Intrinsically disordered proteins are very common and mediate numerous protein-protein and protein-DNA interactions. While it is clear that these interactions are instrumental for the life of the mammalian cell, there is a paucity of data regarding their molecular binding mechanisms. We have here used short peptides as a model system for intrinsically disordered proteins. Linear free-energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the ratelimiting barrier for binding, in a cooperative fashion. This finding suggests that these disordered peptides first form a weak encounter complex with non-native interactions.The data do not support the recent notion that the affinities of intrinsically disordered proteins towards their targets are generally governed by their association rate constants.Instead, we observe the opposite for peptide-PDZ interactions, namely that changes in K d correlate with changes in k off .3

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Because of their prominent roles in cell-cycle regulation and cancer, the interaction between MDM2 and the intrinsically disordered transactivation domain (TAD) of p53 is exceptionally well-studied. However, although there are numerous computational studies on the interaction mechanism, there is a paucity of experimental data regarding the kinetics and mechanism. We have used stopped flow fluorescence to investigate the binding reaction between MDM2 and TAD from p53 as well as from its paralogs p63 and p73, and in particular, focused on the salt dependence of the interaction. The observed kinetics are consistent with a two-state mechanism within the time frame of the stopped flow methodology; thus, any conformational changes including the previously identified MDM2 lid dynamics must occur on a time scale <5 ms at 10 °C. The association rate constants are similar for the three TADs, and differences in the dissociation rate constants determine the various affinities with MDM2. In contrast to previous studies, we found a relatively small ionic-strength dependence for all three interactions, highlighting the large variation in the role of electrostatics among binding reactions of intrinsically disordered proteins (IDPs). The basal association rate constants in the absence of electrostatic interactions were relatively high (≥2 × 10 M s at 10 °C), suggesting that a large number of initial contacts may lead to a productive complex. Our findings support an emerging picture of "conformational funneling" occurring in the initial stages of interactions involving IDPs and that these early binding events can rely on hydrophobic as well as charge-charge interactions.

Abstract. I have integrated 1233 objects with semimajor axes between 4.6 and 5.8 AU 1000 years back and forward in time. A search for Jupiter horseshoe Trojans in transition from tadpole orbits to non-Trojan orbits and objects in temporary horseshoe orbits was made. A total of 13 objects (5 comets, 3 asteroids and 5 Trojans) were found. In a second group, among 337 new Trojans discovered after the primary investigation, 7 transitional candidates were found. Two objects, 2000 HR 24 and 2002 GP 31 , are falsely identified as Trojans. No likely transitional objects were found in the first object set, but all of them have at least temporary captures in orbit types associated with Jupiter's 1:1 mean motion resonance. Trojans in the first set have short observational arcs which can explain the low probability for a Trojan past. Trojans in the second set have longer arcs and 5 of them are good candidates for being in transition from the Trojan area.

Chronic infection by high risk human papillomavirus (HPV) strains may lead to cancer. Expression of the two viral oncoproteins E6 and E7 is largely responsible for immortalization of infected cells. The HPV E6 is a small (approximately 150 residues) two domain protein that interacts with a number of cellular proteins including the ubiquitin ligase E6-associated protein (E6AP) and several PDZ-domain containing proteins. Our aim was to design a high-affinity binder for HPV E6 by linking two of its cellular targets. First, we improved the affinity of the second PDZ domain from SAP97 for the C-terminus of HPV E6 from the high-risk strain HPV18 using phage display. Second, we added a helix from E6AP to the N-terminus of the optimized PDZ variant, creating a chimeric bivalent binder, denoted PDZbody. Full-length HPV E6 proteins are difficult to express and purify. Nevertheless, we could measure the affinity of the PDZbody for E6 from another high-risk strain, HPV16 (Kd = 65 nM). Finally, the PDZbody was used to co-immunoprecipitate E6 protein from HPV18-immortalized HeLa cells, confirming the interaction between PDZbody and HPV18 E6 in a cellular context.