Targeted identification of SUMOylation sites in human proteins using affinity enrichment and paralog-specific reporter ions.

Protein modification by small ubiquitin-like modifier (SUMO) modulates the activities of numerous proteins involved in different cellular functions such as gene transcription, cell cycle, and DNA repair. Comprehensive identification of SUMOylated sites is a prerequisite to determine how SUMOylation regulates protein function. However, mapping SUMOylated Lys residues by mass spectrometry (MS) is challenging because of the dynamic nature of this modification, the existence of three functionally distinct human SUMO paralogs, and the large SUMO chain remnant that remains attached to tryptic peptides.

A comparative analysis of the aggregation behavior of amyloid-β peptide variants.

Aggregated forms of the amyloid-β peptide are hypothesized to act as the prime toxic agents in Alzheimer disease (AD). The in vivo amyloid-β peptide pool consists of both C- and N-terminally truncated or mutated peptides, and the composition thereof significantly determines AD risk. Other variations, such as biotinylation, are introduced as molecular tools to aid the understanding of disease mechanisms.

Mapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction.

Understanding the mechanism of protein folding requires a detailed knowledge of the structural properties of the barriers separating unfolded from native conformations. The S-peptide from ribonuclease S forms its α-helical structure only upon binding to the folded S-protein. We characterized the transition state for this binding-induced folding reaction at high resolution by determining the effect of site-specific backbone thioxylation and side-chain modifications on the kinetics and thermodynamics of the reaction, which allows us to monitor formation of backbone hydrogen bonds and side-chain interactions in the transition state.

Modulation of the peptide backbone conformation by the selenoxo photoswitch.

Photocontrol of the backbone conformation is a useful step forward in regulating the bioactivities of peptides and proteins by means of external signals. In the present work, the selenium analogue of a peptide bond was introduced into tetrapeptides to obtain surprisingly stable selenoxo peptides. Selenoxo peptide bonds allow for a marked increase of cis content in the photostationary state of peptide chains when irradiated with UV light near 290 nm.

Effect of thioxopeptide bonds on alpha-helix structure and stability.

Thioxoamide (thioamide) bonds are nearly isosteric substitutions for amides but have altered hydrogen-bonding and photophysical properties. They are thus well-suited backbone modifications for physicochemical studies on peptides and proteins. The effect of thioxoamides on protein structure and stability has not been subject to detailed experimental investigations up to date.

An essential role for Pin1 in Xenopus laevis embryonic development revealed by specific inhibitors.

The peptidyl prolyl cis/trans isomerase (PPIase) Pin1 plays an important role in phosphorylation-dependent events of the cell cycle. This function is linked to its display of two phosphothreonine/phosphoserine-proline binding motifs, one within the type IV WW domain and a second within the parvulin-like catalytic domain. By microinjection of the compound Ac-Phe-D-Thr(PO3H2)-Pip-Nal-Gln-NH2, which inhibits Xenopus laevis Pin1 with a Ki value of 19.

On the benefit of bivalency in peptide ligand/pin1 interactions.

The human peptidyl prolyl cis/trans isomerase (PPIase) Pin1 has a key role in developmental processes and cell proliferation. Pin1 consists of an N-terminal WW domain and a C-terminal catalytic PPIase domain both targeted specifically to Ser(PO(3)H(2))/Thr(PO(3)H(2))-Pro sequences. Here, we report the enhanced affinity originating from bivalent binding of ligands toward Pin1 compared to monovalent binding.

Structural basis for high-affinity peptide inhibition of human Pin1.

Human Pin1 is a key regulator of cell-cycle progression and plays growth-promoting roles in human cancers. High-affinity inhibitors of Pin1 may provide a unique opportunity for disrupting oncogenic pathways. Here we report two high-resolution X-ray crystal structures of human Pin1 bound to non-natural peptide inhibitors.

A nearly isosteric photosensitive amide-backbone substitution allows enzyme activity switching in ribonuclease s.

psi[CS-NH]4-RNase S, a site specific modified version of RNase S obtained by thioxylation (O/S exchange) at the Ala4-Ala5- peptide bond, was used to evaluate the impact of protein backbone photoswitching on bioactivity. psi[CS-NH](4)-RNase S was yielded by recombination of the S-protein and the respective chemically synthesized thioxylated S-peptide derivative. Comparison with RNase S revealed similar thermodynamic stability of the complex and an unperturbed enzymatic activity toward cytidine 2',3'-cyclic monophosphate (cCMP).

Thermodynamics of phosphopeptide binding to the human peptidyl prolyl cis/trans isomerase Pin1.

Proteins containing phosphorylated Ser/Thr-Pro motifs play key roles in numerous regulatory processes in the cell. The peptidyl prolyl cis/trans isomerase Pin1 specifically catalyzes the conformational transition of phosphorylated Ser/Thr-Pro motifs. Here we report the direct analysis of the thermodynamic properties of the interaction of the PPIase Pin1 with its substrate-analogue inhibitor Ac-Phe-D-Thr(PO3H2)-Pip-Nal-Gln-NH2 specifically targeted to the PPIase active site based on the combination of isothermal titration calorimetry and studies on inhibition of enzymatic activity of wt Pin1 and active site variants.

Isothermal calorimetry as a tool to investigate slow conformational changes in proteins and peptides.

A new calorimetric method has been developed to follow the time course of slow conformational changes during the refolding of denatured proteins. The method is based on the ability of isothermal titration calorimeters (ITC) to detect small amounts of heat continuously over a minute to an hour time range without being disturbed by baseline drift. We benchmarked the method on the basis of the slow kinetic phases resulting from prolyl cis/trans isomerization of oligopeptides.

Nanomolar inhibitors of the peptidyl prolyl cis/trans isomerase Pin1 from combinatorial peptide libraries.

The peptidyl prolyl cis/trans isomerase Pin1 has been implicated in the development of cancer, Alzheimer's disease and asthma, but highly specific and potent Pin1 inhibitors remain to be identified. Here, by screening a combinatorial peptide library, we identified a series of nanomolar peptidic inhibitors. Nonproteinogenic amino acids, incorporated into 5-mer to 8-mer oligopeptides containing a d-phosphothreonine as a central template, yielded selective inhibitors that blocked cell cycle progression in HeLa cells in a dose-dependent manner.

The specific FKBP38 inhibitor N-(N',N'-dimethylcarboxamidomethyl)cycloheximide has potent neuroprotective and neurotrophic properties in brain ischemia.

FK506 and FK506-derived inhibitors of the FK506-binding protein (FKBP)-type peptidylprolyl cis/trans-isomerases (PPIase) display potent neuroprotective and neuroregenerative properties in various neurodegeneration models, showing the importance of neuroimmunophilins as targets for the treatment of acute and chronic neurodegenerative diseases. However, the PPIase activity targeted by active site-directed ligands remains unknown so far. Here we show that neurotrophic FKBP ligands, such as GPI1046 and N-[methyl(ethoxycarbonyl)]cycloheximide, inhibit the calmodulin/Ca(2+) (CaM/Ca(2+))-regulated FKBP38 with up to 80-fold higher affinity than FKBP12.

Direct photomodulation of peptide backbone conformations.

Significant photoswitching ability is found for secondary thioxopeptide bonds and can be used for the photomodulation of the backbone conformation of peptides or proteins.