) micelles, but the oligomeric status of PLB in SR membranes is not known. This work has shown that a mutant of PLB, with all native cysteine residues replaced by alanine (Ala-PLB), runs as a monomer on SDS-PAGE gels, in agreement with previous studies [Karim et al. (2000) Biochemistry 39, 10892-10897]. By contrast, a peptide representing the transmembrane domain of the cysteine-free mutant (TM-Ala-PLB) coexists as pentamers, dimers, and monomers on gels. Solid-state NMR methods were used to examine the size and heterogeneity of Ala-PLB and TM-Ala-PLB labeled with (13)C and (2)H in the transmembrane domain and incorporated into dimyristoylphosphatidylcholine (DMPC) bilayers. Wide line (2)H NMR and (13)C cross-polarization magic-angle spinning (CP-MAS) NMR spectra of Ala-PLB and TM-Ala-PLB revealed two distinct species of each of the proteins in the membranes. In the case of Ala-PLB one species was present initially and a second species emerged after 12 h. Measurements of (1)H-(13)C dipolar couplings for the two species of Ala-PLB showed that the rotational diffusion of one species was relatively rapid, defined by a correlation time (tau(R)) of less than 10 micros, whereas the rotation of the other species was comparatively slow (tau(R) approximately 60 micros). These results suggest that although Ala-PLB runs as a monomer on gels, a mixture of different oligomeric forms of the protein, possibly monomers and pentamers, is present in DMPC bilayers. Caution must therefore be exercised in using SDS-PAGE to draw conclusions about the oligomeric state of PLB variants in lipid bilayers.     

Biphasic control logic of HAMP domain signalling in the Escherichia coli serine chemoreceptor

HAMP domains mediate input-output communication in many bacterial signalling proteins. To explore the dynamic bundle model of HAMP signalling (Zhou et al., Mol. Microbiol. 73: 801, 2009), we characterized the signal outputs of 118 HAMP missense mutants of the serine chemoreceptor, Tsr, by flagellar rotation patterns. Receptors with proline or charged amino acid replacements at critical hydrophobic packing residues in the AS1 and AS2 HAMP helices had locked kinase-off outputs, indicating that drastic destabilization of the Tsr-HAMP bundle prevents kinase activation, both in the absence and presence of the sensory adaptation enzymes, CheB and CheR. Attractant-mimic lesions that enhance the structural stability of the HAMP bundle also suppressed kinase activity, demonstrating that Tsr-HAMP has two kinase-off output states at opposite extremes of its stability range. HAMP mutants with locked-on kinase outputs appeared to have intermediate bundle stabilities, implying a biphasic relationship between HAMP stability and kinase activity. Some Tsr-HAMP mutant receptors exhibited reversed output responses to CheB and CheR action that are readily explained by a biphasic control logic. The findings of this study provide strong support for a three-state dynamic bundle model of HAMP signalling in Tsr, and possibly in other bacterial transducers as well.     

Refinement of the geometry of the retinal binding pocket in dark-adapted bacteriorhodopsin by heteronuclear solid-state NMR distance measurements

The bacterial proton pump bacteriorhodopsin (BR) is a 26.5 kDa seven-transmembrane helical protein. Several structural models have been published at > or =1.55 A resolution. The initial cis-trans isomerization of the retinal moiety involves structural changes within <1 A. To understand the chromophore-protein interactions that are important for light-driven proton transport, very accurate measurements of the protein geometry are required. To reveal more structural details at the site of the retinal, we have, therefore, selectively labeled the tryptophan side chains of BR with (15)N and metabolically incorporated retinal, (13)C-labeled at position 14 or 15. Using these samples, heteronuclear distances were measured with high accuracy using SFAM REDOR magic angle spinning solid-state NMR spectroscopy in dark-adapted bacteriorhodopsin. This NMR technique is applied for the first time to a high-molecular mass protein. Two retinal conformers are distinguished by their different isotropic 14-(13)C chemical shifts. Whereas the C14 position of 13-cis-15-syn-retinal is 4.2 A from [indole-(15)N]Trp86, this distance is 3.9 A in the all-trans-15-anti conformer. This latter distance allows us to check on the details of the active center of BR in the various published models derived from X-ray and electron diffraction data. The experimental approach and the results reported in this paper enforce the notion that distances between residues of a membrane protein binding pocket and a bound ligand can be determined at subangstrom resolution.     

