Equilibrium folding and unfolding dynamics to reveal detailed free energy landscape of src SH3 protein by magnetic tweezers

Src SH3 protein domain is a typical two-state protein which has been confirmed by research of denaturant-induced unfolding dynamics.Force spectroscopy experiments by optical tweezers and atomic force microscopy have measured the force-dependent unfolding rates with different kinds of pulling geometry.However,the equilibrium folding and unfolding dynamics at constant forces has not been reported.Here,using stable magnetic tweezers,we performed equilibrium folding and unfolding dynamic measurement and force-jump measurement of src SH3 domain with tethering points at its N-and C-termini.From the obtained force-dependent transition rates,a detailed two-state free energy landscape of src SH3 protein is constructed with quantitative information of folding free energy,transition state barrier height and position,which exemplifies the capability of magnetic tweezers to study protein folding and unfolding dynamics.

Mutation in a non-force-bearing region of protein L influences force-dependent unfolding behavior

Single-molecule magnetic tweezers(MTs) have revealed multiple transition barriers along the unfolding pathway of several two-state proteins, such as GB1 and Csp. In this study, we utilized MTs to measure the force-dependent folding and unfolding rates of both protein L(PLWT) and its Y47W mutant(PLY47W) where the mutation point is not at the force-bearing β-strands. The measurements were conducted within a force range of 3–120 pN. Notably, the unfolding rates of both PLWT and PWY47W exhibit distinct force sensitivities below 50 pN and above 60 pN, implying a two-barrier free energy landscape. Both PLWT and PLY47W share the same force-dependent folding rate and the same transition barriers,but the unfolding rate of PLY47W is faster than that of PLWT. Our finding demonstrates that the residue outside of the force-bearing region will also affect the force-induced unfolding dynamics.

Catch-bond behavior of DNA condensate under tension

Toroid formation is an important mechanism underlying DNA condensation, which has been investigated extensively by single-molecule experiments in vitro. Here, the de-condensation dynamics of DNA condensates were studied using magnetic tweezers combined with Brownian dynamics simulations. The experimental results revealed a surprising nonmonotonic dependence of the unfolding rate on the force applied under strong adhesion conditions, resembling the catchbond behavior reported in the field of ligand-receptor interactions. Simulation results showed that the different unfolding pathways of DNA condensate under large forces derive from the force-dependent deformation of the DNA toroid, which explains the catch-bond behavior of DNA condensate in the magnetic tweezers experiments. These results challenge the universality of the regular toroidal DNA unwrapping mechanism and provide the most complete description to date of multivalent cation-dependent DNA unwrapping under tension.

A multi-field approach to DNA condensation

DNA condensation is an important process in many fields including life sciences, polymer physics, and applied technology. In the nucleus, DNA is condensed into chromosomes. In polymer physics, DNA is treated as a semi-flexible molecule and a polyelectrolyte. Many agents, including multi-valent cations, surfactants, and neutral poor solvents, can cause DNA condensation, also referred to as coil–globule transition. Moreover, DNA condensation has been used for extraction and gene delivery in applied technology. Many physical theories have been presented to elucidate the mechanism underlying DNA condensation, including the counterion correlation theory, the electrostatic zipper theory, and the hydration force theory. Recently several single-molecule studies have focused on DNA condensation, shedding new light on old concepts. In this document, the multi-field concepts and theories related to DNA condensation are introduced and clarified as well as the advances and considerations of single-molecule DNA condensation experiments are introduced.

Force-dependent unfolding and folding dynamics of protein alpha-catenin modulation domains

α-catenin is an adhesion protein located at the cadherin-based cell-cell adherens junction.α-catenin cross-linksβ-catenin and actin fiber in the adhesion protein complex,and plays an important role in the formation and modulation of cell-cell adhesion.The central modulation domains can be unfolded to expose binding site of vinculin when stretching force is applied.Here,we studied the force-induced unfolding dynamics ofα-catenin modulation domains under different loading rates from which the unfolding distance of M2 and M3 domains is determined to be 5-7 nm,and an unfolding intermediate state is identified.We also found that the folding process of M1-M3 domains goes through different pathways with cooperativity.

