Table 1.

A summary of the biophysical techniques discussed in this review and their application to monitoring protein-protein interactions in DMEs.

Technique	Biophysical basis	Physical observables	Pros	Cons	Drug metabolizing enzyme (DME)	References
X-ray crystallography	Electron diffraction by protein crystal lattice	Atomic bond position and length	Definitive structural assignment of all the residues in a polypeptide	Requires a large amount of protein and limited to a “snapshot” in time	CYP, CPR, GST, SULT, MAO, UGT, and others	Sevrioukova et al., 1997, 1999; Cupp-Vickery et al., 2000; Hall et al., 2001; Podust et al., 2001; Yoshinari et al., 2001; Williams et al., 2003; Scott et al., 2004; Yano et al., 2004; Nagano and Poulos, 2005; Nagano et al., 2005; Grahn et al., 2006; He et al., 2006; Hamdane et al., 2009; Deng et al., 2010; Vincent et al., 2012; Wilderman et al., 2012; Hiruma et al., 2013; Tripathi et al., 2013; Peng et al., 2014; Sugishima et al., 2014; Basudhar et al., 2015; Reed and Backes, 2017; Gill, 1983; Kakuta et al., 1997; Meech and Mackenzie, 1997; Argiriadi et al., 1999; Binda et al., 2001, 2011; Petrotchenko et al., 2001; Polekhina et al., 2001; Abdalla et al., 2002; Miley et al., 2007; Wang and Edmondson, 2007; Schoch et al., 2008; Lewis et al., 2011; Nelson et al., 2013; Matsumoto et al., 2014; Suzuki et al., 2014; Fujiwara et al., 2016; Audet-Delage et al., 2017; Chenge et al., 2017
NMR	Measures changes in magnetic resonance of atomic nuclei (typically 1H)	Provides detailed information about the structure, dynamics, reaction state, and chemical environment of macromolecules	Potential for complete structural assignment of the entire protein; also captures protein dynamics	Large amount of protein required; must be soluble	CYP, CPR (domains only), GST, and SULT	Lian, 1998; McCallum et al., 1999; Mahajan et al., 2006; Kijac et al., 2007; Lampe et al., 2008, 2010; Vallurupalli et al., 2008; Gluck et al., 2009; Raman et al., 2010; Ahuja et al., 2013; Estrada et al., 2013, 2014, 2016; Hiruma et al., 2013; Basudhar et al., 2015; Cook et al., 2016; Zhang et al., 2016
Fluorescence: FRET/BRET	Energy transfer from a donor fluorophore to an acceptor fluorophore through non-radiative dipole–dipole coupling	Intermolecular distance, detection of direct molecular interaction between two proteins	Unique information regarding protein dynamics and specific residues involved in the interaction	(Usually) requires the addition of an exogenous fluorophore	CYP, CPR, GST, UGT	Nisimoto et al., 1983; Schwarze et al., 1983; Wang et al., 1993, 2001; Davydov et al., 1996, 2000, 2010, 2013a,b, 2015; Dietze et al., 1996; Lakowicz, 1999; Lu and Atkins, 2004; Wen et al., 2006; Praporski et al., 2009; Li et al., 2011; Yuan et al., 2015, 2016; Fujiwara et al., 2016
Fluorescence: fluorescence anisotropy	Measurement of the photon emission of a fluorophore along different axis of polarization	Measurement of intermolecular binding constants and reaction kinetics	Allows monitoring of direct interaction in real time	Requires exogenous fluorophore with high quantum yield	CYP, GST	Greinert et al., 1979, 1982; Gut et al., 1985; Schwarz et al., 1993; Gorovits and Horowitz, 1995; Lim et al., 1995; Lakowicz, 1999; Szczesna-Skorupa et al., 2003
Photoaffinity labels/peptide MS	Covalent alkylation of a protein with a photo-reactive group, such as an azide, a diazirine, or benzophenone	Identification of specific sites of protein-protein interaction and distances	Provides direct information on specific sites of interaction	Requires introduction of an exogenous photolabile probe on the protein	CYP	Hodek and Smrcek, 1999; Wen et al., 2005; Gao et al., 2006a; Sulc et al., 2008
Chemical crosslinking/peptide MS	Covalent modification of a protein with a chemically reactive group, such as a malimide, iodoacetamide, or isothiocyanates	Identification of specific sites of protein-protein interaction and distances	Provides direct information on specific sites of interaction	Requires introduction of an exogenous chemically labile probe on the protein	CYP, GST	Cooper, 2002; Gao et al., 2006b; Losel et al., 2008; Reed et al., 2010
SPR/Surface immobilization	Determines the change in refractive index of incident light on a surface bilayer due to resonant oscillation of conduction electrons at the interface	Determination of binding constant (Kd) of interaction, thermodynamic analysis, epitope mapping	Allows for determination of binding constants and mode of interaction	Requires that one protein partner be immobilized on a chip surface	CYP	Ivanov et al., 1997, 1999, 2001; Kuznetsov et al., 2004; Shimada et al., 2005; Pearson et al., 2006; Archakov and Ivanov, 2011; Martin et al., 2015; Bostick et al., 2016; Yablokov et al., 2017
Quartz crystal microbalance conductometric monitoring	Conductometric biosensor coupled with in vitro transcription/translation system for monitoring protein-protein interactions using a quartz crystal microbalance with dissipation monitoring	Disassociation constants (Kd), thermodynamic parameters, complex size	Allows for determination of binding and thermodynamic constants	Requires efficient combined transcription and translation of protein	CYP	Davydov et al., 2013b; Spera et al., 2013