Mutational analysis of the control cable that mediates transmembrane signaling in the Escherichia coli serine chemoreceptor

During transmembrane signaling by Escherichia coli Tsr, changes in ligand occupancy in the periplasmic serine-binding domain promote asymmetric motions in a four-helix transmembrane bundle. Piston displacements of the signaling TM2 helix in turn modulate the HAMP bundle on the cytoplasmic side of the membrane to control receptor output signals to the flagellar motors. A five-residue control cable joins TM2 to the HAMP AS1 helix and mediates conformational interactions between them. To explore control cable structural features important for signal transmission, we constructed and characterized all possible single amino acid replacements at the Tsr control cable residues. Only a few lesions abolished Tsr function, indicating that the chemical nature and size of the control cable side chains are not individually critical for signal control. Charged replacements at I214 mimicked the signaling consequences of attractant or repellent stimuli, most likely through aberrant structural interactions of the mutant side chains with the membrane interfacial environment. Prolines at residues 214 to 217 also caused signaling defects, suggesting that the control cable has helical character. However, proline did not disrupt function at G213, the first control cable residue, which might serve as a structural transition between the TM2 and AS1 helix registers. Hydrophobic amino acids at S217, the last control cable residue, produced attractant-mimic effects, most likely by contributing to packing interactions within the HAMP bundle. These results suggest a helix extension mechanism of Tsr transmembrane signaling in which TM2 piston motions influence HAMP stability by modulating the helicity of the control cable segment.     

Methionine-90-spin-labeled bovine alpha-lactalbumin: electron spin resonance and NMR distance measurements

The unique methionine residue of bovine alpha-lactalbumin was modified by irreversible alkylation with the bromoacetamido nitroxide spin-label 4-(2-bromoacetamido)-2,2,6,6-tetramethylpiperidine-N-oxyl. The line shape of the electron spin resonance (ESR) spectrum was indicative of a fairly mobile spin-label and was sensitive to the calcium-induced conformational change. Paramagnetic broadening of the spin-label ESR lines by a Gd(III) ion substituted at the high-affinity calcium site of the protein yielded a distance between the spin-label and the metal-binding site of 8.0 +/- 1.0 A. The extent of the paramagnetic line broadening by the covalently attached nitroxide spin-label on the proton resonances of several amino acid residues of the protein at 500 MHz allowed estimation of intramolecular distances between the methionine-90 residue and several resolvable protons.     

Signalling substitutions in the periplasmic domain of chemoreceptor Trg induce or reduce helical sliding in the transmembrane domain

We used in vivo oxidative cross-linking of engineered cysteine pairs to assess conformational changes in the four-helix transmembrane domain of chemoreceptor Trg. Extending previous work, we searched for and found a fourth cross-linking pair that spanned the intrasubunit interface between transmembrane helix 1 (TM1) and its partner TM2. We determined the effects of ligand occupancy on cross-linking rate constants for all four TM1-TM2 diagnostic pairs in conditions that allowed the formation of receptor-kinase complexes for the entire cellular complement of Trg. Occupancy altered all four rates in a pattern that implicated sliding of TM2 relative to TM1 towards the cytoplasm as the transmembrane signalling movement in receptor-kinase complexes. Transmembrane signalling can be reduced or induced by single amino acid substitutions in the ligand-binding region of the periplasmic domain of Trg. We determined the effects of these substitutions on conformation in the transmembrane domain and on ligand-induced changes using the diagnostic TM1-TM2 cysteine pairs. Effects on rates of in vivo cross-linking showed that induced signalling substitutions altered the relative positions of TM1 and TM2 in the same way as ligand binding, and reduced signalling substitutions blocked or attenuated the ligand-induced shift. These results provide strong support for the helical sliding model of transmembrane signalling.     

NMR structure and dynamics of a designed water-soluble transmembrane domain of nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828-1833, 2004; Ma et al., PNAS, 105: 16537-42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ~83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T(2) dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.     

Role of a small cytoplasmic domain in the establishment of serine chemoreceptor membrane topology.

