Attention is focused on L-5,5-dimethylproline (dmP) as a substitute to lock L-proline (Pro) in a cis conformation in peptides and proteins, to prevent cis/trans isomerization when a protein with cis X-Pro peptide groups unfolds. Procedures have been developed to obtain optically pure L-dmP and to incorporate this sterically hindered residue as the central one in tripeptides that are suitable for fragment coupling to prepare synthetic proteins. Based on the sequences of residues 92-94 (Tyr-Pro-Asn:YPN) and 113-115 (Asn-Pro-Tyr: NPY) in bovine pancreatic ribonuclease A (RNase A), in which the X-Pro peptide groups are in the cis conformation, the tripeptides Ac-Tyr-dmP-Asn (YdmPN) and Ac-Asn-dmP-Tyr (NdmPY) were synthesized, and their structures were determined by 2D 1 H nuclear magnetic resonance (NMR) spectroscopy. YdmPN was found to exist solely in the cis conformation between 6 and 60 °C, whereas NdmPY was found to have some trans component that increased from about 10% to about 21% as the temperature increased over the range between 6 and 80 °C. Both YdmPN and cis-NdmPY adopt a type VI reverse turn, as does proline. The NMR structures of YdmPN and cis-NdmPY are comparable with the X-ray structures of the corresponding portions of RNase A, and the NMR structure of trans-NdmPY is compatible with the X-ray structure of the isolated tripeptide, Ac-NPY. These results demonstrate that L-dmP is a promising substitute for proline in a variety of protein problems to constrain the X-Pro peptide group to the cis conformation.

The spatial and temporal targeting of proteins or protein assemblies to appropriate sites is crucial to regulate the specificity and efficiency of protein-protein interactions, thus dictating the timing and intensity of cell signaling and responses. The resultant dynamic mass redistribution could be manifested by label free optical biosensor, and lead to a novel and functional optical signature for studying cell signaling. Here we applied this technology, termed as mass redistribution cell assay technology (MRCAT), to study the signaling networks of bradykinin B 2 receptor in A431 cells. Using MRCAT, the spatial and temporal relocation of proteins and protein assemblies mediated by bradykinin was quantitatively monitored in microplate format and in live cells. The saturability to bradykinin, together with the specific and dosedependent inhibition by a B 2 specific antagonist HOE140, suggested that the optical signature is a direct result of B 2 receptor activation. The sensitivity of the optical signature to cholesterol depletion by methyl-b-cyclodextrin argued that B 2 receptor signaling is dependent on the integrity of lipid rafts; disruption of these microdomains hinders the B 2 signaling. Modulations of several important intracellular targets with specific inhibitors suggested that B 2 receptor activation results in signaling via at least dual pathways -G s -and G q -mediated signaling. Remarkably, the two signaling pathways counter-regulate each other. Several critical downstream targets including protein kinase C, protein kinase A, and epidermal growth factor receptor had been identified to involve in B 2 signaling. The roles of endocytosis and cytoskeleton modulation in B 2 signaling were also demonstrated.

Upon addition of thrombin, fibrinopeptides A and B are cleaved off from the N-termini of four chains of fibrinogen (Aalpha Bbeta gamma)2, and sites of polymerization are exposed, resulting in formation of a fibrin clot. For the fibrinogen Aalpha chain, cleavage occurs most prevalently at the Arg16-Gly17 peptide bond. About 25-30% of the human fibrinogen Aalpha chains are phosphorylated in nature at the position of Ser3, but the function for this modification is not understood. Previous NMR studies indicated that the N-terminal portion (1ADSGE5) of unphosphorylated fibrinopeptide A does not interact with the surface of bovine thrombin. Kinetic and NMR studies have now been carried out to assess whether phosphorylation at Ser3 allows the N-terminal segment (1ADSGEGDFLAEGGGVR16) to become anchored on the thrombin surface, leading to formation of a catalytically more efficient enzyme-substrate complex. Kinetic results indicate that phosphorylation leads to an approximately 65% increase in substrate specificity (kcat/Km) toward hydrolysis of fibrinogen Aalpha(1-20). 31P NMR studies reveal that the phosphorylated group does interact with thrombin, and 1H line broadening studies suggest that phosphorylation does promote binding of amino acids 1-5. Two-dimensional transferred nuclear Overhauser effect spectroscopy studies of bound fibrinopeptide A(1-16 Ser3P) indicate that phosphorylation allows new through-space interactions involving amino acid residues 1ADSGE5 to be observed. Computational docking of the peptide onto the X-ray structure of thrombin suggests that the phosphate may interact with basic residues at the rim of the heparin binding site of thrombin. As a result, the phosphate may serve as an anionic linker between the fibrinopeptide and the enzyme thrombin.