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Sparse lipid fluorination enhances the lipids’ 1H signal dispersion, enables clean molecular distinction by 19F NMR, and evinces micelle insertion of proteins via fluorine-induced signal shifts. We present a minimal fluorination scheme, and illustrate the concept on di-(4- fluoro)-heptanoylphosphatidylcholine micelles and solubilised seven-helix transmembrane pSRII protein.

Nucleic acids symposium series 45(3), 1501 - 1515(2017). doi:10.1093/nar/gkw1183, The intrinsically disordered p15$^{PAF}$ regulates DNA replication and repair when interacting with the Proliferating Cell Nuclear Antigen (PCNA) sliding clamp. As many interactions between disordered proteins and globular partners involved in signaling and regulation, the complex between p15$^{PAF}$ and trimeric PCNA is of low affinity, forming a transient complex that is difficult to characterize at a structural level due to its inherent polydispersity. We have determined the structure, conformational fluctuations, and relative population of the five species that coexist in solution by combining small-angle X-ray scattering (SAXS) with molecular modelling. By using explicit ensemble descriptions for the individual species, built using integrative approaches and molecular dynamics (MD) simulations, we collectively interpreted multiple SAXS profiles as population-weighted thermodynamic mixtures. The analysis demonstrates that the N-terminus of p15$^{PAF}$ penetrates the PCNA ring and emerges on the back face. This observation substantiates the role of p15$^{PAF}$ as a drag regulating PCNA processivity during DNA repair. Our study reveals the power of ensemble-based approaches to decode structural, dynamic, and thermodynamic information from SAXS data. This strategy paves the way for deciphering the structural bases of flexible, transient and multivalent macromolecular assemblies involved in pivotal biological processes., Published by Oxford Univ. Press8619, Oxford

Signaling mediated by Notch receptors and their ligands is essential in cell differentiation and morphogenesis in metazoans. As both receptors and ligands are cell-surface expressed proteins, Notch signaling is restricted to nearby interacting cells. The five human ligands of Notch receptors are all single-pass, type I transmembrane proteins consisting of an extracellular region involved in receptor binding and of a 100-150 residue intracellular tail. One of these ligands, Delta-like 4 (DLL4) is a human homologue of Drosophila Delta protein, and plays an important role in the development of blood vessels. The intracellular region of DLL4 (DLL4_IC) is required for receptor/ligand endocytosis, undergoes regulated intra-membrane proteolysis and, through its C-terminal PDZ binding motif, mediates the interaction of DLL4 with Dlg-1, a protein involved in the organization of cell-cell junctions. The sequence of DLL4_IC is very well conserved through evolution but does not encode any domain of known structure. Using a recombinant purified protein expressed from a codon-optimized synthetic gene, we demonstrate through various biophysical methods such as circular dichroism, size-exclusion chromatography, and NMR that DLL4_IC is globally disordered in solution, but can form inter-convertible local secondary structures in response to specific variations in the physico-chemical milieu, as well as in the presence of its target PDZ domain. Most of these conformational changes occur in the functionally relevant C-terminal segment. A computational study on the incidence and location of protein intrinsic disorder in 369 human receptors of the same transmembrane class of DLL4 provides evidence that disorder concentrates in the cytoplasmic tail of these proteins and represents a general phenomenon. In light of these findings, we propose that global disorder in the cytoplasmic tail, in concert with local pre-organization, may play a role in the function of DLL4 as well as in that of other single-pass transmembrane proteins.

The final steps of lagging strand synthesis induce maturation of Okazaki fragments via removal of the RNA primers and ligation. Iterative cycles between Polymerase δ (Polδ) and Flap endonuclease-1 (FEN1) remove the primer, with an intermediary nick structure generated for each cycle. Here, we show that human Polδ is inefficient in releasing the nick product from FEN1, resulting in non-processive and remarkably slow RNA removal. Ligase 1 (Lig1) can release the nick from FEN1 and actively drive the reaction toward ligation. These mechanisms are coordinated by PCNA, which encircles DNA, and dynamically recruits Polδ, FEN1, and Lig1 to compete for their substrates. Our findings call for investigating additional pathways that may accelerate RNA removal in human cells, such as RNA pre-removal by RNase Hs, which, as demonstrated herein, enhances the maturation rate ~10-fold. They also suggest that FEN1 may attenuate the various activities of Polδ during DNA repair and recombination.