The Escherichia coli serine chemoreceptor takes on a simple membrane topology with two transmembrane segments separating cytoplasmically disposed N and C termini from a central periplasmic domain. We investigated the role of the small N-terminal cytoplasmic domain in membrane insertion using alkaline phosphatase gene fusions. Mutations eliminating the positive charge of the domain altered insertion dramatically, with reciprocal effects on hybrids with periplasmic and C-terminal cytoplasmic fusion junctions. Efficient export of the normally cytoplasmic C-terminal domain required that, in addition to the N-terminal changes, a short amphiphatic sequence at the beginning of the C-terminal domain be also absent. These findings document the importance of the positive character of the N-terminal domain in chemoreceptor membrane insertion and imply that partially redundant sequence information controls the orientation of the second transmembrane segment.     

NMR structure and dynamics of the second transmembrane domain of the neuronal acetylcholine receptor beta 2 subunit

Structure and backbone dynamics of a selectively [(15)N]Leu-labeled 28-residue segment of the extended second transmembrane domain (TM2e) of the human neuronal nicotinic acetylcholine receptor (nAChR) beta(2) subunit were studied by (1)H and (15)N solution-state NMR in dodecylphosphocholine micelles. The TM2e structure was determined on the basis of the nuclear Overhauser effects (NOEs) and the hydrogen bond restraints, which were inferred from the presence of H(alpha)(i)-H(N)(i+3), H(alpha)(i)-H(beta)(i+3), and H(alpha)(i)-H(N)(i+4) NOE connectivity and from the slow amide hydrogen exchange with D(2)O. The TM2e structure of the nAChR beta(2) subunit contains a helical region between T4 and K22. Backbone dynamics were calculated using the model-free approach based on the (15)N relaxation rate constants, R(1) and R(2), and on the (15)N-[(1)H] NOE. The data acquired at 9.4 and 14.1 T and calculations using different dynamic models demonstrated no conformational exchange and internal motions on the nanosecond time scale. The global tumbling time of TM2e in micelles was 14.4 +/- 0.2 ns; the NOE values were greater than 0.63 at 9.4 T, and the order parameter, S(2), was 0.83-0.96 for all (15)N-labeled leucine residues, suggesting a restricted internal motion. This is the first report of NMR structure and backbone dynamics of the second transmembrane domain of the human nAChR beta(2) subunit in a membrane-mimetic environment, providing the basis for subsequent studies of subunit interactions in the transmembrane domain complex of the neuronal nAChR.     

Secondary structure and NMR resonance assignments of the C-terminal DNA-binding domain of Uup protein

ATP-binding cassette (ABC) systems belong to a large superfamily of proteins that couple the energy released from ATP hydrolysis to a wide variety of cellular processes, including not only transport of various molecules, but also gene regulation, and DNA repair. Mutations in the bacterial uup gene, which encodes a cytosolic ABC ATPase, lead to an increase in the frequency of precise excision of transposons Tn10 and Tn5, suggesting a role of the Uup protein in DNA metabolism. Uup is a 72?kDa polypeptide which comprises two ABC domains, separated by a 75-residue linker, and a C-terminal domain (CTD) of unknown function. The Uup protein from Escherichia coli has been shown to bind DNA in vitro, and the CTD domain contributes to the DNA-binding affinity. We have produced and purified uniformly labeled ¹⁵N- and ¹⁵N/¹³C Uup CTD domain (region 528?C635), and assigned backbone and side-chains resonances using heteronuclear NMR spectroscopy. Secondary structure evaluation based on backbone chemical shifts is consistent with the presence of three ??-helices, including two long ones (residues 564?C590 and 601?C632), suggesting that Uup CTD may fold as an intramolecular coiled coil motif. This work provides the starting point towards determining the first atomic structure of a non-ATPase domain within the vast REG subfamily of ABC soluble ATPases.     

pH measurements by 31p NMR in bacterial suspensions using phenyl phosphonate as a probe

Phenylphosphonate was used as an internal reference for 31p NMR measurements of E. coli cytoplasmic pH. Phenylphosphonate diffused into the cells allowing determination of pH, independent of magnetic susceptibility differences between intercellular and extracellular pools. Phenylphosphonate was used to measure pH changes during succinate uptake and metabolism, as well as during uncoupling of the transmembrane pH gradient by pentachlorophenol.