Synthetic peptide models for the redox-active disulfide loop of glutaredoxin. Conformational studies

Two cyclic peptide disulfides Boc-Cys-Pro-X-Cys-NHMe (X = L-Tyr or L-Phe) have been synthesized as models for the 14-membered redox-active disulfide loop of glutaredoxin. 'H NMR studies at 270 MHz in chloroform solutions establish a type I 0-turn conformation for the Pro-X segment in both peptides, stabilized by a 4-1 hydrogen bond between the Cys(1) CO and Cys(4) NH groups. Nuclear Overhauser effects establish that the aromatic ring in the X = Phe peptide is oriented over the central peptide unit. In dimethyl sulfoxide solutions two conformational species are observed in slow exchange on the NMR time scale, for both peptides. These are assigned to type I and type I1 p-turn structures with -Pro-Tyr(Phe)-as the corner residues. The structural assignments are based on correlation of NMR parameters with model 14-membered cyclic cystine peptides with Pro-X spacers. Circular dichroism studies based on the -S-Sn- u* transition suggest a structural change in the disulfide bridge with changing solvent polarity, establishing conformational coupling between the peptide backbone and the disulfide linkage in these systems.

Cystine peptides Antiparallel β-sheet conformation of the cyclic biscystine peptide [Boc-Cys-Ala-Cys-NHCH3]2

The crystal structure analysis of the cyclic biscystine peptide [Boc-Cys1-Ala2-Cys3-NHCH3]2 with two disulfide bridges confirms the antiparallel ?-sheet conformation for the molecule as proposed for the conformation in solution. The molecule has exact twofold rotation symmetry. The 22-membered ring contains two transannular NH ? OC hydrogen bonds and two additional NH ? OC bonds are formed at both ends of the molecule between the terminal (CH3)3COCO and NHCH3 groups. The antiparallel peptide strands are distorted from a regularly pleated sheet, caused mainly by the L-Ala residue in which ?=� 155° and ?= 162°. In the disulfide bridge C? (1)-C? (1)-S(1)-(3')-C?(3')-C?(3'), S�S = 2.030 Å, angles C? SS = 107° and 105°, and the torsional angles are �49, �104, +99, �81, �61°, respectively. The biscystine peptide crystallizes in space group C2 with a = 14.555(2) Ã…, b = 10.854(2) Ã…, c = 16.512(2)Ã…, and ?= 101.34(1) with one-half formula unit of C30H52N8O10S4· 2(CH3)2SO per asymmetric unit. Least-squares refinement of 1375 reflections observed with |F| > 3?(F) yielded an R factor of 7.2%.

Crystallization and preliminary X-ray studies of a galactose-specific lectin from the seeds of bitter gourd (Momordica charantia)

A galactose-specific lectin from the seeds of bitter gourd (Momordica charantia) is a four-chain type II ribosome-inactivating protein (RIP) resulting from covalent association through a disulfide bridge between two identical copies of a two-chain unit. The available structural information on such four-chain RIPs is meagre. The bitter gourd lectin was therefore crystallized for structural investigation and the crystals have been characterized. It is anticipated that the structure of the orthorhombic crystals will be analysed using molecular replacement by taking advantage of its sequence, and presumably structural, homology to normal two-chain type II RIPs.

The sequence and structure of snake gourd (Trichosanthes anguina) seed lectin, a three-chain nontoxic homologue of type II RIPs

The sequence and structure of snake gourd seed lectin (SGSL), a nontoxic homologue of type II ribosome-inactivating proteins (RIPs), have been determined by mass spectrometry and X-ray crystallography, respectively. As in type II RIPs, the molecule consists of a lectin chain made up of two beta-trefoil domains. The catalytic chain, which is connected through a disulfide bridge to the lectin chain in type II RIPs, is cleaved into two in SGSL. However, the integrity of the three-dimensional structure of the catalytic component of the molecule is preserved. This is the first time that a three-chain RIP or RIP homologue has been observed. A thorough examination of the sequence and structure of the protein and of its interactions with the bound methyl-alpha-galactose indicate that the nontoxicity of SGSL results from a combination of changes in the catalytic and the carbohydrate-binding sites. Detailed analyses of the sequences of type II RIPs of known structure and their homologues with unknown structure provide valuable insights into the evolution of this class of proteins. They also indicate some variability in carbohydrate-binding sites, which appears to contribute to the different levels of toxicity exhibited by lectins from various sources.

Structural basis for the redox sensitivity of the Mycobacterium tuberculosis SigK-RskA sigma-anti-sigma complex

The host-pathogen interactions in Mycobacterium tuberculosis infection are significantly influenced by redox stimuli and alterations in the levels of secreted antigens. The extracyto-plasmic function (ECF) sigma factor sigma(K) governs the transcription of the serodominant antigens MPT70 and MPT83. The cellular levels of sigma(K) are regulated by the membrane-associated anti-sigma(K) (RskA) that localizes sigma(K) in an inactive complex. The crystal structure of M. tuberculosis sigma(K) in complex with the cytosolic domain of RskA (RskAcyto) revealed a disulfide bridge in the -35 promoter-interaction region of sigma(K). Biochemical experiments reveal that the redox potential of the disulfide-forming cysteines in sigma(K) is consistent with its role as a sensor. The disulfide bond in sigma(K) influences the stability of the sigma(K)-RskA(cyto) complex but does not interfere with sigma(K)-promoter DNA interactions. It is noted that these disulfide-forming cysteines are conserved across homologues, suggesting that this could be a general mechanism for redox-sensitive transcription regulation.

