Use of a copper-chelated lipid speeds up NMR measurements from membrane proteins.

Recent studies have demonstrated the abilities of solid-state NMR techniques to solve atomic-level-resolution structures and dynamics of membrane-associated proteins and peptides. However, high-throughput applications of solid-state NMR spectroscopy are hampered by long acquisition times due to the low sensitivity of the technique. In this study, we demonstrate the use of a paramagnetic copper-chelated lipid to enhance the spin-lattice relaxation and thereby speed up solid-state NMR measurements. Fluid lamellar-phase bicelles composed of a lipid, detergent, and the copper-chelated lipid and containing a uniformly (15)N-labeled antimicrobial peptide, subtilosin A, were used at room temperature. The use of a chelating lipid reduces the concentration of free copper and limits RF-induced heating, a major problem for fluid samples. Our results demonstrate a 6.2-fold speed increase and a 2.7-fold improvement in signal-to-noise ratio for solid-state NMR experiments under magic-angle spinning and static conditions, respectively. Furthermore, solid-state NMR measurements are shown to be feasible even for nanomole concentrations of a membrane-associated peptide.

Membrane permeabilization, orientation, and antimicrobial mechanism of subtilosin A.

Subtilosin A is an antimicrobial peptide produced by the soil bacterium Bacillus subtilis that possesses bactericidal activity against a diverse range of bacteria, including Listeria monocytogenes. Recent structural studies have found that subtilosin A is posttranslationally modified in a unique way, placing it in a new class of bacteriocins. In this study, in order to understand the mechanism of membrane-disruption by subtilosin A, the interaction of the peptide with model phospholipid bilayers is characterized using fluorescence, solid-state NMR and differential scanning calorimetry (DSC) experiments. Our results in this study show that subtilosin A interacts with the lipid head group region of bilayer membranes in a concentration dependent manner. Fluorescence experiments reveal the interaction of subtilosin A with small unilamellar vesicles (SUVs) composed of POPC, POPG and E. coli total lipids, and that at least one edge of the molecule is buried in membrane bilayers. At high concentrations, it induces leakage from SUVs of POPC and POPE/POPG (7:3) mixture. (15)N solid-state NMR data suggests that the cyclic peptide is partially inserted into bilayers, which is in agreement with the fluorescence data. (31)P and (2)H NMR experiments and DSC data support the hypothesis that subtilosin A adopts a partially buried orientation in lipid bilayers, by showing that it induces a conformational change in the lipid headgroup and disordering in the hydrophobic region of bilayers. These results suggest that the lipid perturbation observed in this study may be one of the consequences of subtilosin A binding to lipid bilayers, which results in membrane permeabilization at high peptide concentrations.

NMR solution structure of ImB2, a protein conferring immunity to antimicrobial activity of the type IIa bacteriocin, carnobacteriocin B2.

Bacteriocins produced by lactic acid bacteria are potent antimicrobial compounds which are active against closely related bacteria. Producer strains are protected against the effects of their cognate bacteriocins by immunity proteins that are located on the same genetic locus and are coexpressed with the gene encoding the bacteriocin. Several structures are available for class IIa bacteriocins; however, to date, no structures are available for the corresponding immunity proteins. We report here the NMR solution structure of the 111-amino acid immunity protein for carnobacteriocin B2 (ImB2). ImB2 folds into a globular domain in aqueous solution which contains an antiparallel four-helix bundle. Extensive packing by hydrophobic side chains in adjacent helices forms the core of the protein. The C-terminus, containing a fifth helix and an extended strand, is held against the four-helix bundle by hydrophobic interactions with helices 3 and 4. Most of the charged and polar residues in the protein face the solvent. Helix 3 is well-defined to residue 55, and a stretch of nascent helix followed by an unstructured loop joins it to helix 4. No interaction is observed between ImB2 and either carnobacteriocin B2 (CbnB2) or its precursor. Protection from the action of CbnB2 is only observed when ImB2 is expressed within the cell. The loop between helices 3 and 4, and a hydrophobic pocket which it partially masks, may be important for interaction with membrane receptors responsible for sensitivity to class IIa bacteriocins.

NMR solution structure of the precursor for carnobacteriocin B2, an antimicrobial peptide from Carnobacterium piscicola.

