Project Title: Among the winners of a grant contest within the domain of fundamental research, announced by the SRNSF in 2011, was a Project No 11/14 – “Energy profiles of globular proteins: diverse role and signatures of quantum effects and conformational flexibility through the function and stability”, submitted under the direction of Prof. Dimitri Khoshtariya from I. Beritashvili Center of Experimental Biomedicine (with I. Javakhishvili Tbilisi State University as a partner organization).

The results and an outcome:  In the framework of this project, on the one hand, we continued in-depth theory-based systematic experimental studies of already customary objects – representative globular proteins and, on the other hand, there were designed and accomplished some advanced, unique studies with the involvement of new, more convoluted objects such as structurally and functionally more sophisticated redox-active proteins; also encompassing the biomimetic sensory nanoscopic device composed of copper ions entrapped within the self-assembled monolayer films, with an emphasis on the electron exchange event occurring through them. The results of these studies and respective outcome, regarding the research targets, are specified below:

(A) In the case of a-chymotrypsin (a-CT), we studied an impact of variable concentrations of the organic additive with a dualistic nature, dimethyl sulfoxide (DMSO), on the both, catalytic activity and thermodynamic stability. Our experiments reviled rather complicated character for the impact of DMSO on a-CT around pH values of the optimal catalytic activity. In particular, at pH 8.3 it was found that thermal stability of a-CT at lower concentrations of DMSO additive first increased notably, followed by the subsequent dramatic decrease at elevated DMSO concentrations [1]. We also studied an impact of variable concentrations of the novel organic protic salt, choline dihydrogen phosphate ([ch][dhp]) and disclosed a very pronounced monotonic effect of thermodynamic stabilization [4].

(B) In the framework of already long-standing tradition of the research cooperation with the universities of Erlangen (Germany) and Pittsburgh (USA), by using the method of cyclic voltammetry, we studied electron exchange between the redox-active protein, azurin (Az) and the gold (Au) electrode. Az was immobilized at Au-deposited alkanethiol self assembled monolayer (SAM) films of variable thickness. In these particular studies experiments were performed in the presence of high concentrations (up to 90 % w/w) of the organic salt additive, [ch][dhp] (in ultra-viscous environments), when the protein milieu contains only one or two water molecules per ion pair of the additive. Due to the broadly variable experimental conditions including the additive concentration, temperature and pressure, the dynamical physical characteristics of the electron exchange process were also allowed to vary broadly, disclosing for the firs time in history the new type of mechanistic interplay for a biomoleculat electron exchange at the electrode under the physical condition that factually approaches a glassy state [2].

(C) In cooperation with the University of Erlangen (Germany), by using the method of cyclic voltammetry, we studied the electron exchange mechanisms for myoglobin (Mb) functionalized at Au-deposited SAM films of variable thickness, acting either in the freely diffusing or irreversibly immobilized kinetic regimes. Through the additional variation of temperature and pressure conditions, it has been established that the strength of interaction between Mb and SAM terminal groups determines the kinetic mode of electron exchange, as well as the conformational flexibility of Mb, hence the physical nature of the electron exchange mechanism. In the case of irreversibly (tightly) immobilized Mb, restriction of Mb’s conformational flexibility also restricts intra-globular motion of the heme-coordinated ligand (in our particular case – of a water molecule) that is inseparably coupled to the electron transfer. Consequently, the electron transfer is fast [3,7]. In contrast, when Mb is “free”, its conformational flexibility allows for the substation translocations of a coordinated water, what essentially slows down the electron exchange rate [3,7].

