Retention and splicing complex (RES) - the importance of cooperativity.

One of the great challenges to structural biologists lies in explaining the complexities of the spliceosome - a ribosome-sized molecular machine that is assembled in a step-wise manner and is responsible for pre-mRNA splicing. The spliceosome is both fascinating and difficult to work with, because of its dynamic nature. At each discrete step of splicing tens of proteins come and go orchestrating the functional transition through massive structural rearrangements. The retention and splicing complex (RES) is an important splicing factor interacting with pre-mRNA at the onset of the first transesterification reaction. RES is a specific splicing factor for a number of genes and is important for controlling pre-mRNA retention in the nucleus. RES is a 71kDa heterotrimer composed of the 3 proteins Pml1p, Bud13p and Snu17p. We solved the 3-dimensional structure of the core of the RES complex as well as the 2 dimers, Snu17p-Bud13p and Snu17p-Pml1p. Further biophysical analysis revealed an astounding cooperativity that governs the assembly of this trimeric complex as well as its interaction with pre-mRNA. The more than 100-fold cooperativity originates from the progressive rigidification of Snu17p upon coupled binding-and-folding of protein regions, which are disordered in the unbound state. Our work highlights the role of cooperativity in the spliceosome and poses new questions about the structure and assembly of the spliceosome

Determination of amyloid core structure using chemical shifts.

Amyloid fibrils are the pathological hallmark of a large variety of neurodegenerative disorders. The structural characterization of amyloid fibrils, however, is challenging due to their non-crystalline, heterogeneous, and often dynamic nature. Thus, the structure of amyloid fibrils of many proteins is still unknown. We here show that the structure calculation program CS-Rosetta can be used to obtain insight into the core structure of amyloid fibrils. Driven by experimental solid-state NMR chemical shifts and taking into account the polymeric nature of fibrils CS-Rosetta allows modeling of the core of amyloid fibrils. Application to the Y145X stop mutant of the human prion protein reveals a left-handed beta-helix

Biomolecular condensation of the microtubule-associated protein tau.

Cells contain multiple compartments dedicated to the regulation and control of biochemical reactions. Cellular compartments that are not surrounded by membranes can rapidly form and dissolve in response to changes in the cellular environment. The physicochemical processes that underlie the formation of non-membrane-bound compartments in vivo are connected to liquid-liquid phase separation of proteins and nucleic acids in vitro. Recent evidence suggests that the protein tau, which plays an important role in Alzheimer's disease and other neurodegenerative disorders, phase separates in solution, forms tau phases with microtubules, and associates with phase-separated RNA-binding protein granules in cells. Here we review the experimental evidence that supports the ability of tau to phase separate in solution and form biomolecular condensates in cells. As for other disease-relevant proteins, the physiological and pathological functions of tau are tightly connected - through loss of normal function or gain of toxic function - and we therefore discuss how tau phase separation plays a role for both, and with respect to different cellular functions of tau

Reorientational dynamics of amyloid-β from NMR spin relaxation and molecular simulation.

Amyloid-β (Aβ) aggregation is a hallmark of Alzheimer's disease. As an intrinsically disordered protein, Aβ undergoes extensive dynamics on multiple length and time scales. Access to a comprehensive picture of the reorientational dynamics in Aβ requires therefore the combination of complementary techniques. Here, we integrate 15N spin relaxation rates at three magnetic fields with microseconds-long molecular dynamics simulation, ensemble-based hydrodynamic calculations, and previously published nanosecond fluorescence correlation spectroscopy to investigate the reorientational dynamics of Aβ1-40 (Aβ40) at single-residue resolution. The integrative analysis shows that librational and dihedral angle fluctuations occurring at fast and intermediate time scales are not sufficient to decorrelate orientational memory in Aβ40. Instead, slow segmental motions occurring at ∼5 ns are detected throughout the Aβ40 sequence and reach up to ∼10 ns for selected residues. We propose that the modulation of time scales of reorientational dynamics with respect to intra- and intermolecular diffusion plays an important role in disease-related Aβ aggregation