A Novel Approach Combining Fluorescence-Anisotropy Decays and Microviscometry to Explore the Cotranslational Compaction of Nascent Proteins

Abstract

In recent years, it has become clear that assembly processes based on liquidliquid phase separation (LLPS) of protein and protein-nucleic acid mixtures, acting as membrane-less organelles, play an important role in cellular selfassembly processes. We discuss the combined effects of temperature, pressure, crowding and natural cosolvents on LLPS phenomena of proteins, with a particular focus on the effect of pressure. We studied various systems undergoing LLPS, including lysozyme, crystallin, elastin, LAF1, FUS, and proteins mimicking postsynaptic densities. X-ray and light scattering, microscopy and various spectroscopies were employed to reveal structural changes and mesoscopic phase states of the systems. Corresponding pressure-jump relaxation studies informed about the kinetics of protein droplet formation and their mechanism. Strikingly, for most cases studied thus far, LLPSs are more sensitive to pressure than the folding of proteins, suggesting that organisms inhabiting the deep sea and sub-seafloor sediments, under pressures up to 1 kbar and beyond, have to mitigate this pressure-sensitivity to avoid unwanted destabilization of their functional biomolecular condensates. We found that crowding and particular osmolytes, such as trimethylamine-N-oxide, an osmolyte upregulated in deep-sea fish, can significantly stabilize protein droplets under pressure. We then briefly discuss the effects of aqueous two-phase systems on the pressure dependence of ligand binding and enzymatic reactions as well as the conformational dynamics of nucleic acid structures. These findings are relevant for understanding cellular processes of piezophiles and might have significant bearings on biotechnological applications.

Imaging the rotational mobility of carbon dot-gold nanoparticle conjugates using frequency domain wide-field time-resolved fluorescence anisotropy

“…Single point steady-state and TR-FA measurements are well established and routinely used for various applications in many laboratories. These include studying the dynamic properties of proteins, [18][19][20] estimating the internal viscosities of membranes, 21 performing fluorescencepolarization immunoassays, 22,23 probing molecular proximity manifested by hetero-or homo-FRET assays, 20,24,25 and monitoring cellular activities. 14,26 Nevertheless, since the pioneering work in rotational correlation time imaging of Siegel et al in the time domain (TD) (2003) 4 and Clayton et al in the frequency domain (FD) (2002), 27 only few reports described their extension into two-dimensional (2D) TR-FA imaging (TR-FAIM) in the TD 26,[28][29][30] and almost none in the FD.…”

“…Single point steady-state and TR-FA measurements are well established and routinely used for various applications in many laboratories. These include studying the dynamic properties of proteins, 18 – 20 estimating the internal viscosities of membranes, 21 performing fluorescence-polarization immunoassays, 22 , 23 probing molecular proximity manifested by hetero- or homo-FRET assays, 20 , 24 , 25 and monitoring cellular activities. 14 , 26 Nevertheless, since the pioneering work in rotational correlation time imaging of Siegel et al.…”