SEMPRE: spectral editing mediated by paramagnetic relaxation enhancement.

Paramagnetic relaxation enhancement (PRE) has become a useful and widely applied tool in biomolecular NMR spectroscopy. In particular investigations of large complexes or transient contacts benefit from PRE effects. Frequently such studies involve modification of the biomacromolecules under study. We here present a method for editing NMR spectra by utilizing a soluble gadolinium complex that broadens nuclear spins being at or close to the macromolecule-solvent interface. NOE signals in NOESY spectra are selectively attenuated if surface exposed nuclear spins are involved. HSQC-type spectra with paramagnetic agent contain only signals of the interior of the protein, while the corresponding difference spectra harbor signals allocated to surface spins. Thus, the number of signals can be reduced helping to minimize spectral overlap in large proteins. The method reveals additional information about the localization of spins being helpful for structure determination of large complexes.

Prosequence switching: an effective strategy to produce biologically active E. coli heat-stable enterotoxin STh.

Enterotoxigenic Escherichia coli (ETEC) infections account for the majority of cases of acute secretory diarrhea. The causative agents are enterotoxins secreted by ETEC, among them is the heat-stable enterotoxin, STh. STh is a 19-amino acid peptide containing three disulfide bonds that stimulates fluid secretion in the bowel by binding to the receptor domain of intestinal guanylyl cyclase C (GC-C). Since GC-C agonists have pharmacologic potential for diagnosis and treatment of disorders such as constipation-predominant irritable bowel syndrome (IBS-C), chronic constipation, and colorectal carcinoma, it is crucial to develop methods for the large-scale production of STh and related peptides. Here, we present a strategy for recombinant expression of STh that relies on the use of the prosequence of human uroguanylin to support proper folding and disulfide bond formation. The chimeric protein CysCys-STh consisting of the propeptide of uroguanylin as N-terminus and the STh peptide as C-terminus was expressed in E. coli, and an efficient purification protocol was developed. Trypsin digestion of this protein released the enterotoxin which could be obtained in high purity. NMR and mass spectrometry confirmed the identity and homogeneity of the toxin, and its biological activity was confirmed by a cell-based in vivo assay. The expression scheme introduced here represents a cost-efficient and scalable way of STh production.

Cure and curse: E. coli heat-stable enterotoxin and its receptor guanylyl cyclase C.

Enterotoxigenic Escherichia coli (ETEC) associated diarrhea is responsible for roughly half a million deaths per year, the majority taking place in developing countries. The main agent responsible for these diseases is the bacterial heat-stable enterotoxin STa. STa is secreted by ETEC and after secretion binds to the intestinal receptor guanylyl cyclase C (GC-C), thus triggering a signaling cascade that eventually leads to the release of electrolytes and water in the intestine. Additionally, GC-C is a specific marker for colorectal carcinoma and STa is suggested to have an inhibitory effect on intestinal carcinogenesis. To understand the conformational events involved in ligand binding to GC-C and to devise therapeutic strategies to treat both diarrheal diseases and colorectal cancer, it is paramount to obtain structural information on the receptor ligand system. Here we summarize the currently available structural data and report on physiological consequences of STa binding to GC-C in intestinal epithelia and colorectal carcinoma cells.

The therapeutically anti-prion active antibody-fragment scFv-W226: paramagnetic relaxation-enhanced NMR spectroscopy aided structure elucidation of the paratope-epitope interface.

Antibodies have become indispensable reagents with numerous applications in biological and biotechnical analysis, in diagnostics as well as in therapy. In all cases, selective interaction with an epitope is crucial and depends on the conformation of the paratope. While epitopes are routinely mapped at high throughput, methods revealing structural insights on a rather short timescale are rare. We here demonstrate paramagnetic relaxation-enhanced (PRE) NMR spectroscopy to be a powerful tool unraveling structural information about epitope-orientation in a groove spanned by the complementary determining regions. In particular, we utilize the spin label TOAC, which is fused to the peptidic epitope using standard solid-phase chemistry and which is characterized by a reduced mobility compared to, e.g., spin labels attached to the side-chain functionalities of cysteine or lysine residues. We apply the method to determine the orientation of helix 1 of the prion protein, which is the epitope for the therapeutically anti-prion active scF(v) fragment W226.