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

2012 ◽  
Vol 40 (2) ◽  
pp. 419-423 ◽  
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.

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

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.

scholarly journals Entropy in molecular recognition by proteins

2017 ◽  
Vol 114 (25) ◽  
pp. 6563-6568 ◽  
Author(s):  
José A. Caro ◽  
Kyle W. Harpole ◽  
Vignesh Kasinath ◽  
Jackwee Lim ◽  
Jeffrey Granja ◽  
...  
Keyword(s):  
Molecular Recognition ◽  
Protein Interactions ◽  
Nmr Relaxation ◽  
Quantitative Relationship ◽  
Accessible Surface Area ◽  
Conformational Entropy ◽  
General Role ◽  
High Affinity ◽  
Protein Ligand Interactions ◽  
Ligand Interactions

Molecular recognition by proteins is fundamental to molecular biology. Dissection of the thermodynamic energy terms governing protein–ligand interactions has proven difficult, with determination of entropic contributions being particularly elusive. NMR relaxation measurements have suggested that changes in protein conformational entropy can be quantitatively obtained through a dynamical proxy, but the generality of this relationship has not been shown. Twenty-eight protein–ligand complexes are used to show a quantitative relationship between measures of fast side-chain motion and the underlying conformational entropy. We find that the contribution of conformational entropy can range from favorable to unfavorable, which demonstrates the potential of this thermodynamic variable to modulate protein–ligand interactions. For about one-quarter of these complexes, the absence of conformational entropy would render the resulting affinity biologically meaningless. The dynamical proxy for conformational entropy or “entropy meter” also allows for refinement of the contributions of solvent entropy and the loss in rotational-translational entropy accompanying formation of high-affinity complexes. Furthermore, structure-based application of the approach can also provide insight into long-lived specific water–protein interactions that escape the generic treatments of solvent entropy based simply on changes in accessible surface area. These results provide a comprehensive and unified view of the general role of entropy in high-affinity molecular recognition by proteins.

scholarly journals Structural origins of protein conformational entropy

2021 ◽  
Author(s):  
José A. Caro ◽  
Kathleen G. Valentine ◽  
A. Joshua Wand
Keyword(s):  
Ligand Binding ◽  
Protein Function ◽  
Nmr Relaxation ◽  
Internal Motion ◽  
Side Chains ◽  
Conformational Entropy ◽  
Relaxation Methods ◽  
Detailed Structural Analysis ◽  
Packing Defects ◽  
Single Water Molecule

AbstractThe thermodynamics of molecular recognition by proteins is a central determinant of complex biochemistry. For over a half-century detailed cryogenic structures have provided deep insight into the energetic contributions to ligand binding by proteins1. More recently, a dynamical proxy based on NMR-relaxation methods has revealed an unexpected richness in the contributions of conformational entropy to the thermodynamics of ligand binding2,3,4,5. There remains, however, a discomforting absence of an understanding of the structural origins of fast internal motion and the conformational entropy that this motion represents. Here we report the pressure-dependence of fast internal motion within the ribonuclease barnase and its complex with the protein barstar. Distinctive clustering of the pressure sensitivity correlates with the presence of small packing defects or voids surrounding affected side chains. Prompted by this observation, we performed an analysis of the voids surrounding over 2,500 methyl-bearing side chains having experimentally determined order parameters. We find that changes in unoccupied volume as small as a single water molecule surrounding buried side chains greatly affects motion on the subnanosecond timescale. The discovered relationship begins to permit construction of a united view of the relationship between changes in the internal energy, as exposed by detailed structural analysis, and the conformational entropy, as represented by fast internal motion, in the thermodynamics of protein function.

scholarly journals Interbase-FRET binding assay for pre-microRNAs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mattias Bood ◽  
Anna Wypijewska del Nogal ◽  
Jesper R. Nilsson ◽  
Fredrik Edfeldt ◽  
Anders Dahlén ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Drug Targets ◽  
Structural Changes ◽  
Small Sample Size ◽  
Resonance Energy ◽  
Small Sample ◽  
Titration Calorimetry ◽  
Drug Candidate ◽  
Binding Studies ◽  
Aberrant Expression

AbstractThe aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule inhibitors of miR biogenesis. These inhibitors have the potential to (i) dissect miR mechanisms of action, (ii) discover new drug targets, and (iii) function as new therapeutic agents. Here, we designed Förster resonance energy transfer (FRET)-labeled oligoribonucleotides of the precursor of the oncogenic miR-21 (pre-miR-21) and used them together with a set of aminoglycosides to develop an interbase-FRET assay to detect ligand binding to pre-miRs. Our interbase-FRET assay accurately reports structural changes of the RNA oligonucleotide induced by ligand binding. We demonstrate its application in a rapid, qualitative drug candidate screen by assessing the relative binding affinity between 12 aminoglycoside antibiotics and pre-miR-21. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to validate our new FRET method, and the accuracy of our FRET assay was shown to be similar to the established techniques. With its advantages over SPR and ITC owing to its high sensitivity, small sample size, straightforward technique and the possibility for high-throughput expansion, we envision that our solution-based method can be applied in pre-miRNA–target binding studies.

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