Type IIa bacteriocins, which are isolated from lactic acid bacteria that are useful for food preservation, are potent antimicrobial peptides with considerable potential as therapeutic agents for gastrointestinal infections in mammals. They are ribosomally synthesized as precursors with an N-terminal leader, typically 18-24 amino acid residues in length, which is cleaved during export from the producing cell. We have chemically synthesized the full precursor of carnobacteriocin B2, precarnobacteriocin (preCbnB2), which has a C-terminal amide rather than a carboxyl, and also produced preCbnB2(1-64), which is missing two amino acid residues at the C-terminus (Arg65 and Pro66), via expression in Escherichia coli as a maltose-binding protein fusion that is then cut with Factor Xa. PreCbnB2(1-64) is readily labeled with (15)N and (13)C for NMR studies using the latter approach. Multidimensional NMR analysis of preCbnB2(1-64) shows that, like the parent bacteriocin, it exists as a random coil in water but assumes a defined conformation in water/trifluoroethanol mixtures. In 70 : 30 trifluoroethanol/water, the 3D structure of the preCbnB2 section corresponding to the mature bacteriocin is essentially the same as reported previously by us for carnobacteriocin B2 (CbnB2). This structure maintains the highly conserved alpha-helix corresponding to residues 20-38 of CbnB2 that is believed to be responsible for interaction with a target receptor in sensitive cells, including Listeria monocytogenes. PreCbnB2 also has a second alpha-helix from residues 3-13 (i.e. -15 to -5 relative to CbnB2) in the leader section of the peptide. This helix appears to be conserved in related type IIa bacteriocin precursors based on sequence analysis. It is likely to be a key recognition element for export and processing, and is probably responsible for the considerably reduced antimicrobial activity of preCbnB2. The latter effect may assist the producing cell in avoiding the toxic effects of the bacteriocin. This is the first 3D structure determined for a prebacteriocin from lactic acid bacteria.

Structure of subtilosin A, a cyclic antimicrobial peptide from Bacillus subtilis with unusual sulfur to alpha-carbon cross-links: formation and reduction of alpha-thio-alpha-amino acid derivatives.

The complete primary and three-dimensional solution structures of subtilosin A (1), a bacteriocin from Bacillus subtilis, were determined by multidimensional NMR studies on peptide produced using isotopically labeled [(13)C,(15)N]medium derived from Anabaena sp. grown on sodium [(13)C]bicarbonate and [(15)N]nitrate. Additional samples of 1 were also generated by separate incorporations of [U-(13)C,(15)N]-L-phenylalanine and [U-(13)C,(15)N]-L-threonine using otherwise unlabeled media. The results demonstrate that in addition to having a cyclized peptide backbone (amide between N and C termini), three cross-links are formed between the sulfurs of Cys13, Cys7, and Cys4 and the alpha-positions of Phe22, Thr28, and Phe31, respectively. The stereochemistry of all residues in 1 except for the three modified ones was confirmed to be L by complete desulfurization with nickel boride, acid hydrolysis to the constituent amino acids, and conversion of these to the corresponding pentafluoropropanamide isopropyl esters for chiral GC MS analysis. The stereochemistry at the modified residues was determined by subjecting each of the eight possible stereoisomers of 1 to eight rounds of ARIA structure calculations, starting with the same NMR peak files and assignments. The stereoisomer with the l stereochemistry at Phe22 (alpha-R) and d stereochemistry at Thr28 (alpha-S) and Phe31 (alpha-S) (LDD isomer) fit the NMR data, giving the lowest energy family of structures with the best rmsd. Thus, biochemical formation of the unusual thio links proceeds with net retention of configuration at Phe22, and inversion at Thr28 and Phe31. Model amino acid derivatives bearing a sulfide moiety at the alpha-carbon were synthesized by reaction of the corresponding alpha-alkoxy compounds with benzyl thiol and SnCl(4). Separation of their pure stereoisomers and desulfurization with nickel boride demonstrated that the reduction of such compounds proceeds with epimerization, in contrast to the previously reported retention of stereochemistry for analogous reaction of steroidal sulfides. However, desulfurization of subtilosin A to cyclic peptide 14, which is inactive as an antimicrobial agent, occurs with inversion of stereochemistry at the alpha-carbons of Phe22 and Thr28 and with 4:1 retention at Phe31. This indicates that the desulfurization reaction proceeds via an N-acyl imine and that the structure of the surrounding peptide controls the geometry of reduction. Posttranslational linkage of a thiol to the alpha-carbon of an amino acid residue is unprecedented in ribosomally synthesized peptides or proteins, and very rare in secondary metabolites. Subtilosin A (1) represents a new class of bacteriocins.