Near-Wall Aggregation of Amyloidogenic Aβ 1-40 Peptide: Direct Observation by the FRET

The formation of amyloid fibrils is one of the variants of the self-organization of polypeptide chains. For the amyloid aggregation, the solution must be oversaturated with proteins. The interface of the liquid (solution) and solid (vessel walls) phases can trigger the adsorption of protein molecules, and the resulting oversaturation can initiate conformational transitions in them. In any laboratory experiment, we cannot exclude the presence of surfaces such as the walls of vessels, cuvettes, etc. However, in many works devoted to the study of amyloid formation, this feature is not considered. In our work, we investigated the behavior of the Aβ 1-40 peptide at the water–glass, water–quartz, and water–plastic interface. We carried out a series of simple experiments and showed that the Aβ 1-40 peptide is actively adsorbed on these surfaces, which leads to a significant interaction and aggregation of peptides. This means that the interface can be the place where the first amyloid nucleus appears. We suggest that this effect may also be one of the reasons for the difficulty of reproducing kinetic data when studying the aggregation of the amyloid of the Aβ 1-40 peptide and other amyloidogenic proteins

Residue Folding Degree—Relationship to Secondary Structure Categories and Use as Collective Variable

Recently, we have shown that the residue folding degree, a network-based measure of folded content in proteins, is able to capture backbone conformational transitions related to the formation of secondary structures in molecular dynamics (MD) simulations. In this work, we focus primarily on developing a collective variable (CV) for MD based on this residue-bound parameter to be able to trace the evolution of secondary structure in segments of the protein. We show that this CV can do just that and that the related energy profiles (potentials of mean force, PMF) and transition barriers are comparable to those found by others for particular events in the folding process of the model mini protein Trp-cage. Hence, we conclude that the relative segment folding degree (the newly proposed CV) is a computationally viable option to gain insight into the formation of secondary structures in protein dynamics. We also show that this CV can be directly used as a measure of the amount of α-helical content in a selected segment.

A quantitative model predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions

ABSTRACTN6-methyladenosine (m6A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites. The modification also slows biochemical processes through mechanisms that are not understood. Using temperature-dependent (20°C–65°C) NMR relaxation dispersion, we show that m6A pairs with uridine with the methylamino group in the anti conformation to form a Watson-Crick base pair that transiently exchanges on the millisecond timescale with a singly hydrogen-bonded low-populated (1%) mismatch-like conformation in which the methylamino group is syn. This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with different syn:anti isomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m6A robustly slows duplex annealing without affecting melting at elevated temperatures via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions, and provides an explanation for why the modification robustly slows diverse cellular processes.

Structural dynamics determine voltage and pH gating in human voltage-gated proton channel

Voltage-gated ion channels are key players of electrical signaling in cells. As a unique subfamily, voltage-gated proton (Hv) channels are standalone voltage sensors without separate ion conductive pores. They are gated by both voltage and transmembrane proton gradient (i.e ΔpH), serving as acid extruders in most cells. Amongst their many functions, Hv channels are known to regulate the intracellular pH of human spermatozoa and compensate for the charge and pH imbalances caused by NADPH oxidases in phagocytes. Like the canonical voltage sensors, the Hv channel is a bundle of 4 helices (named S1 through S4), with the S4 segment carrying 3 positively charged Arg residues. Extensive structural and electrophysiological studies on voltage-gated ion channels generally agree on an outwards movement of the S4 segment upon activating voltage, but the real time conformational transitions are still unattainable. With purified human voltage-gated proton (hHv1) channel reconstituted in liposomes, we have examined its conformational dynamics at different voltage and pHs using the single molecule fluorescence resonance energy transfer (smFRET). Here we provided the first glimpse of real time conformational trajectories of the hHv1 voltage sensor and showed that both voltage and pH gradient shift the conformational dynamics of the S4 segment to control channel gating. Our results suggested the biological gating is determined by the conformational distributions of the hHv1 voltage sensor, rather than the conformational transitions between the presumptive ‘resting’ and ‘activated’ conformations. We further identified H140 as the key residue sensing extracellular pH and showed that both the intracellular and extracellular pH sensors act on the voltage sensing S4 segment to enrich the resting conformations. Taken together, we proposed a model that explains the mechanisms underlying voltage and pH gating in Hv channels, which may also serve as a general framework to understand the voltage sensing and gating in other voltage-gated ion channels.

Dynamics in Fip1 regulate eukaryotic mRNA 3'-end processing

Cleavage and polyadenylation factor (CPF/CPSF) is a multiprotein complex essential for mRNA 3ʹ-end processing in eukaryotes. It contains an endonuclease that cleaves pre-mRNAs, and a polymerase that adds a poly(A) tail onto the cleaved 3ʹ-end. Several CPF subunits, including Fip1, contain intrinsically-disordered regions (IDRs). IDRs within multiprotein complexes can be flexible, or can become ordered upon interaction with binding partners. Here, we show that yeast Fip1 anchors the poly(A) polymerase Pap1 onto CPF via an interaction with zinc finger 4 of another CPF subunit, Yth1. We also reconstitute a fully recombinant 850-kDa CPF. By incorporating selectively-labelled Fip1 into recombinant CPF, we could study the dynamics of this single protein within the megadalton complex using nuclear magnetic resonance spectroscopy (NMR). This reveals that a Fip1 IDR that connects the Yth1- and Pap1-binding sites remains highly dynamic within CPF. Together, our data suggest that Fip1 dynamics mediate conformational transitions within the 3ʹ-end processing machinery to coordinate cleavage and polyadenylation.