53 research outputs found
Simple methods to determine the dissociation constant, Kd
No full textThe determination of the dissociation constant (K-d) is pivotal in biochemistry and pharmacology for understanding binding affinities in chemical reactions, which is crucial for drug development and comprehending biological systems. Here, we introduce a single-molecule fluorescence resonance energy transfer-based method for determining K-d, alongside the conventional electrophoretic mobility shift assay method of K-d, offering insights into thermodynamic interactions between proteins and substrates. The single-molecule fluorescence resonance energy transfer approach is highlighted for its ability to accurately measure binding and dissociation kinetics through fluorescence labeling and the intrinsic nature of protein-DNA interactions, representing a significant advancement in the fields of molecular biology and pharmacology. (c) 2024 The Author(s). Published by Elsevier Inc. on behalf of Korean Society for Molecular and Cellular Biology.
Single-Molecule Imaging: A Collagenase Pauses before Embarking on a Killing Spree
No full textSummarySingle-molecule tracking provides new insights into how an ATP-independent endo-proteolytic machine digests collagen fibrils during their remodeling
Allosteric ring assembly and chemo-mechanical melting by the interaction between 5 '-phosphate and lambda exonuclease
No full textPhosphates along the DNA function as chemical energy frequently used by nucleases to drive their enzymatic reactions. Exonuclease functions as a machine that converts chemical energy of the phosphodiester-chain into mechanical work. However, the roles of phosphates during exonuclease activities are unknown. We employed lambda exonuclease as a model system and investigated the roles of phosphates during degradation via single-molecule fluorescence resonance energy transfer (FRET). We found that 5 ' phosphates, generated at each cleavage step of the reaction, chemo-mechanically facilitate the subsequent post-cleavage melting of the terminal base pairs. Degradation of DNA with a nick requires backtracking and thermal fraying at the cleavage site for re-initiation via the formation of a catalytically active complex. Unexpectedly, we discovered that a phosphate of a 5 ' recessed DNA acts as a hotspot for an allosteric trimeric-ring assembly without passing through the central channel. Our study provides new insight into the versatile roles of phosphates during the processive enzymatic reaction.
Unwinding mechanism of SARS-CoV helicase (nsp13) in the presence of Ca2+, elucidated by biochemical and single-molecular studies
No full textThe recent outbreak of COVID-19 has created a serious health crisis with fatFal infectious viral diseases, such as Severe Acute Respiratory Syndrome (SARS). The nsp13, a helicase of coronaviruses is an essential element for viral replication that unwinds secondary structures of DNA and RNA, and is thus considered a major therapeutic target for treatment. The replication of coronaviruses and other retroviruses occurs in the cytoplasm of infected cells, in association with viral replication organelles, called virus-induced cytosolic double-membrane vesicles (DMVs). In addition, an increase in cytosolic Ca2+ concentration accelerates viral replication. However, the molecular mechanism of nsp13 in the presence of Ca2+ is not well understood. In this study, we applied biochemical methods and single-molecule techniques to demonstrate how nsp13 achieves its unwinding activity while performing ATP hydrolysis in the presence of Ca2+. Our study found that nsp13 could efficiently unwind double stranded (ds) DNA under physio-logical concentration of Ca2+ of cytosolic DMVs. These findings provide new insights into the properties of nsp13 in the range of calcium in cytosolic DMVs.
Mechanistic decoupling of exonuclease III multifunctionality into AP endonuclease and exonuclease activities at the single-residue level
No full textBacterial exonuclease III (ExoIII) is a multifunctional enzyme that uses a single active site to perform two conspicuous activities: (i) apurinic/apyrimidinic (AP)-endonuclease and (ii) 3 '-> 5 ' exonuclease activities. The AP endonuclease activity results in AP site incision, while the exonuclease activity results in the continuous excision of 3 ' terminal nucleobases to generate a partial duplex for recruiting the downstream DNA polymerase during the base excision repair process (BER). The key determinants of functional selection between the two activities are poorly understood. Here, we use a series of mutational analyses and single-molecule imaging to unravel the pivotal rules governing these endo- and exonuclease activities at the single amino acid level. An aromatic residue, either W212 or F213, recognizes AP sites to allow for the AP endonuclease activity, and the F213 residue also participates in the stabilization of the melted state of the 3 ' terminal nucleobases, leading to the catalytically competent state that activates the 3 '-> 5 ' exonuclease activity. During exonucleolytic cleavage, the DNA substrate must be maintained as a B-form helix through a series of phosphate-stabilizing residues (R90, Y109, K121 and N153). Our work decouples the AP endonuclease and exonuclease activities of ExoIII and provides insights into how this multifunctional enzyme controls each function at the amino acid level.
