Protocol to identify drug-binding sites in proteins using solution NMR spectroscopy

Srinivasa Penumutchu; Jiuyang Liu; Upendra K Singh; Tatiana G Kutateladze; Yi Zhang

doi:10.1016/j.xpro.2022.101842

Protocol to identify drug-binding sites in proteins using solution NMR spectroscopy

STAR Protoc. 2022 Dec 16;3(4):101842. doi: 10.1016/j.xpro.2022.101842. Epub 2022 Nov 11.

Authors

Srinivasa Penumutchu¹, Jiuyang Liu², Upendra K Singh¹, Tatiana G Kutateladze², Yi Zhang³

Affiliations

¹ Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA.
² Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA.
³ Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA; Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address: yi.zhang26@case.edu.

Abstract

Dusquetide is a next-generation IDR (innate defense regulator) targeting the major autophagy receptor protein SQSTM1/p62 and modulating the innate immune response. Here, we describe a protocol for determining dusquetide-binding sites of p62 by solution NMR spectroscopy. Step-by-step technique details were provided, including sample preparation, NMR experiment setup, data processing, and binding site analysis. This protocol could be applied to characterize other small molecules targeting the ZZ domain of p62 (9 kDa) or other proteins containing ZZ domains. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).

Keywords: Molecular Biology; NMR; Protein Biochemistry; Protein expression and purification.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Binding Sites
Immunity, Innate*
Magnetic Resonance Spectroscopy
Protein Domains
Sequestosome-1 Protein / chemistry
Sequestosome-1 Protein / metabolism

Substances

Sequestosome-1 Protein

Abstract

Publication types

MeSH terms

Substances

Grants and funding