An extended interaction site determines binding between AP180 and AP2 in clathrin mediated endocytosis.

The early phases of clathrin mediated endocytosis are organized through a highly complex interaction network mediated by clathrin associated sorting proteins (CLASPs) that comprise long intrinsically disordered regions (IDRs). AP180 is a CLASP exclusively expressed in neurons and comprises a long IDR of around 600 residues, whose function remains partially elusive. Using NMR spectroscopy, we discovered an extended and strong interaction site within AP180 with the major adaptor protein AP2, and describe its binding dynamics at atomic resolution.

Nuclear magnetic resonance/single molecule fluorescence combinations to study dynamic protein systems.

Many proteins require different structural states or conformations for function, and intrinsically disordered proteins, i.e. proteins without stable three-dimensional structure, are certainly an extreme.

Synergies of Single Molecule Fluorescence and NMR for the Study of Intrinsically Disordered Proteins.

Single molecule fluorescence and nuclear magnetic resonance spectroscopy (NMR) are two very powerful techniques for the analysis of intrinsically disordered proteins (IDPs). Both techniques have individually made major contributions to deciphering the complex properties of IDPs and their interactions, and it has become evident that they can provide very complementary views on the distance-dynamics relationships of IDP systems. We now review the first approaches using both NMR and single molecule fluorescence to decipher the molecular properties of IDPs and their interactions.

Quantitative Description of Intrinsically Disordered Proteins Using Single-Molecule FRET, NMR, and SAXS.

Studying the conformational landscape of intrinsically disordered and partially folded proteins is challenging and only accessible to a few solution state techniques, such as nuclear magnetic resonance (NMR), small-angle scattering techniques, and single-molecule Förster resonance energy transfer (smFRET). While each of the techniques is sensitive to different properties of the disordered chain, such as local structural propensities, overall dimension, or intermediate- and long-range contacts, conformational ensembles describing intrinsically disordered proteins (IDPs) accurately should ideally respect all of these properties. Here we develop an integrated approach using a large set of FRET efficiencies and fluorescence lifetimes, NMR chemical shifts, and paramagnetic relaxation enhancements (PREs), as well as small-angle X-ray scattering (SAXS) to derive quantitative conformational ensembles in agreement with all parameters.