University of Groningen
Single-molecule enzymology with a ClyA nanopore Galenkamp, Nicole
DOI:
10.33612/diss.130258760
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Publication date: 2020
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Galenkamp, N. (2020). Single-molecule enzymology with a ClyA nanopore. University of Groningen. https://doi.org/10.33612/diss.130258760
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Chapter 7
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Chapter 1 provides a general introduction into the history and the working mechanism of proteins. The focus is especially on enzymes and their single-molecule studies. During the last three decades the growth in the field of single-molecule techniques and research has uncovered a wealth of new information about these biological systems, which were hidden by the use of ensemble experiments. With single-molecule experiments, conformational heterogeneities can be detected and transient or rare intermediates can be identified. Also, there is the ability to follow the time series of conformational changes and to reveal parallel reaction pathways. Therefore, using single-molecule methods to study complex dynamic enzyme systems is suitable.
The second part of chapter 1 focusses on using nanopores as a tool to observe single molecules. Nanopores are pore-forming toxins that can be used as single-molecule nanoreactors. By applying a potential across the membrane, an ionic current is passing through the nanopore, and changes in this current can be correlated to analytes that flow inside the pore. As the output signal has a very high resolution, enzyme processes such as the binding of ligands or chemical reactions can be monitored in details, enabling single-molecule enzymology studies with an exquisite richness of detail. Initial work with nanopores focussed on the detection of single molecules, chemical reactions and sequencing of nucleic acids. However, recently more complex macromolecules, such as proteins, are being studied and detected with this technique. Since the first proof-of-principle experiments, this new methodology can now be applied to answer questions about conformational changes and dynamics whereby the native enzyme can be studied directly for extended period of time to reveal their single-molecule stochastic behaviour.
In chapter 2, we developed a nanopore sensor for glucose and asparagine in biological fluids. The native glucose binding protein (GBP) of the substrate binding protein family was internalized in a ClyA nanopore to study the conformational change associated to glucose binding. The open and closed configuration of GBP elicited distinct ionic current blockades due to the large conformational change upon binding of the ligand. The confined environment of the nanopore showed no effect on the binding of the analyte as the dissociation constant was similar to the one measured in bulk experiments. From the ratio of open and closed protein displayed by the current through the nanopore, the glucose levels from pico-liters of untreated samples of blood, sweat and urine could be accurately determined. Additionally, the substrate binding domain 1 (SBD1) protein, which is another member of the Venus flytrap protein family, was added to the set-up to measure also the concentration of asparagine. In this way the glucose and asparagine concentration could be simultaneously determined from a human sweat sample within tens of seconds. This data showed the ability to develop new sensitive, continuous and non-invasive sensors based on the ClyA pore with substrate binding proteins as adaptors. The incorporation of the nanopore into a portable device and the use of various protein adaptors for hundreds different metabolites can become a truly new technology for medical diagnostics.
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In chapter 3, we studied the ligand-induced conformational changes of the enzyme dihydrofolate reductase (DHFR) by nanopore current with a slow-reactive substrate (folate), the cofactors (NADPH, NADP+) and an inhibitor (MTX). By using a modified DHFR that remained trapped inside the ClyA nanopore for seconds, it was possible to observe ligand binding with high sampling frequency. The nanopore measurements revealed different DHFR blockades, all with distinctive affinities for the ligands; NADPH, NADP+, folate and MTX. Upon combining the ligands in the experiments the different blockades could be assigned to at least four separate different ground-state conformers. Only one conformer turned out to be catalytically competent. Unexpectedly, two-third of apo-DHFR molecules adopted a ground-state conformer with reduced or no affinity for its ligands. The conformer equilibrium depended on the experimental conditions. Interconversions between the conformers were accelerated during the sampling of the transition-state conformation of DHFR. This suggests that the progression of the reaction reduces the energy barrier between conformer exchanges. This might be a general feature to enzymatic reaction mechanisms, and can provide a link between the passive and active dynamics on the DHFR catalysis, which has been highly debated. Therefore, the different folding’s of DHFR can now be suggested to be linked to its catalytic cycle and are crucial for efficient catalysis.