Cyclic Peptide Disulfides - Solution And Solid-State Conformation Of Boc-Cys-Pro-Aib-Cys-S-S-Bridge-Nhme,A Disulfide-Bridged Peptide Helix

The solution and solid-state conformations of the peptide disulfide Boc-Cys-Pro-Aib-Cys-NHMe have been determined by NMR spectroscopy and X-ray diffraction. The Cys(4) and methylamide NH groups are solvent shielded in CDCI3 and (CD,),SO, suggesting their involvement in intramolecular hydrogen bonding. On the basis of known stereochemical preferences of Pro and Aib residues, a consecutive @-turn structure is favored in solution. X-ray diffraction analysis reveals a highly folded 310 helical conformation for the peptide, with the S-S bridge lying approximately parallel to the helix axis, linking residues 1 and 4. The backbone conformational angles are Cys(1) 4 = -121.1', $ = 65.6"; Pro(2) 4 = -58.9', 4 = -34.0'; Aib(3) 4 = -61.8', $ = -17.9'; Cys(4) 4 = -70.5', $ = -18.6'. Two intramolecular hydrogen bonds are observed between Cys(1) CO--HN Cys(4) and Pro(2) CO--HNMe. The disulfide bond has a right-handed chirality, with a dihedral angle (xss) of 82'.

Molecular structure of a cyclic tetrapeptide disulfide. A novel 310 helical conformation with an S-S bridge

The crystal structure of the cyclic peptide disulfide Boc-Cys-Pro-Aib-Cys-NHMe has been determined by X-ray diffraction. The peptide crystallizes in the space group P212121, with A = 8.646(1), B = 18.462(2), C = 19.678(3)Å and Z = 4. The molecules adopt a highly folded compact conformation, stabilized by two intramolecular 4→ 1 hydrogen bonds between the Cys (1) and Pro (2) CO groups and the Cys (4) and methylamide NH groups, respectively. The backbone conformational angles for the peptide lie very close to those expected for a 310 helix. The S-S bridge adopts a right handed twist with a dihedral angle of 82°. The structure illustrates the role of stereochemically constrained residues, in generating novel peptide conformations. Aib, α-aminoisobutyric acid; Z, benzyloxycarbonyl; Boc, t-butyloxycarbonyl; OMe, methyl ester; OBz, benzyl ester; NHMe, N-methylamide; Tosyl, p-toluenesulfonyl.

Conformations of disulfide bridges in proteins

The conformational characteristics of disulfide bridges in proteins have been analyzed using a dataset of 22 protein structures, available at a resolution of 2.0 Å, containing a total of 72 disulfide crosslinks. The parameters used in the analysis include (φ, Ψ) values at Cys residues, bridge dihedral angles χss, χ1i, χ1j, χ2i and χ2j the distances Cαi-Cαj and Cβi-Cβj between the Cα and Cβ atoms of Cys(i) and Cys(j). Eight families of bridge conformations with three or more occurrences have been identified on the basis of these stereochemical parameters. The most populated family corresponds to the "left handed spiral" identified earlier by Richardson ((1981) Adv. Protein Chem. 34, 167–330). Disulfide bridging across antiparallel extended strands is observed in α-lytic protease, crambin, and β-trypsin and this structure is shown to be very similar to those obtained in small cystine peptides. Solvent accessible surface area calculations show that the overwhelming majority of disulfide bridges are inaccessible to solvent.

Synthesis of macrocyclic diacyl/dialkyl glycerols containing disulfide tether and studies of their effects upon incorporation in DPPC membranes. Implications in the design of phospholipase A(2) modulators

