An Unusual Cause of Adult Ileoileal Intussusception.

Intestinal intussusception in adults is not considered to be common. Clinical presentations may range from an acute presentation to a chronic one and such wide variations make it challenging to establish the diagnosis on time. Adult intussusceptions usually have an identifiable pathological lead point: commonly a polyp, submucosal lipoma, or other tumors. Our patient, a 42-year-old male, presented to us with features of acute intestinal obstruction. He underwent an emergency laparotomy when intussusception of the ileum was noted; the involved bowel segment was resected. Histopathology showed that the lead point was due to tuberculous lesion. Further investigations showed that the patient had pulmonary tuberculosis (TB), which was not identified till then. The patient was started on antituberculous treatment thereafter and the patient recovered well. We intend to present this case to sensitize the readers to the unusual presentation of intestinal TB as intussusception which should be considered especially in countries with high TB endemicity.

A community resource of experimental data for NMR / X-ray crystal structure pairs.

We have developed an online NMR / X-ray Structure Pair Data Repository. The NIGMS Protein Structure Initiative (PSI) has provided many valuable reagents, 3D structures, and technologies for structural biology. The Northeast Structural Genomics Consortium was one of several PSI centers. NESG used both X-ray crystallography and NMR spectroscopy for protein structure determination. A key goal of the PSI was to provide experimental structures for at least one representative of each of hundreds of targeted protein domain families. In some cases, structures for identical (or nearly identical) constructs were determined by both NMR and X-ray crystallography. NMR spectroscopy and X-ray diffraction data for 41 of these "NMR / X-ray" structure pairs determined using conventional triple-resonance NMR methods with extensive sidechain resonance assignments have been organized in an online NMR / X-ray Structure Pair Data Repository. In addition, several NMR data sets for perdeuterated, methyl-protonated protein samples are included in this repository. As an example of the utility of this repository, these data were used to revisit questions about the precision and accuracy of protein NMR structures first outlined by Levy and coworkers several years ago (Andrec et al., Proteins 2007;69:449-465). These results demonstrate that the agreement between NMR and X-ray crystal structures is improved using modern methods of protein NMR spectroscopy. The NMR / X-ray Structure Pair Data Repository will provide a valuable resource for new computational NMR methods development.

Blind testing of routine, fully automated determination of protein structures from NMR data.

The protocols currently used for protein structure determination by nuclear magnetic resonance (NMR) depend on the determination of a large number of upper distance limits for proton-proton pairs. Typically, this task is performed manually by an experienced researcher rather than automatically by using a specific computer program. To assess whether it is indeed possible to generate in a fully automated manner NMR structures adequate for deposition in the Protein Data Bank, we gathered 10 experimental data sets with unassigned nuclear Overhauser effect spectroscopy (NOESY) peak lists for various proteins of unknown structure, computed structures for each of them using different, fully automatic programs, and compared the results to each other and to the manually solved reference structures that were not available at the time the data were provided. This constitutes a stringent "blind" assessment similar to the CASP and CAPRI initiatives. This study demonstrates the feasibility of routine, fully automated protein structure determination by NMR.

Solution NMR and X-ray crystal structures of membrane-associated Lipoprotein-17 domain reveal a novel fold.

The conserved Lipoprotein-17 domain of membrane-associated protein Q9PRA0_UREPA from Ureaplasma parvum was selected for structure determination by the Northeast Structural Genomics Consortium, as part of the Protein Structure Initiative's program on structure-function analysis of protein domains from large domain sequence families lacking structural representatives. The 100-residue Lipoprotein-17 domain is a "domain of unknown function" (DUF) that is a member of Pfam protein family PF04200, a large domain family for which no members have characterized biochemical functions. The three-dimensional structure of the Lipoprotein-17 domain of protein Q9PRA0_UREPA was determined by both solution NMR and by X-ray crystallography at 2.5 Å. The two structures are in good agreement with each other. The domain structure features three α-helices, α1 through α3, and five ß-strands. Strands ß1/ß2, ß3/ß4, ß4/ß5 are anti-parallel to each other. Strands ß1and ß2 are orthogonal to strands ß3, ß4, ß5, while helix α3 is formed between the strands ß3 and ß4. One-turn helix α2 is formed between the strands ß1 and ß2, while helix α1 occurs in the N-terminal polypeptide segment. Searches of the Protein Data Bank do not identify any other protein with significant structural similarity to Lipoprotein-17 domain of Q9PRA0_UREPA, indicating that it is a novel protein fold.

