The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates

ABSTRACTCatch bonds are a form of mechanoregulation wherein protein-ligand interactions are strengthened by the application of dissociative tension. Currently, the best-characterized examples of catch bonds are between single protein-ligand pairs. The essential AAA (ATPase associated with diverse cellular activities) mechanoenzyme Mdn1 drives two separate steps in ribosome biogenesis, using its MIDAS domain to extract the ubiquitin-like (UBL) domain-containing proteins Rsa4 and Ytm1 from ribosomal precursors. However, it must subsequently release these assembly factors to reinitiate the enzymatic cycle. The mechanism underlying MIDAS-UBL switching between strongly- and weakly-bound states is unknown. Here, we use single-molecule optical tweezers to investigate the force-dependence of MIDAS-UBL binding. Parallel experiments with Rsa4 and Ytm1 show that forces up to ~4 pN, matching the magnitude of force produced by AAA proteins similar to Mdn1, enhance the MIDAS domain binding lifetime up to tenfold, and higher forces accelerate dissociation. Together, our studies indicate that Mdn1’s MIDAS domain forms catch bonds with more than one UBL-substrate, and provide insights into how mechanoregulation may contribute to the Mdn1 enzymatic cycle during ribosome biogenesis.

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A ClyA nanopore tweezer for analysis of functional states of protein-ligand interactions

10.1101/727503 ◽

2019 ◽

Author(s):

Xin Li ◽

Kuohao Lee ◽

Jianhan Chen ◽

Min Chen

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Bound States ◽

Conformational Dynamics ◽

Electrical Current ◽

Label Free ◽

Protein Motions ◽

Protein Ligand Interactions ◽

Ligand Interactions ◽

Molecular Tweezer

AbstractConformational changes of proteins are essential to their functions. Yet it remains challenging to measure the amplitudes and timescales of protein motions. Here we show that the ClyA nanopore can be used as a molecular tweezer to trap a single maltose-binding protein (MBP) within its lumen, which allows conformation changes to be monitored as electrical current fluctuations in real time. The current measurements revealed three distinct ligand-bound states for MBP in the presence of reducing saccharides. Our biochemical and kinetic analysis reveal that these three states represented MBP bound to different isomers of reducing sugars. These findings shed light on the mechanism of substrate recognition by MBP and illustrate that the nanopore tweezer is a powerful, label-free, single-molecule approach for studying protein conformational dynamics under functional conditions.

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Calculating the force-dependent unbinding rate of biological macromolecular bonds from the force-ramp optical trapping assays

10.1101/2021.09.20.461030 ◽

2021 ◽

Author(s):

Apurba Paul ◽

Joshua Alper

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Force Spectroscopy ◽

Optical Tweezer ◽

Stochastic Simulations ◽

Cumulative Distribution ◽

Cellular Functions ◽

Single Molecule Force Spectroscopy ◽

Protein Ligand Interactions ◽

Ligand Interactions

AbstractThe non-covalent biological bonds that constitute protein-protein or protein-ligand interactions play crucial roles in many cellular functions, including mitosis, motility, and cell-cell adhesion. The effect of external force (F) on the unbinding rate (koff(F)) of macromolecular interactions is a crucial parameter to understanding the mechanisms behind these functions. Optical tweezer-based single-molecule force spectroscopy is frequently used to obtain quantitative force-dependent dissociation data on slip, catch, and ideal bonds. However, analyses of this data using dissociation time or dissociation force histograms often quantitatively compare bonds without fully characterizing their underlying biophysical properties. Additionally, the results of histogram-based analyses can depend on the rate at which force was applied during the experiment and the experiment’s sensitivity. Here, we present an analytically derived cumulative distribution function-like approach to analyzing force-dependent dissociation force spectroscopy data. We demonstrate the benefits and limitations of the technique using stochastic simulations of various bond types. We show that it can be used to obtain the detachment rate and force sensitivity of biological macromolecular bonds from force spectroscopy experiments by explicitly accounting for loading rate and noisy data. We also discuss the implications of our results on using optical tweezers to collect force-dependent dissociation data.

