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Protein Labeling and Structure Determination by NMR Spectroscopy

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Biophysical and Computational Tools in Drug Discovery

Part of the book series: Topics in Medicinal Chemistry ((TMC,volume 37))

Abstract

NMR spectroscopy has proven itself to be invaluable in probing the structures of proteins that are not amenable to crystallization. In addition, NMR spectroscopy is of great value in characterizing the weak interactions that form the basis of drug-like action of potential hits. While 2D NMR methods for the characterization of small molecules were developed in the 1970s, in recent times, there has been a significant growth in the macromolecular NMR field, which has potentiated structure determination of several drug target proteins and protein–ligand complexes and discovery of inhibitors through NMR-based screening methods. In this chapter, our aim will be to describe the state-of-the-art methods adopted for isotope labeling of proteins, which will be followed by description of the multidimensional NMR experiments and their utilization for determination of solution structure of the proteins. NMR-based structure determination begins with suitable isotope labeling of proteins using many innovative methods. Once a double or triple isotopically labeled sample has been made, a series of multidimensional NMR experiments are carried out for the assignment of chemical shifts of the backbone and side-chain resonances and for measuring the NOE between identified protons. An ensemble of structures can then be calculated by incorporating the experimentally derived dihedral angle and distance constraints. Refinement of structural quality is done through additional constraints derived from Residual Dipolar Couplings and Paramagnetic Relaxation Enhancements. Using this technique and protocols, structures of several drug target proteins have been successfully determined with high precision. Further, NMR has been used to screen the small molecules that bind to the target protein and to study the structural and dynamics aspects of protein-ligand complexes.

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Abbreviations

CBP:

Cellulose binding protein

COSY :

Correlation spectroscopy

CSI:

Chemical shift index

DOSY :

Diffusion ordered spectroscopy

FBDD:

Fragment-based drug discovery

GST :

Glutathione S-transferase

HMQC :

Heteronuclear multiple quantum coherence

HSQC:

Heteronuclear single quantum coherence

IDP:

Intrinsically disordered protein

ILOE :

Inter-ligand nuclear Overhauser effect

INPHARMA :

Inter-ligand NOEs for pharmacophore mapping

IPTG:

Isopropyl-β-D-1-thiogalactopyranoside

LBT :

Lanthanide-binding peptide tag

MBP:

Maltose binding protein

MTSL:

(1-Oxyl-2,2,5,5-tetramethyl-D3-pyrroline-3-methyl) methanethiosulfonate

NMR:

Nuclear magnetic resonance

NOE :

Nuclear Overhauser effect

NOESY:

Nuclear Overhauser effect spectroscopy

PCS :

Pseudo-contact shift

PRE:

Paramagnetic relaxation enhancement

RDC:

Residual dipolar couplings

SAIL:

Stereo-array isotope labeling

SBDD:

Structure-based drug discovery

Tg:

Toxoplasma gondii

TOCSY :

Total correlation spectroscopy

TROSY :

Transverse relaxation-optimized spectroscopy

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Acknowledgements

AA acknowledges grants from DBT (BT/PR31893/MED/29/1390/2019) and CSIR-CDRI (FBR/MLP2029). This is communication number 10257 from CSIR-Central Drug Research Institute.

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Authors and Affiliations

  1. Biochemistry and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, UP, India

    Surbhi Mundra, Jay Kumar, Diva Maheshwari & Ashish Arora

  2. Institute of Structural and Molecular Biology, University College London, London, UK

    Vaibhav K. Shukla

  3. Department of Chemistry, Mississippi State University, Mississippi State, MS, USA

    Rahul Yadav

  4. Department of Chemistry, State University of New York at Buffalo, Buffalo, NY, USA

    S. V. S. Rama Krishna Pulavarti

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  1. Surbhi Mundra
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  2. Jay Kumar
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Correspondence to Ashish Arora .

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  1. GIPER, Kashipur, India

    Anil Kumar Saxena

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Funding: AA acknowledges grants from DBT (BT/PR31893/MED/29/1390/2019) and CSIR-CDRI (FBR/MLP2029).

Ethical Approval: This manuscript is a review of previously published accounts, as such, no animal or human studies were performed.

Informed Consent: No patients were studied in this chapter.

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Mundra, S. et al. (2021). Protein Labeling and Structure Determination by NMR Spectroscopy. In: Saxena, A.K. (eds) Biophysical and Computational Tools in Drug Discovery. Topics in Medicinal Chemistry, vol 37. Springer, Cham. https://doi.org/10.1007/7355_2021_133

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