SMB

Interactions between a protein and a ligand are essential to all biological processes. Binding and dissociation are the two fundamental steps of ligand–protein interactions, and determine the binding affinity. Intrinsic conformational dynamics of proteins have been suggested to play crucial roles in ligand binding and dissociation. Here, we demonstrate how protein dynamics dictate the binding…

Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and…

We developed a hybrid technique combining optical tweezers and single-molecule three-color fluorescence resonance energy transfer (FRET). In demonstrative experiments, we observed the force-sensitive correlated motion of three helical arms of a Holliday junction and identified the independent unfolding/folding dynamics of two DNA hairpins of the same length. With 3 times the number of observable elements…

Argonaute (Ago) is the catalytic core of small RNA-based gene regulation. Despite plenty of mechanistic studies on Ago, the dynamical aspects and the mechanistic determinants of target mRNA binding and dissociation of Ago–guide strand remain unclear. Here, by using single-molecule fluorescence resonance energy transfer (FRET) assays and Thermus thermophilus Ago (TtAgo), we reveal that the…

Z-DNA binding proteins (ZBPs) specifically recognize and stabilize left-handed double helices, including Z-DNA and Z-RNA. However, the energetics of Z-form stabilization by ZBPs have never been characterized due to the technical limitations of bulk studies, resulting in an unclear understanding of the ZBP operational mechanism at the molecular level. Here, we use single-molecule fluorescence resonance…

Sub-diffraction limit imaging of SnO2 nanowires (NWs) interfacing cells is studied using stochastic optical reconstruction microscopy. This method allows the estimation of the dimension of the NWs with a rather high resolution compared with previous imaging methods. In addition, since this strategy is compatible with biological environments, the NWs can be imaged with cell membrane…

Since its first demonstration about twenty years ago, single-molecule fluorescence resonance energy transfer (FRET) has undergone remarkable technical advances. In this tutorial review, we will discuss two technical advances that increase the information content of the single-molecule FRET measurements: single-molecule multi-color FRET and single-molecule FRET combined with force or torque. Our expectations for future developments…

The specificity of proteases for the residues in and length of substrates is key to understanding their regulatory mechanism, but little is known about length selectivity. Crystal structure analyses of the bacterial aminopeptidase PepS, combined with functional and single-molecule FRET assays, have elucidated a molecular basis for length selectivity. PepS exists in open and closed…

The manner in which Z-DNA is stabilized at high salt concentrations remains unclear. Here, we systematically examine the Z-DNA-stabilizing capabilities of different salts. The strong correlation between the double-stranded DNA denaturation and B-to-Z transition efficiencies indicates that Z-DNA is mainly stabilized by the Hofmeister effect.

Protein dynamics have been suggested to have a crucial role in biomolecular recognition, but the precise molecular mechanisms remain unclear. Herein, we performed single-molecule fluorescence resonance energy transfer measurements for wild-type maltose-binding protein (MBP) and its variants to demonstrate the interplay of conformational dynamics and molecular recognition. Kinetic analysis provided direct evidence that MBP recognizes…

Single-molecule fluorescence imaging has greatly contributed to our understanding of many bio-molecular systems. While reactions occurring in the range of several minutes can be readily studied using conventional single-molecule fluorescence microscopes, data acquisition for longer time scales is hindered by the focal drift of high numerical aperture objectives, which should be corrected in real time….

The viral sensor MDA5 distinguishes between cellular and viral dsRNAs by length-dependent recognition in the range of ∼0.5–7 kb. The ability to discriminate dsRNA length at this scale sets MDA5 apart from other dsRNA receptors of the immune system. We have shown previously that MDA5 forms filaments along dsRNA that disassemble upon ATP hydrolysis. Here,…

Topoisomerase II resolves intrinsic topological problems of double-stranded DNA. As part of its essential cellular functions, the enzyme generates DNA breaks, but the regulation of this potentially dangerous process is not well understood. Here we report single-molecule fluorescence experiments that reveal a previously uncharacterized sequence of events during DNA cleavage by topoisomerase II: nonspecific DNA…

Single‐molecule techniques have been used for only a subset of biological problems because of difficulties in studying proteins that require cofactors or post‐translational modifications. Here, we present a new method integrating single‐molecule fluorescence microscopy and immunopurification to study protein complexes. We used this method to investigate Lin28‐mediated microRNA uridylation by TUT4 (terminal uridylyl transferase 4,…

Hfq is a key regulator involved in multiple aspects of stress tolerance and virulence of bacteria. There has been an intriguing question as to how this RNA chaperone achieves two completely opposite functions—annealing and unwinding—for different RNA substrates. To address this question, we studied the Hfq-mediated interaction of fragments of a non-coding RNA, DsrA, with…