A general method for the preparation of novel disulfide-tethered macrocyclic diacylglycerols (DAGs) has been described. Overall synthesis involved stepwise protection, acylation, and deprotection to yield the bis(omega-bromoacyl) glycerols. In the crucial macrocyclization step, a unique reagent, benzyltriethylammonium tetrathiomolybdate (BTAT), has been used to convert individual bis(omega-bromoacyl) glycerols to their respective macrocyclic disulfides. DAG 6, which had ether linkages between hydrocarbon chains and the glycerol backbone, was also synthesized from an appropriate precursor using a similar protocol. One of the DAGs (DAG 5) had a carbon-carbon tether instead of a disulfide one and was synthesized using modified Glaser coupling. Preparation of alpha-disulfide-tethered DAG (DAG 4) required an alternative method, as treatment of the bisbromo precursor with BTAT gave a mixture of several compounds from which separation of the target molecule was cumbersome. To avoid this problem, the bisbromide was converted to its corresponding dithiocyanate, which on further treatment with BTAT yielded the desired DAG (DAG 4) in good yield. Upon treatment with the reducing agent dithiothreitol (DTT), the DAGs that contain a disulfide tether could be quantitatively converted to their "open-chain" thiol analogues. These macrocyclic DAGs and their reduced "open-chain" analogues have been incorporated in DPPC vesicles to study their effect on model membranes. Upon incorporation of DAG 1 in DPPC vesicles, formation of new isotropic phases was observed by P-31 NMR, These isotropic phases disappeared completely on opening the macrocyclic ring by a reducing agent. The thermotropic properties of DPPC bilayers having DAGs (1-6) incorporated at various concentrations were studied by differential scanning calorimetry. Incorporation of DAGs in general reduced the cooperativity unit (CU) of the vesicles. Similar experiments with reduced "open-chain" DAGs incorporated in a DPPC bilayer indicated a recovery of CU with respect to their macrocyclic "disulfide" counterparts. The effect of inclusion of these DAGs on the activity of phospholipase A(2) (PLA(2)) was studied in vitro. Incorporation of DAC 1 in DPPC membranes potentiated both bee venom and cobra venom PLA(2) activities.

Synthesis of novel disulfide containing macrocyclic diacylglycerols

New diacylglycerols (2-4) containing intramolecular disulfide linkages between pendant acyl chains were synthesized. Due to the differences in the location of disulfide units, the present method allows synthesis of macrocycles that vary in sizes. Copyright.

Disulfide luminescence. Emission characteristics of cyclic tetrapeptide disulfides

Luminescence has been detected in cyclic tetrapeptide disulfides containing only nonaromatic residues. Excitation of the S-S- n-cr transition between 280 and 290 nm leads to.ernission in the region 300-340 nm. The position and intensity of the emission band depends on the stereochemistry of the peptide and polarity of the solvent. Quantum yields ranging from 0.002 to 0.026 have been determined. Disulfide luminescence is quenched by oxygen and enhanced in solutions saturated with nitrogen. Contributions from disulfide linkages should be considered, when analysing the emission spectra of proteins, lacking tryptophan but having a high cystine content.

Conformational analysis of small disulfide loops. Spectroscopic and theoretical studies on a synthetic cyclic tetrapeptide containing cystine

The conformational analysis of the synthetic peptide Boc-Cys-Pro-Val-Cys-NHMe has been carried out, as a model for small disulfide loops, in biologically active polypeptides. 'H NMR studies (270 MHz) establish that the Val(3) and Cys(4) NH groups are solvent shielded, while 13C studies establish an all-trans peptide backbone. Circular dichroism and Raman spectroscopy provide evidence for a right-handed twist of the disulfide bond. Analysis of the vicinal (JaB)c oupling constants for the two Cys residues establishes that XI - *60° for Cys(4), while some flexibility is suggested at Cys( 1). Conformational energy calculations, imposing intramolecular hydrogen bonding constraints, favor a P-turn (type I) structure with Pro(2)-Va1(3) as the corner residues. Theoretical and spectroscopic results are consistent with the presence of a transannular 4 - 1 hydrogen bond between Cys( 1) CO and Cys(4) NH groups, with the Val NH being sterically shielded from the solvent environment.

Easy to build four-terminal AC bridge

A simple four-terminal AC bridge is described which can be used with germanium resistance thermometers down to 1 K. The special features of the bridge are its ease of fabrication and extremely low cost.

Crystal and molecular structure of the quinoxaline antibiotic analog TANDEM (des-N-tetramethyltriostin A)

The crystal structure of TANDEM (des-N-tetramethyltriostin A), a synthetic analogue of the quinoxaline antibiotic triostin A, has been determined independently at -135 and 7 'C and refined to R values of 0.088 and 0.147, respectively. The molecule has approximate 2-fold symmetry, with the quinoxaline chromophores and the disulfide cross-bridge projecting from opposite sides of the peptide ring. The quinoxaline groups are nearly parallel to each other and separated by about 6.5 A. The peptide backbone resembles a distorted antiparallel 13 ribbon joined by intramolecular hydrogen bonds N-H(LVal)--O(L-Ala). At low temperatures, the TANDEM molecule is surrounded by a regular first- and second-order hydration sphere containing 14 independent water molecules. At room temperature, only the first-order hydration shell is maintained. Calculations of the interplanar separation of the quinoxaline groups as a function of their orientation with respect to the peptide ring support the viability of TANDEM to intercalate bifunctionally into DNA.

A simple firing delay control circuit for bridge converters

A simple ramp control firing circuit, suitable for use with fully controlled, line-commutated thyristor bridge circuits, is discussed here. This circuit uses very few components and generates the synchronized firing pulses in a simple way. It operates from a single 15 V Supply and has an inherent pulse inhibit facility. This circuit provides the synchronized firing pulses for both thyristors of the same limb in a bridge. To ensure reliability, wide triggering pulses are used, which are modulated to pass through the pulse transformers1 and demodulated before being fed to the thyristor gates. The use of throe such circuits only for a three-phase bridge is discussed.