Solution nuclear magnetic resonance (NMR) spectroscopy and, in particular, chemical shift perturbation (CSP) titration experiments are ideally suited for characterizing the binding interface of macromolecular complexes. 1H-15 N-HSQC-based CSP studies have become the method of choice due to their simplicity, short time requirements, and not requiring high-level NMR expertise. Nevertheless, CSP studies for characterizing protein–glycosaminoglycan (GAG) interactions have been challenging due to binding-induced aggregation/precipitation and/or poor quality data. In this chapter, we discuss how optimizing experimental variables such as protein concentration, GAG size, and sensitivity of NMR instrumentation can overcome these roadblocks to obtain meaningful structural insights into protein–GAG interactions.
Nuclear magnetic resonance (NMR) Chemical shift perturbation Protein–ligand interactions Glycosaminoglycan Dissociation constant Heparan sulfate Heparin
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Lindahl U, Kjellén L (2013) Pathophysiology of heparan sulphate: many diseases, few drugs. J Intern Med 273:555–571PubMedCrossRefGoogle Scholar
Kamhi E, Joo EJ, Dordick JS, Linhardt RJ (2013) Glycosaminoglycans in infectious disease. Biol Rev Camb Philos Soc 88:928–943PubMedGoogle Scholar
Dominguez C, Boelens R, Bonvin AM (2003) HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc 125:1731–1737PubMedCrossRefGoogle Scholar
Poluri KM, Joseph PR, Sawant KV, Rajarathnam K (2013) Molecular basis of glycosaminoglycan heparin binding to the chemokine CXCL1 dimer. J Biol Chem 288:25143–25153PubMedCentralPubMedCrossRefGoogle Scholar
Gangavarapu P, Rajagopalan L, Kolli D, Guerrero-Plata A, Garofalo RP, Rajarathnam K (2011) The monomer-dimer equilibrium and glycosaminoglycan interactions of chemokine CXCL8 regulate tissue-specific neutrophil recruitment. J Leukoc Biol 91:259–265PubMedCrossRefGoogle Scholar
Markin CJ, Spyracopoulos L (2012) Increased precision for analysis of protein-ligand dissociation constants determined from chemical shift titrations. J Biomol NMR 53:125–138PubMedCrossRefGoogle Scholar
Mittermaier A, Meneses E (2013) Analyzing protein-ligand interactions by dynamic NMR spectroscopy. Methods Mol Biol 1008:243–266PubMedCrossRefGoogle Scholar
Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293PubMedCrossRefGoogle Scholar
Johnson BA, Blevins RA (1994) NMR View: a computer program for the visualization and analysis of NMR data. J Biomol NMR 4:603–614PubMedCrossRefGoogle Scholar
Goddard TD, Kneller DG. SPARKY 3, University of California, San FranciscoGoogle Scholar