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The scaffold protein PSD-95 links postsynaptic receptors to sites of presynaptic neurotransmitter release. Flexible linkers between folded domains in PSD-95 enable a dynamic supertertiary structure. Interdomain interactions within the PSG supramodule, formed by P DZ3, S H3, and G uanylate Kinase domains, regulate PSD-95 activity. Here we combined discrete molecular dynamics and single molecule Förster resonance energy transfer (FRET) to characterize the PSG supramodule, with time resolution spanning picoseconds to seconds. We used a FRET network to measure distances in full-length PSD-95 and model the conformational ensemble. We found that PDZ3 samples two conformational basins, which we confirmed with disulfide mapping. To understand effects on activity, we measured binding of the synaptic adhesion protein neuroligin. We found that PSD-95 bound neuroligin well at physiological pH while truncated PDZ3 bound poorly. Our hybrid structural models reveal how the supertertiary context of PDZ3 enables recognition of this critical synaptic ligand. 

Fluorescent proteins (FPs) have become fundamental tools for live cell imaging. Most FPs currently used are members of the green fluorescent protein super-family, but new fluorophores such as bilin-FPs are being developed and optimized. In particular, the UnaG FP incorporates bilirubin (BR) as a chromophore, enhancing its fluorescence quantum yield by three orders of magnitude relative to that in solution. To investigate the mechanism of this dramatic enhancement and provide a basis for further engineering of UnaG and other tetrapyrrole-based fluorophores, we performed picosecond fluorescence and femtosecond transient absorption measurements of BR bound to UnaG and its N57A site-directed mutant. The dynamics of wt-UnaG, which has a fluorescence QY of 0.51, are largely homogeneous, showing an excited state relaxation of ∼200 ps, and a 2.2 ns excited-state lifetime decay with a kinetic isotope effect (KIE) of 1.1 for D 2 O vs. H 2 O buffer. In contrast, for UnaG N57A (fluorescence QY 0.01) the results show a large spectral inhomogeneity with excited state decay timescales of 47 and 200 ps and a KIE of 1.4. The non-radiative deactivation of the excited state is limited by proton transfer. The loss of direct hydrogen bonds to the endo -vinyl dipyrrinone moiety of BR leads to high flexibility and structural heterogeneity of UnaG N57A, as seen in the X-ray crystal structure.