The E. coli single protein production system for production and structural analysis of membrane proteins.

At present, only 0.9% of PDB-deposited structures are of membrane proteins in spite of the fact that membrane proteins constitute approximately 30% of total proteins in most genomes from bacteria to humans. Here we address some of the major bottlenecks in the structural studies of membrane proteins and discuss the ability of the new technology, the Single-Protein Production system, to help solve these bottlenecks.

Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system.

In the Single Protein Production (SPP) method, all E. coli cellular mRNAs are eliminated by the induction of MazF, an ACA-specific mRNA interferase. When an mRNA for a membrane protein, engineered to have no ACA sequences without altering its amino acid sequence, is induced in the MazF-induced cells, E. coli is converted into a bioreactor producing only the targeted membrane protein. Here we demonstrate that three prokaryotic inner membrane proteins, two prokaryotic outer membrane proteins, and one human virus membrane protein can be produced at very high levels, and assembled in appropriate membrane fractions. The condensed SPP (cSPP) system was used to selectively produce isotope-enriched membrane proteins for NMR studies in up to 150-fold condensed culture without affecting protein yields, providing more than 99% cost saving for isotopes. As a novel application of the cSPP system for studies of membrane proteins prior to purification we also demonstrate, for the first time, fast detergent screening by microcoil NMR and well-resolved NMR spectra of several targeted integral membrane proteins obtained without purification.

Asymmetric insertion of membrane proteins in lipid bilayers by solid-state NMR paramagnetic relaxation enhancement: a cell-penetrating Peptide example.

A novel solid-state NMR technique for identifying the asymmetric insertion depths of membrane proteins in lipid bilayers is introduced. By applying Mn (2+) ions on the outer but not the inner leaflet of lipid bilayers, the sidedness of protein residues in the lipid bilayer can be determined through paramagnetic relaxation enhancement (PRE) effects. Protein-free lipid membranes with one-side Mn (2+)-bound surfaces exhibit significant residual (31)P and lipid headgroup (13)C intensities, in contrast to two-side Mn (2+)-bound membranes, where lipid headgroup signals are mostly suppressed. Applying this method to a cell-penetrating peptide, penetratin, we found that at low peptide concentrations, penetratin is distributed in both leaflets of the bilayer, in contrast to the prediction of the electroporation model, which predicts that penetratin binds to only the outer lipid leaflet at low peptide concentrations to cause an electric field that drives subsequent peptide translocation. The invalidation of the electroporation model suggests an alternative mechanism for intracellular import of penetratin, which may involve guanidinium-phosphate complexation between the peptide and the lipids.

Reversible sheet-turn conformational change of a cell-penetrating peptide in lipid bilayers studied by solid-state NMR.

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The (13)C chemical shifts of (13)C, (15)N-labeled residues in the peptide indicate a reversible conformational change from beta-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C(alpha) and C(beta) sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.

Conformation, dynamics, and insertion of a noncysteine-containing protegrin-1 analogue in lipid membranes from solid-state NMR spectroscopy.

