High Off Rate Research Articles

Site-Directed Chemical Probing to map transient RNA/protein interactions.

RNA-protein interactions are at the bases of many biological processes, forming either tight and stable functional ribonucleoprotein (RNP) complexes (i.e. the ribosome) or transitory ones, such as the complexes involving RNA chaperone proteins. To localize the sites where a protein interacts on an RNA molecule, a common simple and inexpensive biochemical method is the footprinting technique. The protein leaves its footprint on the RNA acting as a shield to protect the regions of interaction from chemical modification or cleavages obtained with chemical or enzymatic nucleases. This method has proven its efficiency to study in vitro the organization of stable RNA-protein complexes. Nevertheless, when the protein binds the RNA very dynamically, with high off-rates, protections are very often difficult to observe. For the analysis of these transient complexes, we describe an alternative strategy adapted from the Site Directed Chemical Probing (SDCP) approach and we compare it with classical footprinting. SDCP relies on the modification of the RNA binding protein to tether an RNA probe (usually Fe-EDTA) to specific protein positions. Local cleavages on the regions of interaction can be used to localize the protein and position its domains on the RNA molecule. This method has been used in the past to monitor stable complexes; we provide here a detailed protocol and a practical example of its application to the study of Escherichia coli RNA chaperone protein S1 and its transitory complexes with mRNAs.

Enantioselective synthesis of adamantylalanine and carboranylalanine and their incorporation into the proteasome inhibitor bortezomib.

The incorporation of adamantylalanine and carboranylalanine at the P2 site of bortezomib is well tolerated and provided potent cell permeable proteasome inhibitors with increased off-rates compared to bortezomib. Adamantylalanine and carboranylalanine were synthesized enantioselectively by an asymmetric Strecker reaction on Ellmans tert-butyl sulfinimines.

Affinity and kinetics of selectin‐carbohydrate interaction

Soluble oligosaccharide mimetics of natural selectin ligands act as competitive inhibitors of leukocyte adhesion during inflammation. We quantified the binding of simple oligosaccharides based on sialyl Lewis-X (sLeX) and complex molecules with the core-2 structure to L- and P-selectin, under static and fluid flow conditions. Surface plasmon resonance (SPR) studies quantified binding kinetics. We observed that: i) The functional group at the anomeric position of carbohydrates play an important role during selectin recognition, since sLeX and sialyl Lewis-a were ~5–7 fold poorer inhibitors of L-selectin mediated cell adhesion compared to their methyl glycosides. ii) Despite their homology to physiological glycans, carbohydrates of GlyCAM-1 and PSGL-1 bound selectins with low-affinity. Thus, besides the carbohydrate portion, the protein core of GlyCAM-1 or the presentation of sugars in clusters on this glycoprotein may contribute to selectin recognition. iii) A novel, neutrally-charged small molecule was identified which blocked L- and P-selectin binding at 30–100 fold lower doses than sLeX. iv) SPR experiments determined that a sLeX-analog (TBC1269) competitively inhibited via steric/allosteric mechanisms, the binding of two anti-P-selectin function blocking antibodies that recognized different epitopes of P-selectin. v) TBC1269 bound P-selectin via both calcium-dependent and -independent mechanisms, with KD of ~0.11mM and high off-rates (>3/s). Overall, our study provides new insight into the kinetics and mechanism of carbohydrate interaction with selectins. Supported by NIH HL63014, HL77258

Novel Interaction Partners of the CD2BP2-GYF Domain

The GYF domain of CD2BP2 serves as an adapter that recognizes proline-rich sequences in intracellular proteins. Although the T cell adhesion molecule CD2 and the core splicing protein SmB/B' were previously shown to interact with CD2BP2-GYF, we are now using a general approach to identify putative GYF domain target sites within the human proteome. The phage display-derived recognition motif for CD2BP2-GYF is PPG(W/F/Y/M/L). SPOT analysis confirmed that the GYF domain interacts with peptides from human proteins containing the consensus site. Epitope mapping by NMR spectroscopy performed for several peptides revealed a conserved binding surface. A direct interaction of the CD2BP2-GYF domain with the novel protein interaction partners PI31 and NPWBP was verified by yeast two-hybrid analysis.