QRB Discovery最新文献

Integrins are critical transmembrane receptors that connect the extracellular matrix (ECM) to the intracellular cytoskeleton, playing a central role in mechanotransduction - the process by which cells convert mechanical stimuli into biochemical signals. The dynamic assembly and disassembly of integrin-mediated adhesions enable cells to adapt continuously to changing mechanical cues, regulating essential processes such as adhesion, migration, and proliferation. In this review, we explore the molecular clutch model as a framework for understanding the dynamics of integrin - ECM interactions, emphasizing the critical importance of force loading rate. We discuss how force loading rate bridges internal actomyosin-generated forces and ECM mechanical properties like stiffness and ligand density, determining whether sufficient force is transmitted to mechanosensitive proteins such as talin. This force transmission leads to talin unfolding and activation of downstream signalling pathways, ultimately influencing cellular responses. We also examine recent advances in single-molecule DNA tension sensors that have enabled direct measurements of integrin loading rates, refining the range to approximately 0.5-4 pN/s. These findings deepen our understanding of force-mediated mechanotransduction and underscore the need for improved sensor designs to overcome current limitations.

Single-stranded nucleic acid (ssNA) binding proteins must both stably protect ssNA transiently exposed during replication and other NA transactions, and also rapidly reorganize and dissociate to allow further NA processing. How these seemingly opposing functions can coexist has been recently elucidated by optical tweezers (OT) experiments that isolate and manipulate single long ssNA molecules to measure conformation in real time. The effective length of an ssNA substrate held at fixed tension is altered upon protein binding, enabling quantification of both the structure and kinetics of protein-NA interactions. When proteins exhibit multiple binding states, however, OT measurements may produce difficult to analyze signals including non-monotonic response to free protein concentration and convolution of multiple fundamental rates. In this review we compare single-molecule experiments with three proteins of vastly different structure and origin that exhibit similar ssNA interactions. These results are consistent with a general model in which protein oligomers containing multiple binding interfaces switch conformations to adjust protein:NA stoichiometry. These characteristics allow a finite number of proteins to protect long ssNA regions by maximizing protein-ssNA contacts while also providing a pathway with reduced energetic barriers to reorganization and eventual protein displacement when these ssNA regions are diminished.

DNA unzipping by nanopore translocation has implications in diverse contexts, from polymer physics to single-molecule manipulation to DNA-enzyme interactions in biological systems. Here we use molecular dynamics simulations and a coarse-grained model of DNA to address the nanopore unzipping of DNA filaments that are knotted. This previously unaddressed problem is motivated by the fact that DNA knots inevitably occur in isolated equilibrated filaments and in vivo. We study how different types of tight knots in the DNA segment just outside the pore impact unzipping at different driving forces. We establish three main results. First, knots do not significantly affect the unzipping process at low forces. However, knotted DNAs unzip more slowly and heterogeneously than unknotted ones at high forces. Finally, we observe that the microscopic origin of the hindrance typically involves two concurrent causes: the topological friction of the DNA chain sliding along its knotted contour and the additional friction originating from the entanglement with the newly unzipped DNA. The results reveal a previously unsuspected complexity of the interplay of DNA topology and unzipping, which should be relevant for interpreting nanopore-based single-molecule unzipping experiments and improving the modeling of DNA transactions in vivo.

Synthetic biology aims to create a viable synthetic cell. However, to achieve this goal, it is essential first to gain a profound understanding of the cellular systems used to build that cell, how to reconstitute those systems in the compartments, and how to track their function. Transcription and translation are two vital cellular systems responsible for the production of RNA and, consequently, proteins, without which the cell would not be able to maintain itself or fulfill its functions. This review discusses in detail how the Protein synthesis Using Recombinant Element (PURE) system and cell lysate are used to reconstitute transcription-translation in vitro. Furthermore, it examines how these systems can be encapsulated in GUVs using the existing methods. It also assesses approaches available to image transcription and translation with a diverse arsenal of fluorescence microscopy techniques and a broad collection of probes developed in recent decades. Finally, it highlights solutions for the challenge ahead, namely the decoupling of the two systems in PURE, and discusses the prospects of synthetic biology in the modern world.

High resolution structures of protein complexes provide a wealth of information on protein structure and function. Databases of these protein structures are also used for artificial-intelligence (AI)-based methods of structural modelling. Despite the wealth of protein structures that have been determined by structural biologists, there are still gaps, or missing pieces in the puzzle of protein structural biology. Highly flexible regions may be missing from protein structures and conformational changes of different protein complex states may not be captured by current databases. In this perspective, I sketch out several ways that cross-linking mass spectrometry can contribute to filling in some of these missing pieces: Identification of cross-linked interactions in highly flexible protein regions not captured by other structural techniques; capturing conformational changes of protein complexes in different functional states; serving as distance constraints in integrative structural modelling and providing structural information of in cellulo proteins. The myriad ways in which cross-linking mass spectrometry contributes to filling in missing pieces in structural biology makes it a powerful technique in structural biology.