In chapter 4 we only focused on the catalytically competent conformer described in chapter 3 to characterize the DHFR catalytic reaction at the single-molecule level. Here, we show that with the ionic current of a single nanopore different conformations of an immobilized DHFR could be sampled upon addition of the ligands to the set-up. The reaction cycle could be followed by sampling the exchange between five slightly different conformations of DHFR with high microsecond resolution. This allowed, for the first time, to measure up to hundred turnovers on one single enzyme. By probing the ternary complexes of the closed and occluded conformation of the enzyme, the single-molecule data revealed the precise hierarchy of binding and the affinity of the enzyme for the different reaction intermediates. The sampling of up to hundreds of consecutive reactions of a single enzyme at physiological conditions further revealed rare and transient intermediates along the reaction pathway as well as disorder in the enzyme. Not all ternary complexes appeared to lead to a reaction and there was a high percentage of non-reactive configuration. Also, DHFR undergoes regular seconds’ long kinetic pauses, which reflects the occupancy of an off-path conformation. Additionally, large rearrangement in the enzyme are observed to get into the closed configuration to perform the reaction although the order of binding of substrates resulted in the occluded conformation of the enzyme. No kinetic isoptope effect (KIE) was observed when the reactive ternary complex was formed, indicating that the protonation of the substrate must precede the hydrogen transfer, and the latter is the rate-limiting step of the reaction. Yet, in the transition-state configuration, the binding of folate shows a KIE of 10, indicating that folate forms a hydrogen bond with the cofactor. This could explain why DHFR cannot react with folate. Besides presenting a full choreography of the catalyzed reaction, the single-molecule analysis revealed that the structure of DHFR is sculpted with multiple ground-state conformational with different affinities for one of the products of the reaction. These observations suggest a mechanism in which the DHFR
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conformers regulate the affinity of the enzyme for its ligands during the different steps of the catalytic cycle. Hereby the free energy of folding is used to improve the efficiency of the catalytic reaction.
In chapter 5, we continued the work of chapter 2, whereby multiple mutations where made to the glucose binding protein to understand the mechanism of binding of the ligand. The glucose binding protein displays a large conformational change when its ligands binds to it, also known as the Venus flytrap mechanism. Multiple proteins from the same family has been researched before to understand the role of conformational selection and induced-fit model in these proteins. We measured the intrinsic dynamic kinetics and kinetic binding rates for glucose for multiple GBP mutants at the single-molecule level. The results in this study were used to propose a model for the binding of glucose, in which glucose enters the binding pocket of the protein, and then interacts with an aromatic residue present in the active site, which then clamps the sugar in the right orientation. By entering the active site, the sugar changes the physical-chemical environment there and displaces the water molecules present. This then induces the switch to the closed conformation of the protein. The sugar is then bound in the active site with an extensive network of hydrogen bonds and the two aromatic residues. These interactions secures the ligand in the binding site and keeps the protein in the closed conformation. However, further research is required to verity our proposed hypothesis. In additional we also used these mutated forms of the protein in the protein design of GBP as a biosensor. We were able to find mutants that showed a reduced affinity for glucose which are promising to use as a biosensor to measure glucose in the physiological conditions.
Lastly, in chapter 6 I put the results described in this thesis into a broader perspective. I describe how the DHFR reaction can be further studied to understand the enzyme in even more details. Additionally, I propose several mutations to the E.coli enzyme to research and suggest studying DHFR from a different species to probe the evolutionary aspects of enzyme dynamics. Further research is also proposed to further develop the single-molecule nanopore technique to make it a more standardized approach for studying proteins and enzymes. Highly dynamic enzymes such as adenylate kinase and cyclophilin A are possible target enzymes to test in the nanopore, also because probing the different conformations in these enzymes has been shown to be a difficult task with other single-molecule techniques. By working close together with computer scientists, the experimental data can be supported by theories and computational models. Computational approaches can also contribute to improving the nanopore data analysis. In addition, this nanopore technique can be further developed by having an even larger choice of nanopores, particularly larger diameter nanopores, so that every size and shape of nanopore is available to fit any enzyme in. Furthermore, by extending the use of nanopores for the detection of small analytes and by incorporating it into a portable device, this technique can be the next frontier in personalized healthcare and can result in a wearable biosensor in the future.
