Unraveling the dynamic nature of protein–graphene oxide interactions

Abstract

The globular protein ubiquitin interacts with graphene oxide and undergoes dynamic and reversible association–dissociation as revealed by NMR spectroscopy.

Modulation of protein–graphene oxide interactions with varying degrees of oxidation

“…To characterize and quantify the dynamic aspects of the interaction of ubiquitin with various types of GO at the residue level, we recorded 2D [ 15 N, 1 H] Heteronuclear Single Quantum Coherence (HSQC) NMR spectra and amide proton relaxation times T 2 ( 1 H N ) of the protein sample in the presence and absence of each GO sample at pH 6. 25,37,38 In a 2D [ 15 N, 1 H] HSQC NMR spectrum each cross-peak corresponds to a directly bonded 1 H-15 N spin pair (with the exception of proline residues); thus the individual behaviour of each residue of ubiquitin can be probed using the intensity of its own cross-peak belonging to that amide bond. 37 Two types of titrations were performed, forward titration and reverse titration.…”

“…37 Two types of titrations were performed, forward titration and reverse titration. 25,37,39,40 In the former (forward) titration, from a stock of 1 mg ml À1 , successive amounts of a particular GO sample (20 ml, 30 ml, 40 ml, 50 ml and 60 ml corresponding to a nal concentration of each GO sample of 38.4 mg ml À1 , 56.6 mg ml À1 , 74.1 mg ml À1 , 90.9 mg ml À1 and 107.1 mg ml À1 aer considering the dilution of the whole volume) were added to 500 ml of the uniformly 15 N-labeled ubiquitin (100 mM) dissolved in 50 mM phosphate buffer supplemented with 150 mM NaCl and 5% 2 H 2 O at pH 6. This is referred to as 'forward titration'.…”

“…The modication involves the introduction of an additional delay during the rst INEPT and acquisition of two spectra (two-point HSQC), each with an additional delay time (16 ms in the rst (referred to as "with delay spectrum" and 20 ms in the second (referred to as "without delay spectrum"))), while keeping all the parameters constant. 25,37 The residue specic exponential decay of the signals of amide protons ( 1 H N ) as observed in a 2D [ 15 N, 1 H] HSQC NMR experiment due to the transverse relaxation of spins can be written as:…”

“…varies, as calculated theoretically from the reverse titration (as described in the ESI †). 25,37 1 mg of G_0 binds $3 mg of Ubq 1 mg of G_30 binds $5.7 mg of Ubq 1 mg of G_80 binds $4.3 mg of Ubq 1 mg of G_100 binds $5.7 mg of Ubq The difference in the loading capacity of the different GO samples is conrmed by the BCA assay. 43 This variable loading capacity of different GO samples is dependent upon their surface charge which in turn depends on the degree of oxidation.…”

“…35 This feature renders NMR a powerful spectroscopic technique for probing structural and dynamic perturbations in the protein backbone upon its interaction with GO. 25 In the present work, using NMR spectroscopy in combination with other techniques such as isothermal titration calorimetry (ITC), zeta potential (z) measurements, FTIR spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), CD spectroscopy, Atomic Force Microscopy (AFM) and Raman spectroscopy, we have studied the residue specic interaction of a globular protein human ubiquitin with GO prepared with different oxidation degrees. 36 We have chosen human ubiquitin as the model globular protein in the present study because it is found in almost all eukaryotes and is an important regulatory protein involved in the ubiquitin proteasome pathway.…”