(D) In cooperation with the University of Greensboro (NC, USA) by using the method of fast scan cyclic voltammetry, we also studied the electron exchange mechanism for the redox active enzyme, glucose oxidase (GOx) that was functionalized at polymer-treated and carbon nanotube- (CNT) modified glassy carbon (GC) electrodes, In general, biological function of GOx is much more complex than the simple electron exchange. In the absence of its natural substrate, glucose, its co-factor, flavin adenine dinucleotide (FAD) exchanges two electrons with the GC electrode which is coupled with the translocations of two protons. Our studies revealed that the polymer-activated CNT ending parts (factually, acting as nanoelectrodes) penetrate into the active sites of GOx, leading to the direct electronic “wiring” between otherwise intra-globularly buried FAD moieties and GC electrodes, after which the abovementioned two-electron process takes place [6].

(E) Furthermore, we studied the mechanism of an electron exchange process within the biomimetic device composed of an Au-electrode coated by the cysteine SAM and copper ions entrapped at the SAM-electrolyte border. Kinetic studies of electron exchange using the cyclic voltammetry  technique under the variable temperature and pressure conditions reveled the strong impact of a glass-forming environment of an interfacial SAM layer, manifested through the kinetic signatures typical for a nonergodic and nonlinear milieu response [5].

(F) We have also undertaken preliminary studies of a supramolecular complex composed of Mb and the cromophoric ligand, proflavine (Pf), by the method of computer modeling. The purpose of this work was finding of the optimal condition for future experimental photophysical studies of electron transfer between these entities. These preliminary studies revealed the well-pronounced attached (docked) structure with a remarkable free energy minimum regarding the corresponding conformational space.

The results obtained in the framework of the accountant project have been published as 7 original research papers in peer reviewed, reputable international scientific journals:

 

1. Tretyakova, M. Shushanyan, T. Partskhaladze, M. Makharadze, R. van Eldik & D.E. Khoshtariya, Simplicity within the complexity: bilateral impact of DMSO on the functional and unfolding patterns of a-chymotrypsin. Biophysical Chem. (Elsevier B.V.), 2013, v.175, p.17-27.
2. Uchaneishvili, M. Makharadze, M. Shushanyan, R. van Eldik & D.E. Khoshtariya, Notable stabilization of α-chymotrypsin by the protic ionic additive, [ch][dhp]: Calorimetric evidence for a fine enthalpy/entropy balance. ISRN Biophysics (Hindawi Publishers), 2014, Article No. 834189 (6 p.)
3. E. Khoshtariya, T.D. Dolidze, T. Tretyakova, D.H. Waldeck and R. van Eldik, Electron transfer with azurin at Au/SAM junctions in contact with a protic ionic melt: Impact of glassy dynamics. Phys. Chem. Chem. Phys. (Royal Cemical Society, UK) 2013, v.15, p. 16515-16526.
4. E. Khoshtariya, T.D. Dolidze, M. Shushanyan, & R. van Eldik, Long-range electron transfer with myoglobin immobilized at Au/mixed-SAM junctions: Mechanistic impact of the strong protein confinement. J. Phys. Chem. B. (American Chemical Society, USA), 2014, v.118, p.692-706.
5. E. Khoshtariya, T.D. Dolidze, T. Tretyakova, & R. van Eldik, Electron transfer with self-assembled copper ions in Au-deposited biomimetic films: mechanistic ‘anomalies’ disclosed by temperature- and pressure-assisted fast-scan voltammetry. J. Phys. D: Appl. Phys. (IOP Publishing, UK), 2015, v.48, Article No. 513699, (11 p.).
6. Liu, T.D. Dolidze, S. Singhal, D.E. Khoshtariya, & J. Wei, New Evidence for A Quasi-Simultaneous Proton-Coupled Two-Electron Transfer and Direct Wiring for Glucose Oxidase Captured by The Carbon Nanotube-Polymer Matrix. J. Phys. Chem. C (American Chemical Society, USA), 2015, Submitted.
7. D. Dolidze, M. Shushanyan, & D.E. Khoshtariya, Electron transfer with myoglobin in free and strongly confined regimes: disclosing diverse mechanistic role of the Fe-coordinated water by temperature- and pressure-assisted voltammetric studies. J. Coord. Chem. (Taylor and Francis, UK), 2015, v.68, p.3164-3180.