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Calculating the force-dependent unbinding rate of biological macromolecular bonds from force-ramp optical trapping assays

10.21203/rs.3.rs-933021/v1 ◽

2021 ◽

Author(s):

Apurba Paul ◽

Joshua Alper

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Force Spectroscopy ◽

Optical Tweezer ◽

Stochastic Simulations ◽

Cumulative Distribution ◽

Cellular Functions ◽

Single Molecule Force Spectroscopy ◽

Protein Ligand Interactions ◽

Ligand Interactions

Abstract The non-covalent biological bonds that constitute protein-protein or protein-ligand interactions play crucial roles in many cellular functions, including mitosis, motility, and cell-cell adhesion. The effect of external force (𝐹) on the unbinding rate (𝑘off(𝐹)) of macromolecular interactions is a crucial parameter to understanding the mechanisms behind these functions. Optical tweezer-based single-molecule force spectroscopy is frequently used to obtain quantitative force-dependent dissociation data on slip, catch, and ideal bonds. However, analyses of this data using dissociation time or dissociation force histograms often quantitatively compare bonds without fully characterizing their underlying biophysical properties. Additionally, the results of histogram-based analyses can depend on the rate at which force was applied during the experiment and the experiment’s sensitivity. Here, we present an analytically derived cumulative distribution function-like approach to analyzing force-dependent dissociation force spectroscopy data. We demonstrate the benefits and limitations of the technique using stochastic simulations of various bond types. We show that it can be used to obtain the detachment rate and force sensitivity of biological macromolecular bonds from force spectroscopy experiments by explicitly accounting for loading rate and noisy data. We also discuss the implications of our results on using optical tweezers to collect force-dependent dissociation data.

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Calculating the force-dependent unbinding rate of biological macromolecular bonds from force-ramp optical trapping assays

Scientific Reports ◽

10.1038/s41598-021-03690-1 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Apurba Paul ◽

Joshua Alper

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Force Spectroscopy ◽

Optical Tweezer ◽

Stochastic Simulations ◽

Cumulative Distribution ◽

Cellular Functions ◽

Single Molecule Force Spectroscopy ◽

Protein Ligand Interactions ◽

Ligand Interactions

AbstractThe non-covalent biological bonds that constitute protein–protein or protein–ligand interactions play crucial roles in many cellular functions, including mitosis, motility, and cell–cell adhesion. The effect of external force ($$F$$ F ) on the unbinding rate ($${k}_{\text{off}}\left(F\right)$$ k off F ) of macromolecular interactions is a crucial parameter to understanding the mechanisms behind these functions. Optical tweezer-based single-molecule force spectroscopy is frequently used to obtain quantitative force-dependent dissociation data on slip, catch, and ideal bonds. However, analyses of this data using dissociation time or dissociation force histograms often quantitatively compare bonds without fully characterizing their underlying biophysical properties. Additionally, the results of histogram-based analyses can depend on the rate at which force was applied during the experiment and the experiment’s sensitivity. Here, we present an analytically derived cumulative distribution function-like approach to analyzing force-dependent dissociation force spectroscopy data. We demonstrate the benefits and limitations of the technique using stochastic simulations of various bond types. We show that it can be used to obtain the detachment rate and force sensitivity of biological macromolecular bonds from force spectroscopy experiments by explicitly accounting for loading rate and noisy data. We also discuss the implications of our results on using optical tweezers to collect force-dependent dissociation data.

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Arranging Small Molecules with Subnanometer Precision on DNA Origami Substrates for the Single‐Molecule Investigation of Protein–Ligand Interactions

Small Structures ◽

10.1002/sstr.202000038 ◽

2020 ◽

Vol 1 (1) ◽

pp. 2000038

Author(s):

Jingyuan Huang ◽

Antonio Suma ◽

Meiying Cui ◽

Guido Grundmeier ◽

Vincenzo Carnevale ◽

...

Keyword(s):

Small Molecules ◽

Single Molecule ◽

Dna Origami ◽

Protein Ligand Interactions ◽

Ligand Interactions

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Integrated Method to Attach DNA Handles and Functionally Select Proteins to Study Folding and Protein-Ligand Interactions with Optical Tweezers

Scientific Reports ◽

10.1038/s41598-017-11214-z ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 8

Author(s):

Yuxin Hao ◽

Clare Canavan ◽

Susan S. Taylor ◽

Rodrigo A. Maillard

Keyword(s):

Optical Tweezers ◽

Integrated Method ◽

Protein Ligand Interactions ◽

Ligand Interactions

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Modern Biophysical Approaches to Study Protein–Ligand Interactions

Biophysical Reviews and Letters ◽

10.1142/s1793048018300013 ◽

2018 ◽

Vol 13 (04) ◽

pp. 133-155

Author(s):

Priyanka Biswas

Keyword(s):