Disulfide-bonded beta-hairpin structures are common among antimicrobial peptides. Disulfide bonds are known to be important for antimicrobial activity, but the underlying structural reason is not well understood. We have investigated the membrane-bound structure of a disulfide-deleted analogue of the antimicrobial peptide protegrin-1, in which the four Cys residues were replaced by Ala. The secondary structure, dynamics, and topology of this Ala-PG1 peptide in the membrane were determined by using magic-angle-spinning NMR spectroscopy. Conformation-dependent (13)C isotropic chemical shifts of multiple (13)C-labeled residues were obtained from 1D cross-polarization and direct-polarization spectra, and from 2D J-coupling-mediated (13)C-(13)C correlation spectra. Most labeled residues exhibited two conformations: a random coil and a beta-sheet structure. The dual-conformation property was present in both anionic lipid bilayers, which mimic the bacterial membrane, and zwitterionic cholesterol-containing bilayers, which mimic the eukaryotic cell membrane. The mobility of the peptide was measured by using a 2D C-H dipolar-shift correlation experiment. The random-coil fraction was highly mobile whereas the beta-sheet component was rigid. (1)H spin diffusion from the lipid chains to the peptide indicates that the beta-sheet component was well inserted into the anionic membrane, but surface bound in the cholesterol-containing neutral membrane. Thus, the removal of disulfide bonds changed some PG-1 molecules to highly mobile random coils that were poorly associated with the lipid membrane, but other molecules retained a beta-sheet conformation and had a similar membrane-binding topology to the parent peptide. Thus, the reduced antimicrobial activity of Ala-PG1 was largely due to the reduced number of insertion-competent beta-sheet molecules, rather than uniformly weakened activity of identically structured peptides.

Side-chain conformation of the M2 transmembrane peptide proton channel of influenza a virus from 19F solid-state NMR.

The M2 transmembrane peptide (M2TMP) of the influenza A virus forms a tetrameric helical bundle that acts as a proton-selective channel important in the viral life cycle. The side-chain conformation of the peptide is largely unknown and is important for elucidating the proton-conducting mechanism and the channel stability. Using a 19F spin diffusion NMR technique called CODEX, we have measured the oligomeric states and interhelical side chain-side chain 19F-19F distances at several residues using singly fluorinated M2TMP bound to DMPC bilayers. 19F CODEX data at a key residue of the proton channel, Trp41, confirm the tetrameric state of the peptide and yield a nearest-neighbor interhelical distance of approximately 11 A under both neutral and acidic pH. Since the helix orientation is precisely known from previous 15N NMR experiments and the backbone channel diameter has a narrow allowed range, this 19F distance constrains the Trp41 side-chain conformation to t90 (chi1 approximately 180 degrees , chi2 approximately 90 degrees ). This Trp41 rotamer, combined with a previously measured 15N-13C distance between His37 and Trp411, suggests that the His37 rotamer is t-160. The implication of the proposed (His37, Trp41) rotamers to the gating mechanism of the M2 proton channel is discussed. Binding of the antiviral drug amantadine to the peptide does not affect the F-F distance at Trp41. Interhelical 19F-19F distances are also measured at residues 27 and 38, each mutated to 4-19F-Phe. For V27F-M2TMP, the 19F-19F distances suggest a mixture of dimers and tetramers, whereas the L38F-M2TMP data indicate two tetramers of different sizes, suggesting side chain conformational heterogeneity at this lipid-facing residue. This work shows that 19F spin diffusion NMR is a valuable tool for determining long-range intermolecular distances that shed light on the mechanism of action and conformational heterogeneity of membrane protein oligomers.

Membrane-dependent oligomeric structure and pore formation of a beta-hairpin antimicrobial peptide in lipid bilayers from solid-state NMR.

