Lapidus Lab

The Lapidus Lab studies protein folding and aggregation on the nanosecond to millisecond timescales. We use a combination of standard biophysical techniques as well as state-of-the-art methods in microfluidics and laser spectroscopy. Our main interests are understanding the complexity of protein folding at the earliest times and how unfolded protein dynamics affect a protein's propensity to aggregate.

 

 

Observing Reconfiguration in Unfolded Proteins

Tryptophan (W) is excited to a long-lived triplet state that is quenched on contact with cysteine (C). The rates we measure give us an estimate of the volume of the chain and the rate of reconfiguration.





Unfolded proteins reconfigure into many different conformations. Some of these conformations are aggregation competent (red) and some are not (black). If two aggregation competent monomers meet by random translational diffusion, they can stick together to form an oligomer (cyan) or one of them can reconfigure and the complex comes apart. k_O is usually very slow. If reconfiguration (k_1)is the same timescale as bimolecular association (k_bi), then aggregation is more likely than if k_1 is much faster or much slower than k_bi.

The video below shows the different regimes of reconfiguration. Cyan is the dangerous middle regime, red is the safe fast regime and blue is the safe slow regime.



Microfluidic Mixers for Observing Folding in Microseconds

Mixing two solutions is a common way to prompt protein folding, but conventional (stopped-flow) mixers are limited by turbulence to measuring folding after ~1 ms. Turbulence can be eliminated by scaling down the device to microns and mixing can be complete in less than 10 microseconds. The mixer above can be used to observe folding with intrinsic Trp fluorescence, Fluorescence Resonance Energy Transfer (FRET) or Fast Photochemical Oxidation of Proteins (FPOP).

For folding probes that rely on optical absorption we use a serpentine-shaped mixer that forces two liquid streams to turn several corners. Mixing is complete in ~250 microseconds. This mixer can be used to observe folding with Circular Dichroism (CD), Trp-Cys contact quenching and infrared absorption (FTIR).