Common intermediates and kinetics, but different energetics, in the assembly of SNARE proteins

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are evolutionarily conserved machines that couple their folding/assembly to membrane fusion. However, it is unclear how these processes are regulated and function. To determine these mechanisms, we characterized the folding energy and kinetics of four representative SNARE complexes at a single-molecule level using high-resolution optical tweezers. We found that all SNARE complexes assemble by the same step-wise zippering mechanism: slow N-terminal domain (NTD) association, a pause in a force-dependent half-zippered intermediate, and fast C-terminal domain (CTD) zippering. The energy release from CTD zippering differs for yeast (13 kBT) and neuronal SNARE complexes (27 kBT), and is concentrated at the C-terminal part of CTD zippering. Thus, SNARE complexes share a conserved zippering pathway and polarized energy release to efficiently drive membrane fusion, but generate different amounts of zippering energy to regulate fusion kinetics.

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Single-Molecule Studies of Protein Folding with Optical Tweezers

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-013118-111442 ◽

2020 ◽

Vol 89 (1) ◽

pp. 443-470 ◽

Cited By ~ 5

Author(s):

Carlos Bustamante ◽

Lisa Alexander ◽

Kevin Maciuba ◽

Christian M. Kaiser

Keyword(s):

Protein Folding ◽

Optical Tweezers ◽

Single Molecule ◽

Cellular Protein ◽

Folding Energy ◽

Precise Control ◽

Single Molecule Studies ◽

Force Range ◽

And Function

Manipulation of individual molecules with optical tweezers provides a powerful means of interrogating the structure and folding of proteins. Mechanical force is not only a relevant quantity in cellular protein folding and function, but also a convenient parameter for biophysical folding studies. Optical tweezers offer precise control in the force range relevant for protein folding and unfolding, from which single-molecule kinetic and thermodynamic information about these processes can be extracted. In this review, we describe both physical principles and practical aspects of optical tweezers measurements and discuss recent advances in the use of this technique for the study of protein folding. In particular, we describe the characterization of folding energy landscapes at high resolution, studies of structurally complex multidomain proteins, folding in the presence of chaperones, and the ability to investigate real-time cotranslational folding of a polypeptide.

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Molecular Motors: Force and Movement Generated by Single Myosin II Molecules

Physiology ◽

10.1152/nips.01389.2002 ◽

2002 ◽

Vol 17 (5) ◽

pp. 213-218 ◽

Cited By ~ 3

Author(s):

Caspar Rüegg ◽

Claudia Veigel ◽

Justin E. Molloy ◽

Stephan Schmitz ◽

John C. Sparrow ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Molecular Motors ◽

Molecular Motor ◽

Myosin Ii ◽

Force Production ◽

Myosin Isoforms ◽

Single Molecule Level ◽

Recent Developments ◽

Muscle Myosin

Muscle myosin II is an ATP-driven, actin-based molecular motor. Recent developments in optical tweezers technology have made it possible to study movement and force production on the single-molecule level and to find out how different myosin isoforms may have adapted to their specific physiological roles.

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Studying Nucleic Acid–Drug Interactions at the Single-Molecule Level Using Optical Tweezers

Methods for Studying Nucleic Acid/Drug Interactions ◽

10.1201/b11691-6 ◽

2016 ◽

pp. 154-177

Keyword(s):

Nucleic Acid ◽

Drug Interactions ◽

Optical Tweezers ◽

Single Molecule ◽

Single Molecule Level ◽

Nucleic Acid Drug

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Energetic dependencies dictate folding mechanism in a complex protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914366116 ◽

2019 ◽

Vol 116 (51) ◽

pp. 25641-25648 ◽

Cited By ~ 7

Author(s):

Kaixian Liu ◽

Xiuqi Chen ◽

Christian M. Kaiser

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Large Scale ◽

Elongation Factor ◽

Multidomain Protein ◽

Domain Interactions ◽

The Stability ◽

Domain Iii ◽

And Function ◽

Terminal Domains

Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.

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Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

10.1101/2020.07.31.231274 ◽

2020 ◽

Author(s):

Keith J Mickolajczyk ◽

Patrick M M Shelton ◽

Michael Grasso ◽

Xiaocong Cao ◽

Sara R Warrington ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Helicase Activity ◽

Structural Protein ◽

Rna Dependent Rna Polymerase ◽

Velocity Increase ◽

Single Molecule Level ◽

On Demand ◽

Dependent Behavior ◽

Stimulation Of

The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

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How Microbes Use Force To Control Adhesion

Journal of Bacteriology ◽

10.1128/jb.00125-20 ◽

2020 ◽

Vol 202 (12) ◽

Author(s):

Albertus Viljoen ◽

Johann Mignolet ◽

Felipe Viela ◽

Marion Mathelié-Guinlet ◽

Yves F. Dufrêne

Keyword(s):

Single Molecule ◽

Molecular Mechanisms ◽

Flow Chamber ◽

Microbial Adhesion ◽

Single Molecule Level ◽

Force Microscopy ◽

Atomic Force ◽

Adhesive Interactions ◽

And Function ◽

Mechanisms Of Adhesion

ABSTRACT Microbial adhesion and biofilm formation are usually studied using molecular and cellular biology assays, optical and electron microscopy, or laminar flow chamber experiments. Today, atomic force microscopy (AFM) represents a valuable addition to these approaches, enabling the measurement of forces involved in microbial adhesion at the single-molecule level. In this minireview, we discuss recent discoveries made applying state-of-the-art AFM techniques to microbial specimens in order to understand the strength and dynamics of adhesive interactions. These studies shed new light on the molecular mechanisms of adhesion and demonstrate an intimate relationship between force and function in microbial adhesins.

