Lanthanide-binding peptides and the enzymes that Might Have Been.

The trivalent lanthanide ions are chemically similar to Ca(II) ions, making them useful Ca analogs for a multitude of applications. In addition, Ln(III) ions are efficient catalysts of hydrolysis due to their much stronger Lewis acidity relative to Ca(II) ions. Ln-binding peptides thus offer both the opportunity to study known Ca sites as well as to explore new biological functions with an entire family of spectroscopically rich and reactive ions. This review discusses Ln-binding peptides in three roles: (i) as models of Ca-protein structure and function, (ii) as spectroscopic tags for protein expression and characterization and (iii) as designed artificial endonucleases. The creation of hydrolytically active Ln peptides that can fold, bind, cleave and discriminate among substrates shows that the design of Ln enzymes can be accomplished, and they will serve as versatile biochemical tools to investigate protein folding, structure and nuclease function.

Chimeric HTH motifs based on EF-hands.

The design of a new peptide construct from two structurally equivalent basis motifs is reported. A chimera was designed from the helical regions of a helix-turn-helix (HTH) domain, incorporating the consensus EF-hand Ca-binding loop at the turn. Two 33-residue peptides were constructed: one (P3, designed) includes the 12-residue consensus EF-hand loop, while the other (P2, control) contains the reversed EF-hand loop sequence. The Eu(III) and Ca(II) binding properties of P2 and P3 were investigated by circular dichroism and NMR. The designed peptide (P3) is 25% helical in its Eu(III)-saturated form, and 14% helical with excess Ca(II). Both the free and Eu-bound peptides have inherent solution structure, as demonstrated by the helicity induced by the addition of trifluoroethanol solvent. While Eu(III) binding stabilizes the structure of P3, it destabilizes the structure of P2. The NMR titration of P3 with Eu(III) resulted in new resonances characteristic of Ca-bound EF-hand loops. As observed for isolated EF-hands, the resonances appear within the first 0.5 equivalents of Eu(III) added, suggesting that one metal ion organizes two equivalents of peptide to fold into the back-to-back dimer structure of native EF-hands. The EuP3 chimera, but not EuP2, has significant affinity for supercoiled plasmid DNA, causing a gel shift at concentrations as low as 10 microM EuP3 (50 microM base pairs). These results show our chimeric peptide combines the characteristics of the parent motifs, maintaining both metal binding and DNA affinity.

Lanthanide-mediated DNA hydrolysis.

Lanthanide ions are remarkably effective catalysts for the hydrolytic cleavage of phosphate ester bonds, including the robust bonds of DNA. This makes Ln(III) and Ce(IV) ions attractive candidates for developing selective and efficient artificial nucleases, which could have many biochemical and clinical applications. Both small-molecule-based and biopolymer-based lanthanide complexes are being pursued.

Differential DNA recognition by the enantiomers of 1-Rh(MGP)2 phi: a combination of shape selection and direct readout.

The enantiomers of the symmetric metallointercalator complex 1-Rh(MGP)2phi5+ [MGP = 4-(guanidylmethyl)-1,10-phenanthroline; phi = phenanthrenequinone diimine] bound to DNA decamer duplexes containing their respective 6 bp recognition sequences have been investigated using 1H NMR. Shape selection due to the chirality of the metal center and hydrogen-bonding contacts of ancillary guanidinium groups to 3'-G N7 atoms define the recognition by complexes which bind by intercalation to duplex DNA. The titration of Lambda-Rh into the self-complementary decamer containing the recognition sequence (5'-GACATATGTC-3', L1) resulted in one symmetric bound conformation observed in the 1H NMR spectrum, indicating that the DNA duplex retains its symmetry in the presence of the metal complex. Upfield chemical shifts of duplex imino protons and the disruption of the NOE base-sugar contacts defined the central T5-A6 intercalation site. The downfield shift of the G8 imino proton supports the conclusion that the pendant guanidinium arms make simultaneous H-bonding contacts to the N7 atoms of 3'-G8 bases on either side of the site. A variable-temperature study of a partially titrated sample (2:3 Lambda-Rh/L1) showed the exchange rate (kobs) at 298 K to be 68 s-1 and the activation barrier to exchange (DeltaG of association) to be 2.7 kcal/mol, a value comparable to the stacking energy of one base step. The results presented coupled with biochemical data are therefore consistent with binding models in which Lambda-1-Rh(MGP)2phi5+ (Lambda-Rh) traps the recognition site 5'-CATATG-3' in an unwound state, permitting intercalation centrally and hydrogen bonding to guanines at the first and sixth base pair positions. The data suggest a different model of binding and recognition by Delta-Rh. The titration of Delta-Rh into a DNA decamer containing the 6 bp recognition site (D1, 5'-CGCATCTGAC-3'; D2, 5'-GTCAGATGCG-3') resulted in two, distinct conformers, in slow exchange on the NMR time scale. The rate of exchange between the two conformers (kobs) at 298 K is 37 s-1, most likely due to partial dissociation between binding modes. The slower rate relative to Lambda-Rh association reflects the relative rigidity of the D1 and/or D2 sequence in comparison to L1. NOE cross-peaks between the intercalating phi ligand and protons of T5-C6, as well as the upfield shifts observed for imino protons at this step, serve to define the central T5-C6 step as the single site of intercalation. The downfield shift of the 3'-G imino protons indicates the complex makes hydrogen bond contacts with these bases. The complex, which is too small to span a 6 bp B-form DNA sequence, nonetheless makes major groove contacts with 3'-G bases to either side of the site. Notably, both 3'-guanine bases are necessary to impart site specificity and slow dissociation kinetics with the 5'-CATCTG-3' site, as evidenced by the extremely exchange-broadened two-dimensional NOESY spectra of Delta-Rh bound to modified duplexes containing N7-deazaguanine at either G8 or G18; the loss of one major groove contact completely abolishes specificity for 5'-CATCTG-3'. DNA chemical shifts upon binding and intermolecular NOE contacts therefore support a model in which Delta-Rh intercalates in one of two canted binding conformations. Within this model, each intercalation mode allows one guanidinium-guanine hydrogen bond at a time, while bringing the other arm close to the phosphate backbone.

