The overall goal of this interdisciplinary protocol is to enhance our fundamental understanding of the impact of solvent dynamics on enzyme catalysis. This method can help answer key questions in the biocatalysis field. Such as, how solvent reorganization drives enzyme dynamics and catalysis.
The main advantage of this technique is that it allows for the engineering of tunnels, or motives of water transport, which affects specificity, activity, and thermodynamics. Visual demonstration of this method is critical, as the interdisciplinary steps are difficult to learn because they bridge several sub-disciplines of science. To begin, download a PDB structure of the protein of interest from the Protein Databank.
Prepare the structure by removing excess subunits and then adding missing hydrogen atoms, an explicit solvent, using the cell neutralization and pkA prediction experiment in YASARA. For membrane-bound triterpene cyclases, suitable dimensions of the waterbox are 91 x 67 x 77 Angstroms. Adjust the protonation state of the catalytically active amino acids manually.
Minimize the structure, by the energy minimization command, using the AMBER force-field suite. Use the particle mesh ewald approach, to account for long-range electrostatic interactions. Set the cutoff for van der Waals'interactions, according to standard settings.
Following simulated annealing and molecular dynamics, repair the simulation snapshots, by deleting all water molecules and eventual ligand substrates, using the edit, delete, residue command. Superpose each snapshot individually on the original PDB structure, by the superpose object command, to ensure that all structures share the same spatial position. Save each snapshot prepared in this way as a new PDB file, using the file, save as, PDB command.
Use the prepared snapshots as inputs to the software CAVER 3.0. Visualize the tunnels, generated by CAVER 3.0, in a molecular graphic software program, using the macro shown in the text protocol. Identify the highest ranked tunnels, according to the header ID"listed in the output summary text file, generated from the CAVER 3.0 software.
Using the obtained model, identify amino acids lining the highest ranked tunnels that display a small, side chain pointing toward the channel interior by visual inspection. Identify single amino acid substitutions, that will block the tunnel by increased steric hindrance, using the swap residue command. Verify that the tunnel is obstructed by visual inspection, and then by repeating the protocol thus far.
Here, the introduced tryptophan is shown in magenta, and the swapped serine is shown for comparison. Prepare a potassium-phosphate, resuspension buffer, detergent buffer, and reaction buffer as listed in the text protocol. For squalene-hopene cyclases, or other membrane-bound enzymes with a lower pH optimum, use a citrate-containing resuspension buffer.
Weigh a frozen cell pellet in a glass beaker. Resuspend the cells in resuspension buffer, using a homogenizer to a final concentration of 0.3 gram-cells/milliliter. Then, lyse the resuspended cells by ultrasonication with an amplitude of 80%and a pulse of one second on, and one second off.
Use three repeating cycles of 50 seconds each. Add detergent buffer to a final concentration of 0.2%detergent. Equilibrate, by end over end mixing, at four degrees Celsius for one hour.
Recover the membrane protein fraction, by saving the supernatant after a single centrifugation step at 39, 000 gs, for 50 minutes at four degrees Celsius. Make a fresh, five millimolar stock solution of substrate in reaction buffer. Obtain a homogeneous emulsion by ultrasonication at 30%amplitude for two to five minutes.
Next, equilibrate a ThermoMixer at the desired reaction temperature. Verify the temperature inside a glass vial containing water by an external thermometer. For single-substrate kinetics, dilute the emulsified substrate into at least five glass vials, to the same final substrate concentration.
Pre incubate the glass vials in the ThermoMixer for 10 minutes. Start the reaction by adding the enzyme. A total reaction volume of one milliliter is suitable.
Use a shaking speed of at least 1200 RPM. Stop the reactions at different time points by adding 500 microliters of ethyl acetate, spiked with 100 micromolar decanol as an integration standard. Extract the reaction mixtures by vortexing, and manually shaking the glass vials vigorously for one minute.
Then, centrifuge the glass vials at 9, 600 gs, in a tabletop centrifuge, for 10 minutes at room temperature. Transfer the top layer to an empty tube. Add an additional 500 microliters of extraction solvent to the reaction tubes, and repeat the extraction procedure.
Dry the extracted reaction mixtures with sodium sulfate. Vortex for five seconds and let the tubes rest for 10 minutes. Perform a final centrifugation step at 9, 600 gs for one minute at room temperature, using a tabletop centrifuge.
Then, transfer the extracted samples to gas chromatography, or GC, vials. Repeat the procedure for each enzyme variant of interest, using at least four different initial concentrations of the substrate for single-substrate kinetics, and four different temperatures for both single-substrate and competitive kinetics. After performing GC analysis, as described in the text protocol, integrate the peak areas of the products and transform product peak areas into the corresponding product concentrations, by utilizing the area of the integration standard.
Plot the concentration of each product, versus the reaction time. Perform linear regression of the data points corresponding to less than 10%conversion. The initial reaction speed is given by the slope of the fitted line.
For one-pot relative kinetics, with multiple substrates, obtain the relative apparent K-cat over Km values, using the equation listed in the text protocol, following the cyclization reaction over time. The influence of solvent reorganization on the temperature dependence of apparent K-cat over Km values is shown. Here are representative results of thermodynamical analysis using transition state theory and linear fit of the experimental data to the transition-state theory equation for single-substrate kinetics.
The impact of solvent dynamics, on the thermodynamic parameters of activation, obtained from the experimental thermodynamical analysis is substantial. Enzyme variants with impaired water channels, display fundamentally different substrate specificities, caused by complex changes in relative activation entropies and enthalpies, as shown here. After its development, this technique paved the way for researchers in the field of biocatalysis, to explore the role of water in driving enzyme dynamics and catalysis, which is currently poorly understood.