We used solid-state NMR spectroscopy to investigate the oligomeric structure and insertion of protegrin-1 (PG-1), a beta-hairpin antimicrobial peptide, in lipid bilayers that mimic either the bacterial inner membrane [palmitoyloleoylphosphatidyl ethanolamine and palmitoyloleoylphosphatidylglycerol (POPE/POPG) bilayers] or the red blood cell membrane [neutral palmitoyloleoylphosphatidylcholine (POPC)/cholesterol bilayers]. (1)H spin diffusion from lipids to the peptide indicates that PG-1 contacts both the lipid acyl chains and the headgroups in the anionic membrane but resides far from the lipid chains in the POPC/cholesterol bilayer. (19)F spin diffusion data indicates that 75% of the beta-hairpins have homodimerized N strands and C strands in the anionic membrane. The resulting (NCCN)(n) multimer suggests a membrane-inserted beta-barrel enclosing a water pore. The lipids surrounding the beta-barrel have high orientational disorder and chain upturns, thus they may act as fillers for the pore. These results revise several features of the toroidal pore model, first proposed for magainin and subsequently applied to PG-1. In the POPC/cholesterol membrane, the N and C strands of PG-1 cluster into tetramers, suggesting the formation of beta-sheets on the membrane surface. Thus, the membrane composition plays a decisive role in defining the assembly and insertion of PG-1. The different oligomeric structures of PG-1 help to explain its greater toxicity for bacteria than for eukaryotic cells.

Membrane-disruptive abilities of beta-hairpin antimicrobial peptides correlate with conformation and activity: a 31P and 1H NMR study.

The membrane interaction and solution conformation of two mutants of the beta-hairpin antimicrobial peptide, protegrin-1 (PG-1), are investigated to understand the structural determinants of antimicrobial potency. One mutant, [A(6,8,13,15)] PG-1, does not have the two disulfide bonds in wild-type PG-1, while the other, [Delta(4,18) G10] PG-1, has only half the number of cationic residues. 31P solid-state NMR lineshapes of uniaxially aligned membranes indicate that the membrane disorder induced by the three peptides decreases in the order of PG-1 > [Delta(4,18) G10] PG-1>>[A(6,8,13,15)] PG-1. Solution NMR studies of the two mutant peptides indicate that [Delta(4,18) G10] PG-1 preserves the beta-hairpin fold of the wild-type peptide while [A(6,8,13,15)] PG-1 adopts a random coil conformation. These NMR results correlate well with the known activities of these peptides. Thus, for this class of peptides, the presence of a beta-hairpin fold is more essential than the number of cationic charges for antimicrobial activity. This study indicates that 31P NMR lineshapes of uniaxially aligned membranes are well correlated with antimicrobial activity, and can be used as a diagnostic tool to understand the peptide-lipid interactions of these antimicrobial peptides.

Solid-state NMR investigation of the selective disruption of lipid membranes by protegrin-1.

The interaction of a beta-hairpin antimicrobial peptide, protegrin-1 (PG-1), with various lipid membranes is investigated by (31)P, (2)H, and (13)C solid-state NMR. Mixed lipid bilayers containing anionic lipids and cholesterol are used to mimic the bacterial and mammalian cell membranes, respectively. (31)P and (2)H spectra of macroscopically oriented samples show that PG-1 induces the formation of an isotropic phase in anionic bilayers containing phosphatidylglycerol. Two-dimensional (31)P exchange experiments indicate that these isotropic lipids are significantly separate from the residual oriented lamellar bilayers, ruling out toroidal pores as the cause for the isotropic signal. (1)H spin diffusion experiments show that PG-1 is not exclusively bound to the isotropic phase but is also present in the residual oriented lamellar bilayers. This dynamic and morphological heterogeneity of the anionic membranes induced by PG-1 is supported by the fact that (13)C T(2) relaxation times measured under cross polarization and direct polarization conditions differ significantly. In contrast to the anionic membrane, the zwitterionic phosphatidylcholine (PC) membrane does not form an isotropic phase in the presence of PG-1 but shows significant orientational disorder. The addition of cholesterol to the PC bilayer significantly reduces this orientational disorder. The (13)C T(2) relaxation times of the PC lipids in the presence of both cholesterol and PG-1 suggest that the peptide may decrease the dynamic heterogeneity of the cholesterol-containing membrane. The observed selective interaction of PG-1 with different lipid membranes is consistent with its biological function and may be caused by its strong cationic and amphipathic structure.