Colorimetric detection of immobilised PCR products generated on a solid support.

By extending functional primers attached to a solid phase and incorporating a digoxigenin label, it is possible to visualise PCR products as discrete spots on specific regions of a solid support after colorimetric detection. The technique has been used for the detection of the point mutation associated with porcine malignant hyperthermia.

Simultaneous detection of a sex-specific sequence and the Ryr1 point mutation in porcine genomic DNA.

The advantages are becoming increasingly apparent of designing livestock breeding programmes around the detection of specific sequences in genomic DNA using amplification by the polymerase chain reaction (PCR). Furthermore, by subjecting the products of such reactions to restriction enzyme digestion, important information conveyed by single-base substitutions can be retrieved and used in marker-assisted selection. The potential for the rapid diagnosis of several DNA markers simultaneously would seem to offer particular benefits in the field of in vitro fertilisation and embryo transfer, where only a few cells constitute the source of the DNA, and where keeping the duration of the tests to a minimum is imperative. However, where the markers to be detected fall into different categories, different kinds of amplification reactions may need to be combined. The present study with porcine DNA combines a one-step multiplex PCR test for sex-determination with a specialised PCR reaction designed to diagnose the Ryrl or 'halothane' genotype. A total of seven primers have been utilised to amplify by, firstly, a control sequence related to the Zfx/y genes present in both sexes, secondly to amplify a Y chromosome sex-specific sequence related to the Sry gene and lastly, to detect either allele of the Ryr1 mutation associated with porcine stress syndrome and pale, soft exudative meat. The presence of PCR products of characteristic size on agarose gel electrophoresis gives a visual read-out of animal sex and halothane genotype. Although primarily a model system, the test may have direct applications in the context of embryo transfer, sperm separation technology and also in the characterisation of pork samples undergoing sensory evaluation by meat scientists.

Use of mutagenically separated PCR for the detection of the mutation associated with porcine stress syndrome.

A point mutation in the Ryr1 gene encoding the ryanodine receptor in porcine skeletal muscle is associated with enhanced growth characteristics and leanness but also with porcine stress syndrome and pale, soft exudative meat in some animals. The current diagnostic test for the mutation is based on the polymerase chain reaction (PCR), followed by a restriction enzyme digestion step, prior to agarose gel electrophoresis. Using a technique known as mutagenically separated PCR (MS-PCR), a one-step procedure for the identification of the point mutation associated with porcine stress syndrome has been developed. This removes the requirement of the current PCR-based test for restriction enzyme digestion, is consequently quicker to perform, and may lend itself more readily to automation. DNA from blood samples from a series of animals were genotyped using both the conventional test and MS-PCR, and complete agreement between the two methods was obtained.

Visual recovery using small dilating eye drops.

It is well established that reduced size dilating eye drops of 1% tropicamide and 10% phenylephrine (micro drops) are effective for clinical purposes. Excellent pupil dilatation (mydriasis) is achieved and pupil constriction does not occur in response to light. In this study, the effect of micro drops of 1% tropicamide on distance and near visual recovery was compared with standard drops in a group of 20 healthy volunteers. For each person studied, one eye was selected at random to be tested first with the standard drop size, and then after a minimum of one week, the same eye was again tested using a drop of the same drug one fifth standard size. An iris photograph, Snellen visual acuity at 6 m, and reading visual acuity was obtained for each test procedure: before drop instillation and at 30 min, 1, 2 and 4 h after drug instillation. Use of the micro drops caused a small but statistically significant improvement in the rate of recovery of distance and near visual acuity. These findings, allied to the known beneficial effects of reduced systemic absorption using micro drops, lend further weight to the argument that mydriasis may be achieved more safely, with fewer side effects, and with earlier return of normal vision when reduced size drops are used. It is hoped that practical micro drop dispensers will be developed.