Despite major efforts toward its eradication, cholera remains a major health threat and economic burden in many low- and middle-income countries. Between outbreaks, the bacterium responsible for the disease, Vibrio cholerae, survives in aquatic environmental reservoirs, where it commonly forms biofilms, for example, on zooplankton. N-acetyl glucosamine-binding protein A (GbpA) is an adhesin that binds to the chitinaceous surface of zooplankton and breaks its dense crystalline packing thanks to its lytic polysaccharide monooxygenase (LPMO) activity, which provides V. cholerae with nutrients. In addition, GbpA is an important colonization factor associated with bacterial pathogenicity, allowing the binding to mucins in the host intestine. Here, we report the discovery of a cation-binding site in proximity of the GbpA active site, which allows Ca²⁺, Mg²⁺, or K⁺ binding close to its carbohydrate-binding surface. In addition to the X-ray crystal structures of cation-LPMO complexes (to 1.5 Å resolution), we explored how the presence of ions affects the stability and activity of the protein. Calcium and magnesium ions were found to bind to GbpA specifically, with calcium ions - abundant in natural sources of chitin - having the strongest effect on protein stability. When the cation-binding site was rendered non-functional, a decrease in activity was observed, highlighting the importance of the structural elements stabilized by calcium. Our findings suggest a cation-binding site specific to GbpA and related LPMOs that may fine-tune binding and activity for its substrates during environmental survival and host infection.

Host-virus interactions are critically important for various stages of the viral replication cycle. The reliance of viruses on the host factors for their entry, replication, and maturation processes can be exploited for the development of antiviral therapeutics. Thus, the identification and characterization of such viral-host dependency factors has been an attractive area of research to provide novel antiviral targets. Traditional proteomic efforts based on affinity purification of protein complexes from cell lysates are limited to detecting strong and stable interactions. In this perspective, we discuss the integration of two latest proteomic techniques, based on in situ proximity labelling and chemical crosslinking methods, to uncover host-virus protein-protein interactions in living cells.

The TRiC/CCT complex assists in the folding of approximately 10% of cytosolic proteins through an ATP-driven conformational cycle, playing a crucial role in maintaining protein homeostasis. Despite our understanding of ATP-driven TRiC ring closing and substrate folding, the process and mechanisms underlying TRiC ring-opening and substrate release remain largely unexplored. In this study, by determining an ensemble of cryo-EM structures of yeast TRiC in the presence of ADP, including three intermediate transition states, we present a comprehensive picture of the TRiC ring-opening process. During this process, CCT3 detects the loss of γ-phosphate and initiates with the dynamics of its apical protrusion, and expands to the outward leaning of the consecutive CCT6/8/7/5 subunits. This is followed by significant movements of CCT2, CCT4, and especially CCT1 subunits, resulting in the opening of the TRiC rings. We also observed an unforeseen temporary separation between the two rings in the CCT2 side, coordinating the release of the originally locked CCT4 N-terminus, which potentially participates in the ring-opening process. Collectively, our study reveals a stepwise TRiC ring-opening mechanism, provides a comprehensive view of the TRiC conformational landscape, and sheds lights on its subunit specificity in sensing nucleotide status and substrate release. Our findings deepen our understanding of protein folding assisted by TRiC and may inspire new strategies for the diagnosis and treatment of related diseases.

In situ structural biology with cryo-electron tomography (cryo-ET) and subtomogram averaging (StA) is evolving as a major method to understand the structure, function, and interactions of biological molecules in cells in a single experiment. Since its inception, the method has matured with some stellar highlights and with further opportunities to broaden its applications. In this short review, I want to provide a personal perspective on the developments in cryo-ET as I have seen it for the last ~20 years and outline the major steps that led to its success. This perspective highlights cryo-ET with my eyes as a junior researcher and my view on the present and past developments in hardware and software for in situ structural biology with cryo-ET.

The molecular mechanism of olfaction, namely, how we smell with limited olfactory receptors to recognize exceedingly diverse and large numbers of scents remains unknown despite the recent advances in chemistry, chemical, structural, and molecular biology. Olfactory receptors are notoriously difficult to study because they are fully embedded in the cell membrane. After decades of efforts and significant funding, there are only three olfactory receptor structures known. To understand olfaction, we carried out the structural bioinformatic study of six human olfactory receptors including OR51E1, OR51E2, OR52cs, OR1A1, OR1A2, TAAR9, and their AlphaFold3 predicted water-soluble QTY variants with odorants. We applied the QTY code to replace leucine (L) with glutamine (Q), isoleucine (I) and valine (V) with threonine (T), and phenylalanine (F) with tyrosine (Y) only in the transmembrane helices. Therefore, these QTY variants become water-soluble. We also present the superimposed structures of native olfactory receptors and their water-soluble QTY variants. The superimposed structures show remarkable similarity with RMSDs between 0.441 and 1.275 Å despite significant changes to the protein sequence of the transmembrane domains (43.03%-50.31%). We also show the differences in hydrophobicity surfaces between the native olfactory receptors and their QTY variants. Furthermore, we also used AlphaFold3 and molecular dynamics to study the odorant octanoate with OR1A2 and spermidine with TAAR9. Our bioinformatics studies provide insight into the differences between the hydrophobic helices and hydrophilic helices, and will likely further stimulate designs of water-soluble integral transmembrane proteins and other aggregated proteins.