Theory and applications of supercycled symmetry-based recoupling sequences in solid-state NMR

We present the theoretical principles of supercycled symmetry-based recoupling sequences in solid-state magic-angle-spinning NMR. We discuss the construction procedure of the SR26 pulse sequence, which is a particularly robust sequence for double-quantum homonuclear dipole-dipole recoupling. The supercycle removes destructive higher-order average Hamiltonian terms and renders the sequence robust over long time intervals. We demonstrate applications of the SR26 sequence to double-quantum spectroscopy, homonuclear spin counting, and determination of the relative orientations of chemical shift anisotropy tensors

Structure and Mode-of-Action of the Two-Peptide (Class-IIb) Bacteriocins

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15–30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure–function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix–helix structure involving helix–helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix–helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death

NMR structures and mutational analysis of the two peptides constituting the bacteriocin plantaricin S

The structure of the individual peptides of the two-peptide bacteriocin plantaricin S, an antimicrobial peptide produced by a Lactobacillus plantarum strain, has been determined in DPC micelles. The two peptides of plantaricin S, Pls-α and Pls-β, form an α-helix from and including residue 8 to 24 with a less structured region around residue 16-19 and an amphiphilic α-helix from and including residue 7 to 23, respectively. Activity assays on single amino acid-substituted GxxxG and GxxxG-like motifs show that substituting the Ser and Gly residues in the G9xxxG13 motif in Pls-α and the S17xxxG21 motif in Pls-β reduced or drastically reduced the antimicrobial activity. The two-peptide bacteriocin muricidin contains GxxxG-like motifs at similar positions and displays 40-50% amino acid identity with plantaricin S. Activity assays of combinations of the peptides that constitute the bacteriocins plantaricin S and muricidin show that some combinations are highly active. Furthermore, sequence alignments show that the motifs important for plantaricin S activity align with identical motifs in muricidin. Based on sequence comparison and activity assays, a membrane-inserted model of plantaricin S in which the two peptides are oriented antiparallel relative to each other and where the GxxxG and GxxxG-like motifs important for activity come close in space, is proposed

A Hydrophobic Patch in the Competence-Stimulating Peptide, a Pneumococcal Competence Pheromone, Is Essential for Specificity and Biological Activity

Induction of competence for natural genetic transformation in Streptococcus pneumoniae depends on pheromone-mediated cell-cell communication and a signaling pathway consisting of the competence-stimulating peptide (CSP), its membrane-embedded histidine kinase receptor ComD, and the cognate response regulator ComE. Extensive screening of pneumococcal isolates has revealed that two major CSP variants, CSP1 and CSP2, are found in members of this species. Even though the primary structures of CSP1 and CSP2 are about 50% identical, they are highly specific for their respective receptors, ComD1 and ComD2. In the present work, we have investigated the structural basis of this specificity by determining the three-dimensional structure of CSP1 from nuclear magnetic resonance data and comparing the agonist activity of a number of CSP1/CSP2 hybrid peptides toward the ComD1 and ComD2 receptors. Our results show that upon exposure to membrane-mimicking environments, the 17-amino-acid CSP1 pheromone adopts an amphiphilic α-helical configuration stretching from residue 6 to residue 12. Furthermore, the pattern of agonist activity displayed by the various hybrid peptides revealed that hydrophobic amino acids, some of which are situated on the nonpolar side of the α-helix, strongly contribute to CSP specificity. Together, these data indicate that the identified α-helix is an important structural feature of CSP1 which is essential for effective receptor recognition under natural conditions

1H, 13C, 15N backbone assignment of the human heat-labile enterotoxin B-pentamer and chemical shift mapping of neolactotetraose binding

The major virulence factor of enterotoxigenic Escherichia coli is the heat-labile enterotoxin (LT), an AB5 toxin closely related to the cholera toxin. LT consists of six subunits, the catalytically active A-subunit and five B-subunits arranged as a pentameric ring (LTB), which enable the toxin to bind to the epithelial cells in the intestinal lumen. LTB has two recognized binding sites; the primary binding site is responsible for anchoring the toxin to its main receptor, the GM1-ganglioside, while the secondary binding site recognizes blood group antigens. Herein, we report the 1H, 13C, 15N main chain assignment of LTB from human isolates (hLTB; 103 a.a. per subunit, with a total molecular mass of 58.5 kDa). The secondary structure was predicted based on 13C′, 13Cα, 13Cβ, 1HN and 15N chemical shifts and compared to a published crystal structure of LTB. Neolactotetraose (NEO) was titrated to hLTB and chemical shift perturbations were measured. The chemical shift perturbations were mapped onto the crystal structure, confirming that NEO binds to the primary binding site of hLTB and competes with GM1-binding. Our new data further lend support to the hypothesis that binding at the primary binding site is transmitted to the secondary binding site of the toxin, where it may influence the binding to blood group antigens. This research was first published in Biomolecular NMR Assignments. © Springer Verlag