Theoretical study of the nonlinear force-loading control in single-molecule stretching experiments

Force spectrum measurements with constant loading rates are widely used in single-molecule manipulation experiments to study the mechanical stability and force response of biomolecules.Force-dependent transition rates can be obtained from the transition force distribution,but it is limited to the force range with non-zero force distribution.Although constant loading rate control can be realized with magnetic tweezers,the loading rate range is limited due to the slow movement of permanent magnets.Non-linear exponential and exponential squared force loading functions are more feasible in magnetic tweezers,while there is no theoretical result available for these two kinds of non-linear force loading functions.In this study,we solved the unfolding process of a protein following Bell's model under nonlinear exponential and exponential squared force loading functions,which offer a broader range of unfolding force distribution compared to the traditional constant loading rate experiments.Furthermore,we derived two force loading functions,which can produce uniform unfolding force distribution.This research contributes fundamental equations for the analysis of experimental data obtained through single-molecule manipulation under nonlinear force loading controls,paving the way for the use of nonlinear force control in magnetic tweezer experiments.

Reading Time and DNA Sequence Preference of TET3 CXXC Domain Revealed by Single-Molecule Profiling

Recognition of CpG dinucleotide DNA in epigenetic information flow plays a pivotal role for cellular differentiation and development.The TET3 CXXC domain binds to CpG DNA,serving a basic epigenetic information reading mechanism.During the selective recognition of a CpG motif by a CXXC domain from crowded binding sites in a gene sequence,the protein-DNA interactions are beyond CpG dinu-cleotide.However,the selective binding dynamics of CpG within a long DNA context by epigenetic enzymes have been rarely exploit-ed,which is hard for ensemble methods to probe.Here,we used single-molecule magnetic tweezers to quantitatively examine the dynamics of TET3's CXXC domain on a Hoxa9 promoter DNA.Our single-molecule binding profile revealed that CXXC-DNA interactions involve both CpG motifs and their flanking sequences.The residence time of TET3 CXXC differs by about 1000 times in five distin-guished CpG clusters in the context of a CpG island.Moreover,we performed multi-state hidden Markov modeling analysis on the zip-ping/unzipping dynamics of a CpG hairpin,discovering TET3 CXXC's preference on CpG motifs regarding the-2 to+2 flanking bases.Our results shed light on the selective binding dynamics of a CXXC on a gene sequence,facilitating studies on epigenetic information reading mechanisms.

Characterizing in situ poroelastic properties of cytoplasm by the translation of a rigid spherical inclusion

Poroelasticity of cytoplasm is a rate-and size-dependent biphasic material behavior that reflects the normal activities and pathological states of cells,mainly caused by the migration of fluid molecules and the deformation of porous solid skeleton(protein scaffold).While micro/nano-indentation tests have been extensively used to characterize the poroelasticity of a cell,characterizing the in situ poroelasticity of cytoplasm remains elusive.In this study,based on the theory of the translation of a rigid spherical inclusion,we proposed a new method to characterize the in situ poroelasticity of cytoplasm.Based on data from optical/magnetic tweezers tests,we estimated three key poroelasticity parameters-shear modulus,Poisson ratio and diffusion coefficient-of cytoplasm for a variety of cells,including cardiomyocytes,endothelial cells of bovine capillary,and fibroblasts.The proposed method provides a powerful tool for in situ measurement of poroelastic properties of cytoplasm via optical/magnetic tweezers.

The kinetics of force-dependent hybridization and strand-peeling of short DNA fragments

Deoxyribonucleic acid(DNA) carries the genetic information in all living organisms. It consists of two interwound single-stranded(ss) strands, forming a double-stranded(ds) DNA with a right-handed double-helical conformation. The two strands are held together by highly specific basepairing interactions and are further stabilized by stacking between adjacent basepairs. A transition from a dsDNA to two separated ssDNA is called melting and the reverse transition is called hybridization. Applying a tensile force to a dsDNA can result in a particular type of DNA melting, during which one ssDNA strand is peeled away from the other. In this work, we studied the kinetics of strand-peeling and hybridization of short DNA under tensile forces. Our results show that the force-dependent strand-peeling and hybridization can be described with a simple two-state model. Importantly, detailed analysis of the force-dependent transition rates revealed that the transition state consists of several basepairs dsDNA.