Atomic cranks and levers control sugar ring conformations
No full textIn this paper we review the conformational analysis of sugar rings placed under tension during mechanical manipulations of single polysaccharide molecules with the atomic force microscope and during steered molecular dynamics simulations. We examine the role of various chemical bonds and linkages between sugar rings in inhibiting or promoting their conformational transitions by means of external forces. Small differences in the orientation of one chemical bond on the sugar ring can produce significantly different mechanical properties at the polymer level as exemplified by two polysaccharides: cellulose, composed of beta-1 -> 4-linked D-glucose, and amylose, composed of alpha-1 -> 4-linked D-glucose. In contrast to beta-glucose rings, which are mechanically stable and produce simple entropic elasticity of the chain, a-glucose rings flip under tension from their chair to a boat-like structure and these transitions produce deviations of amylose elasticity from the freely jointed chain model. We also examine the deformation of two mechanically complementary 1 -> 6-linked polysaccharides: pustulan, a beta-1 -> 6-linked glucan, and dextran, a alpha-1 -> 6-linked glucan. Forced rotations about the C-5-C-6 bonds govern the elasticity of pustulan, and complex conformational transitions that involve simultaneous C-5-C-6 rotations and chair-boat transitions govern the elasticity of dextran. Finally, we discuss the likelihood of various conformational transitions in sugar rings in biological settings and speculate on their significance.
Direct Detection of Inter‐residue Hydrogen Bonds in Polysaccharides by Single‐Molecule Force Spectroscopy
No full textThe elasticity of single amylose chains in solvents that promote or inhibit the formation of inter-residue hydrogen bonds was examined by AFM (see picture). Hydrogen bonds were found to rigidify the amylose chain in solvents of low dielectric constant, and the (Chemical Equation Presented) strength of inter-residue hydrogen bonds in sugars can be measured.
Nanomechanical Fingerprints of UV Damage To DNA
No full textThe nanomechanical fingerprints elasticity of UV-damage to DNA in a dose- and sequence-dependent manner, using Atomic Force Microscopy (AFM)-based single-molecule force spectroscopy, was analyzed. The force spectrograms results indicate a significant differences in the elasticity of UV-treated individual DNA molecules as compared to the untreated DNA. The UV treatment shortens the B-S DNA transition length below 70% of the original contour length measured for the untreated DNA. The elasticity of individual duplexes exposed to various doses of radiations of UV light shows that the shortening of the B-S transition is equivalent to that recorded on UV-irradiated DNA. The results also show that UV radiation affects the mechanics of DNA double helix at a single-molecule level in a dose-dependent manner and the irradiated synthetic DNA shows a damage saturation in a sequence-dependent manner.
Single-molecule analysis reveals three phases of DNA degradation by an exonuclease
No full textlambda exonuclease degrades one strand of duplex DNA in the 5'-to-3' direction to generate a 3' overhang required for recombination. Its ability to hydrolyze thousands of nucleotides processively is attributed to its ring structure, and most studies have focused on the processive phase. Here we have used single-molecule fluorescence resonance energy transfer ( FRET) to reveal three phases of l exonuclease reactions: the initiation, distributive and processive phases. The distributive phase comprises early reactions in which the 3' overhang is too short to stably engage with the enzyme. A mismatched base is digested one-fifth as quickly as a Watson-Crick-paired base, and multiple concatenated mismatches have a cooperatively negative effect, highlighting the crucial role of base pairing in aligning the 5' end toward the active site. The rate-limiting step during processive degradation seems to be the post-cleavage melting of the terminal base pair. We also found that an escape from a known pausing sequence requires enzyme backtracking.
Reversible and Controllable Nanolocomotion of an RNA-Processing Machinery
No full textMolecular motors have inspired many avenues of research For nanotechnology but most molecular motors studied so far allow only unidirectional movement. The archaeal RNA-exosom,e is a reversible motor that can either polymerize or degrade an RNA strand, depending on the chemical environments. We developed a single molecule fluorescence assay to analyze the real time locomotion of this nanomachine on RNA. Despite the multimeric structure, the enzyme followed the Michaelis-Menten kinetics with the maximum speed of similar to 3 nucleotides/s, showing that the three catalytic cylinders do not fire cooperatively. We also demonstrate rapid directional switching on demand by fluidic control. When the two reaction speeds are balanced on average, the enzyme shows a memory of the previous reaction it catalyzed and stochastically switches between primarily polymerizing and primarily degrading behaviors. The processive, reversible, and controllable locomotion propelled by this nanomachine has a promising potential in environmental sensing, diagnostic, and cargo delivery applications.
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