The rebinding kinetics is dependent the local concentration,, that is calculated as that of one molecule within a half-sphere with radius, r. The different binding modes in panel a are designated by ‘AAaa’ for the partially bound complexes (green), by ‘aAAa’ for the bivalently bound complex (red) and by ‘aaAAaa’ for ‘ternary’ complex with two partly bound ligands (blue).
#Scaffold protein assembly full
( a) Thermodynamic scheme for homobivalent ligand, ‘aa’- target, ‘AA’, interactions (see also Figure 4-figure supplement 1 for full scheme). ( f) Representative images showing time dependent PICK1 dissociation from LKV and LKI. LKI is fitted to a one-state dissociation with a half-life of 431 ± 16 min. LKV is fitted to a two-state dissociation with estimated fast and slow half-life of 21 ± 8 and 373 ± 51 min., respectively. ( e) Representative PICK1 dissociation curves from SCMS expressing LKV or LKI (points are means ±SD). ( d) Representative images showing time dependent PICK1 binding to LKV and LKI. Half maximum binding values are 24 ± 14 min for LKV, and 11 ± 4 min for LKI (means ±s.e.m, n = 3). ( c) Representative PICK1 binding to SCMS expressing LKV or LKI as a function of incubation time. ( b) Representative images demonstrating concentration dependent binding of PICK1 to SCMS expressing LKV, LKI and LKA constructs. ( i) Quantification of concentration dependent binding of PICK1 to TAC-YFP-GluA2 (K d *=73 ± 19 nM) (n = 3). ( g) Schematic representation of TAC-YFP-GluA2 constructs ( h) Representative end points of PICK1 concentrations series. ( f) Quantification of concentration dependent binding of PICK1 on SCMS’s expressing SF-GluA2 (black) (K d *=67 ± 6 nM), or SF-GluA2 +A (red), (n = 3, ****p≤0.0001). ( e) Expression levels of SF-GluA2 and SF-GluA2 +A in SCMS were similar (p=0.66). ( d) Schematic representation of SF-GluA2 constructs labeled with anti-FLAG M1-alexa 488 antibody. ( b) Representative confocal images demonstrating concentration dependent binding of PICK1 (red) to SCMS expressing SF-GluA2 (green), or ( c) endpoints for SF-GluA2 +A. Binding was quantified by measuring the intensity of PICK1, I PICK1, relative to the intensity of the receptor, I rec, in a region of interest (dashed white lines). SCMS were incubated with buffer containing fluorescently labeled scaffolding protein (red). Single bilayers were readily distinguished from undisrupted cells or organelles by their lack of three-dimensional structure.
( a) SCMS were prepared from HEK293 Grip tite cells transfected with a fluorescently labeled (green) membrane protein of interest (left) by pressing a pre-coated glass coverslip onto the cells (middle) and subsequently detaching the apical plasma membranes from the remains of the cells (right). Our data supported by simulations suggest that intrinsic PDZ domain affinities are finely tuned and encode specific cellular responses, enabling multiplexed cellular functions of PDZ scaffolds.īiochemistry chemical biology none post synaptic density scaffold proteins synaptic structure. Interestingly, discrete changes in the intrinsic PICK1 PDZ affinity did not affect overall binding strength but instead revealed dual scaffold modes for PICK1. The kinetics of the binding were remarkably slow and binding strength two-three orders of magnitude higher than the intrinsic affinity for the isolated PDZ interaction. Our data demonstrate how multivalent protein-protein and protein-lipid interactions provide critical avidity for the strong binding between the PDZ domain scaffold proteins, PICK1 and PSD-95, and their cognate transmembrane binding partners. Here, we investigate assembly of PDZ scaffolds using supported cell membrane sheets, a unique experimental setup enabling direct access to the intracellular face of the cell membrane. PDZ domain scaffold proteins are molecular modules orchestrating cellular signalling in space and time.
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