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

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

10.1101/2021.08.30.458255 ◽

2021 ◽

Author(s):

Keith J. Mickolajczyk ◽

Paul Dominic B. Olinares ◽

Brian T. Chait ◽

Shixin Liu ◽

Tarun M. Kapoor

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Ribosome Biogenesis ◽

Bound States ◽

Weakly Bound ◽

Aaa Atpase ◽

Protein Ligand Interactions ◽

Single Protein ◽

Ligand Interactions ◽

Catch Bonds

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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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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Conformational dynamics and thermodynamics of protein–ligand binding studied by NMR relaxation

Biochemical Society Transactions ◽

10.1042/bst20110750 ◽

2012 ◽

Vol 40 (2) ◽

pp. 419-423 ◽

Cited By ~ 46

Author(s):

Mikael Akke

Keyword(s):

Ligand Binding ◽

Bound States ◽

Conformational Dynamics ◽

Nmr Relaxation ◽

Titration Calorimetry ◽

Conformational Entropy ◽

Chain Order ◽

Ligand Interactions ◽

Galectin 3 ◽

Relaxation Experiments

Protein conformational dynamics can be critical for ligand binding in two ways that relate to kinetics and thermodynamics respectively. First, conformational transitions between different substates can control access to the binding site (kinetics). Secondly, differences between free and ligand-bound states in their conformational fluctuations contribute to the entropy of ligand binding (thermodynamics). In the present paper, I focus on the second topic, summarizing our recent results on the role of conformational entropy in ligand binding to Gal3C (the carbohydrate-recognition domain of galectin-3). NMR relaxation experiments provide a unique probe of conformational entropy by characterizing bond-vector fluctuations at atomic resolution. By monitoring differences between the free and ligand-bound states in their backbone and side chain order parameters, we have estimated the contributions from conformational entropy to the free energy of binding. Overall, the conformational entropy of Gal3C increases upon ligand binding, thereby contributing favourably to the binding affinity. Comparisons with the results from isothermal titration calorimetry indicate that the conformational entropy is comparable in magnitude to the enthalpy of binding. Furthermore, there are significant differences in the dynamic response to binding of different ligands, despite the fact that the protein structure is virtually identical in the different protein–ligand complexes. Thus both affinity and specificity of ligand binding to Gal3C appear to depend in part on subtle differences in the conformational fluctuations that reflect the complex interplay between structure, dynamics and ligand interactions.

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Label-free DNA biosensing by topological light confinement

Nanophotonics ◽

10.1515/nanoph-2021-0396 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Gianluigi Zito ◽

Gennaro Sanità ◽

Bryan Guilcapi Alulema ◽

Sofía N. Lara Yépez ◽

Vittorino Lanzio ◽

...

Keyword(s):

Single Molecule ◽

Bound States ◽

Production Control ◽

Point Of Care ◽

Low Cost ◽

Label Free ◽

Large Area ◽

Light Confinement ◽

Binding Event ◽

The Continuum

Abstract Large-area and transparent all-dielectric metasurfaces sustaining photonic bound states in the continuum (BICs) provide a set of fundamental advantages for ultrasensitive biosensing. BICs bridge the gap of large effective mode volume with large experimental quality factor. Relying on the transduction mechanism of reactive sensing principle, herein, we first numerically study the potential of subwavelength confinement driven by topological decoupling from free space radiation for BIC-based biosensing. Then, we experimentally combine this capability with minimal and low-cost optical setup, applying the devised quasi-BIC resonator for PNA/DNA selective biosensing with real-time monitoring of the binding event. A sensitivity of 20 molecules per micron squared is achieved, i.e. ≃0.01 pg. Further enhancement can easily be envisaged, pointing out the possibility of single-molecule regime. This work aims at a precise and ultrasensitive approach for developing low-cost point-of-care tools suitable for routine disease prescreening analyses in laboratory, also adaptable to industrial production control.

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Understanding the Role of Conformational Dynamics in Protein-Ligand Interactions Using NMR Relaxation Methods

Encyclopedia of Magnetic Resonance ◽

10.1002/9780470034590.emrstm1363 ◽

2014 ◽

pp. 255-266 ◽

Cited By ~ 1

Author(s):

Asli Ertekin ◽

Francesca Massi

Keyword(s):

Conformational Dynamics ◽

Nmr Relaxation ◽

Relaxation Methods ◽

Protein Ligand Interactions ◽

Ligand Interactions

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Insight to Functional Conformation and Noncovalent Interactions of Protein-Protein Assembly Using MALDI Mass Spectrometry

Molecules ◽

10.3390/molecules25214979 ◽

2020 ◽

Vol 25 (21) ◽

pp. 4979

Author(s):

Marco Giampà ◽

Elvira Sgobba

Keyword(s):

Mass Spectrometry ◽

Conformational Changes ◽

Protein Interactions ◽

Noncovalent Interactions ◽

Conformational Dynamics ◽

High Sensitivity ◽

Maldi Mass Spectrometry ◽

Noncovalent Interaction ◽

Ionization Mass ◽

Label Free

Noncovalent interactions are the keys to the structural organization of biomolecule e.g., proteins, glycans, lipids in the process of molecular recognition processes e.g., enzyme-substrate, antigen-antibody. Protein interactions lead to conformational changes, which dictate the functionality of that protein-protein complex. Besides biophysics techniques, noncovalent interaction and conformational dynamics, can be studied via mass spectrometry (MS), which represents a powerful tool, due to its low sample consumption, high sensitivity, and label-free sample. In this review, the focus will be placed on Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) and its role in the analysis of protein-protein noncovalent assemblies exploring the relationship within noncovalent interaction, conformation, and biological function.

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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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Conformational Dynamics and the Energetics of Protein−Ligand Interactions: Role of Interdomain Loop in Human Cytochrome P450 Reductase†

Biochemistry ◽

10.1021/bi700596s ◽

2007 ◽

Vol 46 (28) ◽

pp. 8244-8255 ◽

Cited By ~ 23

Author(s):

Alex Grunau ◽

Kalotina Geraki ◽

J. Günter Grossmann ◽

Aldo Gutierrez

Keyword(s):

Cytochrome P450 ◽

Conformational Dynamics ◽

Cytochrome P450 Reductase ◽

P450 Reductase ◽

Protein Ligand Interactions ◽

Human Cytochrome ◽

Ligand Interactions

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Conformational flexibility of adenine riboswitch aptamer in apo and bound states using NMR and an X-ray free electron laser

Journal of Biomolecular NMR ◽

10.1007/s10858-019-00278-w ◽

2019 ◽

Vol 73 (8-9) ◽

pp. 509-518

Author(s):

Jienv Ding ◽

Monalisa Swain ◽

Ping Yu ◽

Jason R. Stagno ◽

Yun-Xing Wang

Keyword(s):

Conformational Changes ◽

Free Electron ◽

Free Electron Laser ◽

Messenger Rna ◽

Bound States ◽

Conformational Dynamics ◽

Thermal Fluctuation ◽

Binding Pocket ◽

X Ray ◽

Aptamer Domain

Abstract Riboswitches are structured cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the add adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the add adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall molecule exhibits a motion at kex ~ 319 s−1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with kex~ 83 s−1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 s−1. The µs–ms motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation, ~ 50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.

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