The effect of molecular crowding on the structure and function of Escherichia coli prolyl-transfer RNA synthetase (Ec ProRS), a member of the aminoacyl-transfer RNA synthetase family, has been investigated using a combined experimental and theoretical method. Ec ProRS is a multidomain enzyme; coupled-domain dynamics are essential for efficient catalysis. To gain insight into the mechanistic detail of the crowding effect, kinetic studies were conducted with varying concentrations and sizes of crowders. In parallel, spectroscopic and quantum chemical studies were employed to probe the ''soft interactions'' between crowders and protein side chains. Finally, the dynamics of the dimeric protein was examined in the presence of crowders using a long-duration (70 ns) classical molecular dynamic simulations. The results of the simulations revealed a shift in the conformational ensemble, which is consistent with the preferential exclusion of cosolutes. The ''soft interactions'' model of the crowding effect also explained the alteration in kinetic parameters. In summary, the study found that the effects of molecular crowding on both conformational dynamics and catalytic function are correlated in the multidomain Ec ProRS, an enzyme that is central to protein synthesis in all living cells. This study affirmed that large and small cosolutes have considerable impacts on the structure, dynamics, and function of modular proteins and therefore must be considered for stabilizing protein-based pharmaceuticals and industrial enzymes.

Prolyl-tRNA synthetases (ProRSs) catalyze the covalent attachment of proline onto cognate transfer ribonucleic acids (tRNAs), an indispensable step for protein synthesis in all living organisms. ProRSs are modular enzymes and the "prokaryotic-like" ProRSs are distinguished from "eukaryotic-like" ProRSs by the presence of an editing insertion domain (INS) inserted between motifs 2 and 3 of the main catalytic domain. Earlier studies suggested that the presence of coupled-domain dynamics could contribute to catalysis; however, the role that the distal and highly mobile INS domain plays in catalysis at the synthetic active site is not completely understood. In the present study, a combination of theoretical and experimental approaches has been used to elucidate the precise role of INS domain dynamics. Quantum mechanical/molecular mechanical simulations were carried out to model catalytic prolyl-adenylate formation by Enterococcus faecalis ProRS. The energetics of the adenylate formation by the wild-type enzyme was computed and contrasted with variants containing active site mutations as well as a deletion mutant lacking the INS domain. The combined results revealed that two distinct types of dynamics contribute to the enzyme's catalytic power. One set of motions is intrinsic to the INS domain and leads to conformational preorganization that is essential for catalysis. A second type of motion, stemming from the electrostatic reorganization of active site residues, impacts the height and width of the energy profile and has a critical role in fine-tuning the substrate orientation to facilitate reactive collisions. Thus, motions in a distal domain can preorganize the active site of an enzyme to optimize catalysis.

Quinone reductases belong to the family of flavin-dependent oxidoreductases. With the redox active cofactor, flavin adenine dinucleotide, quinone reductases are known to utilize a ‘ping-pong’ kinetic mechanism during catalysis in which a hydride is bounced back and forth between flavin and its two substrates. However, the continuation of this catalytic cycle requires product displacement steps, where the product of one redox half-cycle is displaced by the substrate of the next half-cycle. Using improved hybrid quantum mechanical/molecular mechanical simulations, both the catalytic hydride transfer and the product displacement reactions were studied in NRH:quinone oxidoreductase 2. Initially, the self-consistent charge-density functional tight binding theory was used to describe flavin ring and the substrate atoms, while embedded in the molecular mechanically-treated solvated active site. Then, for each step of the catalytic cycle, a further improvement of energetics was made using density functional theory-based corrections. The present study showcases an integrated interplay of solvation, protonation, and protein matrix-induced polarization as the driving force behind the thermodynamic wheel of the ‘ping-pong’ kinetics. Reported here is the first-principles model of the ‘ping-pong’ kinetics that portrays how cyclic changes in the active site polarization and dynamics govern the oscillatory hydride transfer and product displacement in this enzyme.

The effect of macromolecular crowding on the structure and function of Escherichia coli prolyl-tRNA synthetase (Ec ProRS) has been investigated using a combined experimental and theoretical method. Ec ProRS is a multi-domain enzyme; coupled-domain dynamics is essential for efficient catalysis. To gain an insight into the mechanistic detail of the crowding effect, kinetic studies were conducted with varying concentrations and sizes of crowders. In parallel, spectroscopic and quantum chemical studies were employed to probe the "soft-interactions" between crowders and protein side chains. Finally, the dynamics of the dimeric protein was examined in the presence of crowders using a long-duration (70 ns) classical molecular dynamic simulations. The results of the simulations revealed a significant shift in the conformational ensemble, which is consistent with the "soft-interactions" model of the crowding effect and explained the observed alteration in kinetic parameters. Collectively, the present study demonstrated that the effects of molecular crowding on both conformational dynamics and catalytic function, are correlated. This is the first report where molecular crowding has been found to impact the conformational ensemble in the multi-domain Ec ProRS, a member of aminoacyl-tRNA synthetase family, which is central to protein synthesis in all living cells. The present study affirmed that the effect of crowders should be considered while investigating the structure-dynamics-function relationship in modular enzymes.All crowding agents were purchased from Sigma Aldrich, except for polyethylene glycol (PEG) 8000 (Fisher Scientific). Proline (≥ 99%) was also from Sigma Aldrich. Both [γ-32 P] ATP and [ 32 P] PP i were purchased through Perkin Elmer, Shelton, CT. Overexpression and purification of Ec ProRSWild-type (WT) Ec ProRS was overexpressed in SG13009 (pREP4) competent cells using 0.1 mM isopropyl β-D-thiogalactoside for 4 hours at 37 °C. Histidine-tagged WT Ec ProRS was purified using Talon cobalt affinity resin column; 100 mM imidazole was used to elute the protein (24,25). The Bio-Rad protein assay (Bio-Rad Laboratories) was used to determine total concentration of protein. An active-site titration was performed to determine the concentration of active protein (26). Enzyme kineticsCrowding agent concentration variation. The ATP-PP i exchange assay was performed, following the protocol described elsewhere, to examine the effect of increasing concentrations of crowding agents on proline activation (eq.1) by Ec ProRS (27). In ATP-PP i exchange assay, radiolabeled PP i ( 32 PP i ) and non-radiolabeled ATP were used and the percent product (Pro-AMP) formation at 20 minutes post-initiation of the reaction was measured in the presence of crowding agents. In the present study, the amount of Pro-AMP formed was indirectly measured by monitoring the amount of 32 P-ATP formed via the reverse reaction of eq. 1 (ProRS + Pro + ATP ⇌ ProRS·(Pro-AMP) + PP i ). The percent product formation was calculated from the ratio of the Pro-A...

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