Single Molecule ◽

Analytical Ultracentrifugation ◽

Correlation Spectroscopy ◽

Titration Calorimetry ◽

Resonance Fluorescence ◽

Biological Interactions ◽

Protein Ligand Interactions ◽

Dual Polarization Interferometry ◽

Ligand Interactions

Protein–ligand interactions act as a pivot to the understanding of most of the biological interactions. The study of interactions between proteins and cellular molecules has led to the establishment and identification of various important pathways that control biological systems. Investigators working in different fields of biological sciences have an intrinsic interest in this field and complement their findings by the application of different biophysical approaches and tools to quantify protein–ligand interactions that include protein–small molecules, protein–DNA, protein–RNA, protein–protein both in vitro and in vivo. In this paper, the various biophysical techniques that can be employed to study such interactions will be discussed. In addition to native gel electrophoresis and fluorescence-based methods, more details will be discussed, on the broad range of modern day biophysical tools such as Circular Dichroism, Fourier Transform Infrared (FTIR) Spectroscopy, Isothermal Titration Calorimetry, Analytical Ultracentrifugation, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy, Differential Scanning Fluorimetry, Nuclear Magnetic Resonance, Mass Spectroscopy, Single Molecule Spectroscopy, Dual Polarization Interferometry, Micro Scale Thermophoresis and Electro–switchable Biosensors that can be used to study the different aspects of protein–ligand interactions.

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Solvent Isotope Effects in Atomic Force Spectroscopy

Microscopy and Microanalysis ◽

10.1017/s143192760003035x ◽

2001 ◽

Vol 7 (S2) ◽

pp. 856-857

Author(s):

Jeffrey G. Forbes ◽

John P. Santos

Keyword(s):

Deuterium Oxide ◽

Isotope Effects ◽

Protein Ligand Interactions ◽

Atomic Force ◽

Single Protein ◽

Ligand Interactions ◽

Solvent Isotope ◽

Functionalized Tips ◽

Glass Surfaces ◽

Solvent Isotope Effects

The utility of the atomic force microscope (AFM) for measuring forces is now well established. The AFM is capable of measuring forces from tens of piconewtons to tens of nanonewtons. Systems ranging from the adhesion of clean or functionalized tips interacting with different surface, to singlemolecule measurements of the force required to rupture protein-ligand interactions, to the forced unfolding of single protein molecules have been studied. The adhesion of a clean silicon nitride tip to a clean glass surface is one of the simplest systems to study and was the first system to demonstrate quantized adhesion. AFM cantilevers will adhere to glass surfaces in the presence of water and the force required to separate the surfaces is on the order of 1-6 nN at low pH. The adhesion mechanism is believed to be due to hydrogen bonds formed between the silanol groups present on both the glass and AFM tip. Deuterium oxide (D2O) has physical properties significantly different from that of H2O; the density, melting temperature and heat capacity are all greater than that of water. These and the differences between H2O and D2O can be attributed to the lower vibrational frequency of the OD bond and the fact that O-D…O bonds are about lkJ/mol stronger than O-H…O bonds.

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Force spectroscopy studies on protein-ligand interactions: A single protein mechanics perspective

FEBS Letters ◽

10.1016/j.febslet.2014.04.009 ◽

2014 ◽

Vol 588 (19) ◽

pp. 3613-3620 ◽

Cited By ~ 28

Author(s):

Xiaotang Hu ◽

Hongbin Li

Keyword(s):

Force Spectroscopy ◽

Protein Mechanics ◽

Protein Ligand Interactions ◽

Single Protein ◽

Ligand Interactions ◽

Spectroscopy Studies

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19F-NMR in Target-based Drug Discovery

Current Medicinal Chemistry ◽

10.2174/0929867326666190610160534 ◽

2019 ◽

Vol 26 (26) ◽

pp. 4964-4983 ◽

Cited By ~ 7

Author(s):

CongBao Kang

Keyword(s):

Drug Discovery ◽

Conformational Changes ◽

Protein Structures ◽

19F Nmr ◽

Binding Affinities ◽

Protein Ligand Interactions ◽

Compound Libraries ◽

Ligand Interactions ◽

Reference Ligand ◽

Hit Identification

Solution NMR spectroscopy plays important roles in understanding protein structures, dynamics and protein-protein/ligand interactions. In a target-based drug discovery project, NMR can serve an important function in hit identification and lead optimization. Fluorine is a valuable probe for evaluating protein conformational changes and protein-ligand interactions. Accumulated studies demonstrate that 19F-NMR can play important roles in fragment- based drug discovery (FBDD) and probing protein-ligand interactions. This review summarizes the application of 19F-NMR in understanding protein-ligand interactions and drug discovery. Several examples are included to show the roles of 19F-NMR in confirming identified hits/leads in the drug discovery process. In addition to identifying hits from fluorinecontaining compound libraries, 19F-NMR will play an important role in drug discovery by providing a fast and robust way in novel hit identification. This technique can be used for ranking compounds with different binding affinities and is particularly useful for screening competitive compounds when a reference ligand is available.

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