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Broken force dispersal network in tip-links by the mutations at the Ca2+-binding residues induces hearing-loss

Biochemical Journal ◽

10.1042/bcj20190453 ◽

2019 ◽

Vol 476 (16) ◽

pp. 2411-2425 ◽

Cited By ~ 1

Author(s):

Jagadish P. Hazra ◽

Amin Sagar ◽

Nisha Arora ◽

Debadutta Deb ◽

Simerpreet Kaur ◽

...

Keyword(s):

Hearing Loss ◽

Inner Ear ◽

Single Molecule ◽

Force Sensor ◽

Force Sensors ◽

Single Molecule Level ◽

Conformational Variability ◽

Atomic Force ◽

Wide Range ◽

And Function

Abstract Tip-link as force-sensor in hearing conveys the mechanical force originating from sound to ion-channels while maintaining the integrity of the entire sensory assembly in the inner ear. This delicate balance between structure and function of tip-links is regulated by Ca2+-ions present in endolymph. Mutations at the Ca2+-binding sites of tip-links often lead to congenital deafness, sometimes syndromic defects impairing vision along with hearing. Although such mutations are already identified, it is still not clear how the mutants alter the structure-function properties of the force-sensors associated with diseases. With an aim to decipher the differences in force-conveying properties of the force-sensors in molecular details, we identified the conformational variability of mutant and wild-type tip-links at the single-molecule level using FRET at the endolymphatic Ca2+ concentrations and subsequently measured the force-responsive behavior using single-molecule force spectroscopy with an Atomic Force Microscope (AFM). AFM allowed us to mimic the high and wide range of force ramps (103–106 pN s−1) as experienced in the inner ear. We performed in silico network analysis to learn that alterations in the conformations of the mutants interrupt the natural force-propagation paths through the sensors and make the mutant tip-links vulnerable to input forces from sound stimuli. We also demonstrated that a Ca2+ rich environment can restore the force-response of the mutant tip-links which may eventually facilitate the designing of better therapeutic strategies to the hearing loss.

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Profound Nanoscale Structural and Biomechanical Changes in DNA Helix upon Treatment with Anthracycline Drugs

International Journal of Molecular Sciences ◽

10.3390/ijms21114142 ◽

2020 ◽

Vol 21 (11) ◽

pp. 4142

Author(s):

Aleksandra Kaczorowska ◽

Weronika Lamperska ◽

Kaja Frączkowska ◽

Jan Masajada ◽

Sławomir Drobczyński ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Regulatory Elements ◽

Dna Molecules ◽

Anthracycline Antibiotics ◽

Single Molecule Level ◽

Force Microscopy ◽

Atomic Force ◽

Microscopy Analysis ◽

Dna Regulatory Elements

In our study, we describe the outcomes of the intercalation of different anthracycline antibiotics in double-stranded DNA at the nanoscale and single molecule level. Atomic force microscopy analysis revealed that intercalation results in significant elongation and thinning of dsDNA molecules. Additionally, using optical tweezers, we have shown that intercalation decreases the stiffness of DNA molecules, that results in greater susceptibility of dsDNA to break. Using DNA molecules with different GC/AT ratios, we checked whether anthracycline antibiotics show preference for GC-rich or AT-rich DNA fragments. We found that elongation, decrease in height and decrease in stiffness of dsDNA molecules was highest in GC-rich dsDNA, suggesting the preference of anthracycline antibiotics for GC pairs and GC-rich regions of DNA. This is important because such regions of genomes are enriched in DNA regulatory elements. By using three different anthracycline antibiotics, namely doxorubicin (DOX), epirubicin (EPI) and daunorubicin (DAU), we could compare their detrimental effects on DNA. Despite their analogical structure, anthracyclines differ in their effects on DNA molecules and GC-rich region preference. DOX had the strongest overall effect on the DNA topology, causing the largest elongation and decrease in height. On the other hand, EPI has the lowest preference for GC-rich dsDNA. Moreover, we demonstrated that the nanoscale perturbations in dsDNA topology are reflected by changes in the microscale properties of the cell, as even short exposition to doxorubicin resulted in an increase in nuclei stiffness, which can be due to aberration of the chromatin organization, upon intercalation of doxorubicin molecules.

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PRINCIPLES OF SINGLE-MOLECULE MANIPULATION AND ITS APPLICATION IN BIOLOGICAL PHYSICS

International Journal of Modern Physics B ◽

10.1142/s021797921230006x ◽

2012 ◽

Vol 26 (13) ◽

pp. 1230006 ◽

Cited By ~ 3

Author(s):

WEI-HUNG CHEN ◽

JONATHAN D. WILSON ◽

SITHARA S. WIJERATNE ◽

SARAH A. SOUTHMAYD ◽

KUAN-JIUH LIN ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Biological Molecules ◽

Force Detection ◽

Biomolecular Systems ◽

Single Molecule Level ◽

Detection Techniques ◽

Atomic Force ◽

Manipulation System ◽

Single Molecule Manipulation

Recent advances in nanoscale manipulation and piconewton force detection provide a unique tool for studying the mechanical and thermodynamic properties of biological molecules and complexes at the single-molecule level. Detailed equilibrium and dynamics information on proteins and DNA have been revealed by single-molecule manipulation and force detection techniques. The atomic force microscope (AFM) and optical tweezers have been widely used to quantify the intra- and inter-molecular interactions of many complex biomolecular systems. In this article, we describe the background, analysis, and applications of these novel techniques. Experimental procedures that can serve as a guide for setting up a single-molecule manipulation system using the AFM are also presented.

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