The importance of the compact disordered state in the fuzzy interactions between intrinsically disordered proteins.

The intrinsically disordered C-terminal domain (CTD) of protein 4.1G is able to specifically bind a 26-residue intrinsically disordered region of NuMA, forming a dynamic fuzzy complex. As one of a few cases of extremely fuzzy interactions between two intrinsically disordered proteins/regions (IDPs/IDRs) without induced folding, the principle of the binding is unknown. Here, we combined experimental and computational methods to explore the detailed mechanism of the interaction between 4.1G-CTD and NuMA. MD simulations suggest that the kinetic hub states in the structure ensemble of 4.1G-CTD are favorable in the fuzzy complex. The feature of these hub states is that the binding 'hot spot' motifs ßA and ßB exhibit ß strand propensities and are well packed to each other. The binding between 4.1G-CTD and NuMA is disrupted at low pH, which changes the intramolecular packing of 4.1G-CTD and weakens the packing between ßA and ßB motifs. Low pH conditions also lead to increased hydrodynamic radius and acceleration of backbone dynamics of 4.1G-CTD. All these results underscore the importance of tertiary structural arrangements and overall compactness of 4.1G-CTD in its binding to NuMA, i.e. the compact disordered state of 4.1G-CTD is crucial for binding. Different from the short linear motifs (SLiMs) that are often found to mediate IDP interactions, 4.1G-CTD functions as an intrinsically disordered domain (IDD), which is a functional and structural unit similar to conventional protein domains. This work sheds light on the molecular recognition mechanism of IDPs/IDRs and expands the conventional structure-function paradigm in protein biochemistry.

Principles of fluorescence correlation spectroscopy applied to studies of biomolecular liquid-liquid phase separation.

Fluorescence correlation spectroscopy (FCS) investigates the temporal relationship of fluctuating fluorescence signals reflecting underlying molecular processes occurring in a solution sample or a single live cell. This review article introduces the principles of two basic and most used FCS techniques: fluorescence auto-correlation spectroscopy (FACS) and fluorescence cross-correlation spectroscopy (FCCS). Combined, FACS and FCCS techniques can quantitatively analyze multiple properties of molecule or nanoparticle samples, including molar concentration, diffusion coefficient and hydrodynamic radius, homo- or hetero-interaction, fluorescence brightness, etc. Not surprisingly, FCS techniques have long been used to investigate molecular mechanisms of biomolecular phase separation, first in the lipid bilayer and more recently in cell cytosol and nucleoplasm. The latter applications are especially exciting since a whole new class of membraneless cellular organelles have been discovered, which are proposed to be results of biomolecule liquid-liquid phase separation (LLPS). LLPS research can benefit significantly from the multifunctionality and single-molecule sensitivity of a variety of FCS techniques, particularly for live-cell studies. This review illustrates how FACS and FCCS techniques can be used to investigate multiple aspects of the molecular mechanisms of LLPS, and summarizes FCS applications to LLPS research in vivo and in vitro.

A two-dimensional fingerprint nanoprobe based on black phosphorus for bio-SERS analysis and chemo-photothermal therapy.

Flake-shaped nanohybrids based on black phosphorus (BP) have been developed as multifunctional theranostic nanoplatforms for drug delivery, phototherapy and bioimaging. In this work, we report a facile strategy for fabrication of black phosphorus-Au nanoparticle hybrids (BP-AuNPs), which reveal an extraordinary near-infrared (NIR) photothermal transduction efficiency and drug delivery capacity. The applications of the nanocomposites as therapeutic agents for high-performance chemo-photothermal tumor therapy are accomplished in vitro and in vivo. BP-AuNPs also exhibit wonderful surface-enhanced Raman scattering (SERS) activity under NIR laser excitation with a low Raman background, allowing BP-AuNPs to be used as a promising two-dimensional (2D) fingerprint nanoprobe for bio-SERS analysis. The cellular component identification and label-free live-cell bioimaging based on this type of 2D SERS substrate are generally investigated, which open up promising new perspectives in nanomedicine, including diagnosis, imaging and therapy.