Notes & Tips Thermodynamics of the cyclophilin-A/cyclosporin-A interaction: A direct comparison of parameters determined by surface plasmon resonance using Biacore T100 and isothermal titration calorimetry Martin A. Wear, Malcolm D. Walkinshaw * The Centre for Translational and Chemical Biology, The University of Edinburgh, Michael Swann Building, King’s Buildings, Mayfield Road, Edinburgh EH9 3JR, UK Received 4 August 2006 Available online 22 September 2006 Surface plasmon resonance (SPR) 1 is regularly exploited in the kinetic analysis of biomolecular interactions and small-molecule drug-discovery/hit-validation studies [1,2]. SPR instruments uniquely allow the measurement of kinetic and thermodynamic data specifically associated with com- plex formation and dissociation. Such SPR data can be used to ‘‘thermodynamic profile’’ interactions [3–5] enhancing the correlation of solution binding measurements with structur- al features and the assignment of proportional energetic con- tributions to individual functional groups, the basis of the whole-structure-based design approach to engineered thera- peutics and drug design [6,7]. Here we compare thermody- namic parameters determined by SPR using Biacore T100 (a new SPR biosensor from Biacore AB (Uppsala, Sweden)) and by isothermal titration calorimetry (ITC), the standard method of determining equilibrium thermodynamics [8], for the interaction between human cyclophilin-A (CypA) and its natural inhibitor, a hydrophobic cyclic undecapep- tide, cyclosporin-A (CsA) [9]. The CypA/CsA structure has been solved [10,11] and the biochemistry of the interaction well characterized [9,12–14], thus it allowed us to rationalize the thermodynamic data with regards to the the molecular mechanism and structure and enabled a more accurate assessment and comparison of the parameters obtained from SPR and ITC. To assess the ability of Biacore T100 to produce quality equilibrium thermodynamic data, we generated a highly active ( P85% activity) and stable SPR sensor surface of His-CypA using a protocol slightly modified from that described [14]. His-CypA was covalently stabilized, using standard amine coupling chemistry, during capture/orienta- tion on a Ni 2+ –nitrilotriacetic acid sensor (see supplementa- ry material for full experimental details). SPR experiments with CsA were performed (in triplicate) at temperatures ranging from 16 to 35 °C. CsA concentrations from 13.1 to 209.6 nM, were injected over the sensor surface at 50 ll min 1 for 300 s. Surface regeneration between experiments was performed by dissociating any formed complex in running buffer for 30 min followed by a further 5-min stabilization period (CypA is intolerant to more rigorous regeneration [14]). The apparent on- (k + ) and off-rate (k  ) constants and the equilibrium dissociation constant (K dT100 )(Supplementary Table 1) were calculated by global fitting of a 1:1 binding model to the sensorgrams (Supple- mentary Fig. 1A shows typical fitted data) using software provided with the instrument. Over the 16 to 35 °C temperature range, the off-rate is increased 70-fold compared to a 12-fold increase in the on-rate (inset Supplementary Fig. 1A, Supplementary Table 1), resulting in a 4-fold loss of affinity from 10 to 40 nM (Supplemen- tary Fig. 1B, Supplementary Table 1). Little effect on the constants as a function of temperature due to mass transport considerations was seen. Notably, this is first time that the on- and off-rates, defined as a function of temperature, have been reported for CypA binding CsA. For simple equilibrium thermodynamics, the van’t Hoff equation states that a plot of ln K d versus 1/T should be a straight line, with a slope of DH°/R and an ordinate intercept of DS°/R. However, nonlinearity indicates temperature-depen- dent enthalpy and entropy changes upon complex formation; i.e., the heat capacities of the reactants differ from the heat capacity of the complex. In the simplest case, DC p may be 0003-2697/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.08.038 * Corresponding author. E-mail address: M.Walkinshaw@ed.ac.uk (M.D. Walkinshaw). 1 Abbreviations used: SPR, surface plasmon resonance; ITC, isothermal titration calorimetry; CYPA, cyclophilin-A; CSA, cyclosporin-A. www.elsevier.com/locate/yabio ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 359 (2006) 285–287