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Research has been conducted on backbone amide hydrogen bonds. The effect of amide to ester substitution on protein structure stability has been investigated via the study of sequential single ester substitution introduced into the hydrophobic core of GCN4-derived sequence at a and d positions, and the results suggest that backbone engineering via ester substitution can be used in probing backbone hydrogen bond strength.

The synthesis and biological evaluation of penicillamine6-5- tert-butylproline7-oxytocin analogs and comparison with their proline7-oxytocin counterparts has led to the discovery of two potent oxytocin (OT) antagonists: [dPen1,Pen6]-oxytocin ( 1, pA2 = 8.22, EC50 = 6.0 n M) and [dPen1,Pen6,5- tBuPro7]-oxytocin ( 2, pA2 = 8.19, EC50 = 6.5 n M). In an attempt to understand the conformational requirements for their biological activity, spectroscopic analyses of 1 and 2 were performed using 1H NMR, laser Raman and CD techniques. In H2O, oxytocin analogs 1 and 2 exhibited cis-isomer populations of 7% and 35%, respectively. Measurement of the amide proton temperature coefficients revealed solvent shielded hydrogens for Gln4 and Pen6 in the major trans-conformer of 1 as well as for Gln4 in the minor cis-conformer of 2. Few long-distance NOEs were observed, suggesting conformational averaging for analogs 1 and 2 in water; moreover, a lower barrier (16.6 ± 0.2 kcal/mol) for isomerization of the amide N-terminal to 5- tBuPro7 relative to OT was calculated from measuring the coalescence temperature of the Gly9 backbone NH signals in the NMR spectra of 2. Observed bands in the Raman spectra of 1 and 2 correspond to Cβ-S-S-Cβ dihedral angles of +110-115° and ±90°, respectively. In water, acetonitrile and methanol, the CD spectra for 1 exhibited a positive maximum around 236-239 nm; in trifluoroethanol, the spectra shifted and a negative maximum was observed at 240 nm. The CD spectra of 2 were unaffected by solvent changes and exhibited a negative maximum at 236-239 nm. The CD and Raman data both suggested that a conformation having a right-handed screw sense about the disulfide and a χCS−SC dihedral angle value close to 115° was favored for analog 1 in water, methanol and acetonitrile, but not trifluoroethanol, where a ±90° angle was favored. Analog 2 was more resilient to conformational change about the disulfide, and adopted a preferred disulfide geometry corresponding to a ±90° χCS−SC dihedral angle. Monte Carlo conformational analysis of analogs 1 and 2 using distance restraints derived from NMR spectroscopy revealed two prominent conformational minima for analog 1 with disulfide geometries around +114° and +116°. Similar analysis of analog 2 revealed one conformational minimum with a disulfide geometry around +104°. In sum, the conformation about the disulfide in [dPen1,Pen6]-OT ( 1) was shown to be contingent on environment and in TFE, adopted a geometry similar to that of [dPen1,Pen6,5- tBuPro7]-OT ( 2) which appeared to be stabilized by hydrophobic interactions between the 5- tBuPro7 (5 R)- tert-butyl group, the Leu8 isopropyl sidechain and the Pen6β-methyl substituents. In light of the conformational rigidity of 2 about the disulfide bond, and the similar geometry adopted by 1 in TFE, a S-S dihedral angle close to +110° may be a prerequisite for their binding at the receptor. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.

Inhibitor of apoptosis (IAP) proteins are antiapoptotic regulators that block cell death in response to diverse stimuli. They are expressed at elevated levels in human malignancies and are attractive targets for the development of novel cancer therapeutics. Herein, we demonstrate that small-molecule IAP antagonists bind to select baculovirus IAP repeat (BIR) domains resulting in dramatic induction of auto-ubiquitination activity and rapid proteasomal degradation of c-IAPs. The IAP antagonists also induce cell death that is dependent on TNF signaling and de novo protein biosynthesis. Additionally, the c-IAP proteins were found to function as regulators of NF-kappaB signaling. Through their ubiquitin E3 ligase activities c-IAP1 and c-IAP2 promote proteasomal degradation of NIK, the central ser/thr kinase in the noncanonical NF-kappaB pathway.