Mutational Analysis of the Class IIa Bacteriocin Curvacin A and Its Orientation in Target Cell Membranes▿

To analyze the orientation in target cell membranes of the pediocin-like bacteriocin (antimicrobial peptide) curvacin A, 55 variants were generated by site-directed mutagenesis and their potencies against four different target cells determined. The result suggest that the somewhat hydrophilic short central helix (residues 19 to 24), along with the N-terminal β-sheet-like structure (residues 1 to 16), inserts in the interface region of the target cell membrane, with Ala22 close to the hydrophobic core of the membrane. The following hinge region, with Gly28 as an important residue, may then form a turn wherein Gly28 becomes positioned near the border between the interface and the hydrophobic regions, thus permitting the longer and more-hydrophobic C-terminal helix (residues 29 to 41) to insert into the hydrophobic core of the membrane. This helix contains three glycine residues (G33, G37, and G40) that form a putative helix-helix-interacting GxxxGxxG motif. The replacement of any of these glycines with a larger residue was very detrimental, suggesting their possible involvement in helix-helix interactions with a membrane-embedded receptor protein

Symmetry-based 29Si dipolar recoupling magic angle spinning NMR spectroscopy: a new method for investigating three-dimensional structures of zeolite frameworks

A new 29Si solid-state MAS NMR experiment is described for investigating the framework structures of pure silica zeolites. The symmetry-based homonuclear dipolar recoupling sequence SR26411 has been incorporated into a two-dimensional NMR experiment to probe the Si-O-Si bonding connectivities and long-range Si-Si distances in zeolite frameworks. This dipolar recoupling sequence is shown to have a number of advantages over the J-coupling-based INADEQUATE experiment. For the clathrasil Sigma-2, it is demonstrated that there is excellent agreement between experimental double-quantum build-up curves obtained from a series of two-dimensional double-quantum correlation spectra and simulated curves which consider all Si-Si distances out to 8 Å. This result suggests that this experiment could be used to solve zeolite frameworks with unknown structures

A robust pulse sequence for the determination of small homonuclear dipolar couplings in magic-angle spinning NMR

We present a new solid-state NMR pulse sequence that accomplishes efficient broad-band dipolar recoupling in systems with small dipolar couplings and large chemical shift anisotropies. The method involves a supercycled symmetry-based recoupling sequence incorporated in a constant-interval data acquisition strategy. The supercycle removes destructive higher-order average Hamiltonian terms, and makes the method more robust at long time intervals. We demonstrate 38.4% double-quantum filtering efficiency on diammonium [1,4-13C2]-fumarate in which the internuclear distance, as estimated by X-ray diffraction, is 387.8 pm. The estimated 13C-13C dipolar coupling was -136.5±5.1 Hz, corresponding to an internuclear distance of 382.5±4 pm

Identification of a High Affinity Fc gamma RIIA-binding Peptide That Distinguishes Fc gamma RIIA from Fc gamma RIIB and Exploits Fc gamma RIIA-mediated Phagocytosis and Degradation

FcγRIIA is a key activating receptor linking immune complex formation with cellular effector functions. FcγRIIA has 93% identity with an inhibitory receptor, FcγRIIB, which negatively regulates FcγRIIA. FcγRIIA is important in the therapeutic action of several monoclonal antibodies. Binding molecules that discriminate FcγRIIA from FcγRIIB may optimize receptor activity and serve as a lead for development of therapeutics with FcγRIIA as a key target. Here we report the use of phage display libraries to select short peptides with distinct FcγRIIA binding properties. An 11-mer peptide (WAWVWLTETAV) was characterized that bound FcγRIIA with a Kd of 500 nm. It mediated cell internalization and degradation of a model antigen. The peptide-binding site on FcγRIIA was shown to involve Phe163 and the IgG binding amino acids Trp90 and Trp113. It is thus overlapping but not identical to that of IgG. Neither activating receptors FcγRI and FcγRIII, nor FcγRIIB, all of which lack Phe163, bound the peptide. This research was originally published in: Journal of Biological Chemistry. © the American Society for Biochemistry and Molecular Biology