The Molten Globule State of Maltose-Binding Protein: Structural and Thermodynamic Characterization by EPR Spectroscopy a
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Applied Magnetic Resonance
ORIGINAL PAPER
The Molten Globule State of Maltose‑Binding Protein: Structural and Thermodynamic Characterization by EPR Spectroscopy and Isothermal Titration Calorimetry Chen Nickolaus1 · Carolyn Vargas2 · Jörg Reichenwallner3,5 · Mohammed Chakour1 · Benjamin Selmke1 · Rusha Chakraborty4 · Raghavan Varadarajan4 · Sandro Keller2 · Wolfgang E. Trommer1 Received: 28 June 2020 / Revised: 21 July 2020 © The Author(s) 2020
Abstract Employing site-directed spin labeling (SDSL), the structure of maltose-binding protein (MBP) had previously been studied in the native state by electron paramagnetic resonance (EPR) spectroscopy. Several spin-labeled double cysteine mutants were distributed all over the structure of this cysteine-free protein and revealed distance information between the nitroxide residues from double electron–electron resonance (DEER). The results were in good agreement with the known X-ray structure. We have now extended these studies to the molten globule (MG) state, a folding intermediate, which can be stabilized around pH 3 and that is characterized by secondary but hardly any tertiary structure. Instead of clearly defined distance features as found in the native state, several additional characteristics indicate that the MG structure of MBP contains different polypeptide chain and domain orientations. MBP is also known to bind its substrate maltose even in MG state although with lower affinity. Additionally, we have now created new mutants allowing for spin labeling at or near the active site. Our data confirm an already preformed ligand site structure in the MG explaining its substrate binding capability and thus most probably serving as a nucleation center for the final native structure.
* Wolfgang E. Trommer [email protected]‑kl.de 1
Department of Chemistry, TU Kaiserslautern, Erwin‑Schrödinger‑Str. 54, 67663 Kaiserslautern, Germany
2
Molecular Biophysics, TU Kaiserslautern, Erwin‑Schrödinger‑Str. 13, 67663 Kaiserslautern, Germany
3
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
4
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India
5
Present Address: Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada
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Abbreviations ANS 8-Anilinonaphthalene-1-sulfonic acid CW Continuous wave DEER Double electron–electron resonance DQC Double quantum coherence EPR Electron paramagnetic resonance IDP Intrinsically disordered protein ITC Isothermal titration calorimetry MBP Maltose-binding protein MG Molten globule MTS (1-Oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl) methanethiosulfonate SDSL Site-directed spin labeling
1 Introduction Maltose-binding protein (MBP) in E. coli is a single-chain polypeptide of 370 amino acid residues devoid of cofactors. It is composed of two domains with the active site located in a cleft between the domains primarily binding maltose. Substrate binding is accompanied by a conformational change of the
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