University of Groningen
Single-molecule enzymology with a ClyA nanopore Galenkamp, Nicole
DOI:
10.33612/diss.130258760
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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 6
Concluding discussion and perspectives
Ever since the discovery of the existence of proteins and enzymes, researchers have tried to unravel their structures, mechanisms and, more recently, to understand the role of enzyme dynamics. Thanks to decades of studies, there is now a good general understanding on how
enzymes work. However, most of enzyme studies have been performed using ensemble measurements, which average over an entire population of enzymes. Although that gave a
good understanding of the mechanism of enzyme reactions, many details have not been elucidated yet. This includes dynamic processes such as the existence of conformational heterogeneities between enzymes like variety in turnover number and the present of transient
or rare intermediates, which are related to the catalytic function of enzymes.
The results in this thesis clearly demonstrates that nanopores currents is a powerful new technology that could significantly contribute and even transform our knowledge of proteins and enzymes. To establish the single-molecule nanopore technique into a more standardized approach for studying the dynamics of proteins and enzyme catalysis, there is a large range of
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6.1 Biosensors
In chapter 2 of this thesis, we show that both the glucose binding protein (GBP) and substrate binding domain 1 (SBD1) can be trapped in the ClyA nanopore. The open and the substrate-bound conformations were reflected by the ionic current signal, and their kinetic parameters obtained from the nanopore measurements matched well with values from ensemble studies. With this method, we could quantify accurately metabolites in pico-liters aliquots of human blood, sweat, urine and saliva, simultaneously. As both proteins had a different blocked current signal, both proteins could be distinguished from each other.
Although the biosensor technology has evolved tremendously in the last years, the number of devices that remained as proof-of-concept is quite high and the main biorecognition element used in these devices is still mostly an enzyme, which can loses its activity. Therefore, to sense a whole lot of biomolecules, the biosensor industry, has not reached its full potential yet. Due to the great sensitivity and selectivity of periplasmic binding proteins, this protein family is an ideal candidate to use for the further development of biosensors. Due to their Venus flytrap mechanism, the binding of its ligand results in a relatively large conformational change in the protein. This will results in distinct ionic current blockades between the open and closed conformation in the nanopore. Currently, there are 302 periplasmic binding protein structures deposited in the Protein Data Bank, which bind a wide variety of ligand including amino acids, dipeptides, lipids, vitamins, drugs, metabolic products and hormones.1 As most of these ligands are biomarkers for certain diseases2–5, these protein are suitable to be used as a biosensor. Thus, by expanding the range of protein adaptors that can be trapped in the ClyA, a panel of small analytes can be detected from complex biological samples.
Except for this class of protein also other classes of proteins or biological sensing elements, such as DNA aptamers, can be considered when expending the panel of sensing elements in the nanopore. Also, the determination of protease activity is a powerful tool to detect cancer6, intoxication7 or virus infections8 and was already shown to be possible with nanopores. Furthermore, enzymes in combination with nanopores have been used to detect very small amounts of DNA, for genetic disorder9 or early-stage cancer diagnosis10. The effective detection of protein modifications is a prerequisite for proteomics and protein sequencing with nanopores. Additionally, the ClyA can be implemented in a parallelized microfluidic device. This strategy will enable large-scale integration of multiple sensing proteins in ClyA pore to detect the concentration of multiple analytes at the same time from the same biological sample.
To further create a portable biosensor, ClyA could also be integrated into the MinION™ of Oxford Nanopores Technologies. This portable device is currently only used for sequencing DNA and RNA in real-time. However, this device could be further miniaturised into a wearable biosensor, which could provide for the next frontier device in personalised medicine and healthcare. However, to eventually result in a wearable biosensor for in vivo biomonitoring,
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clearly still many technological advances must be overcome. This includes improving aspects as the innovative fabrication technique.
6.2 Mutated forms of dihydrofolate reductase
Proteins can respond drastically to mutations, even when these occur at sites far away from the active sites or binding pockets. Therefore, site-directed mutagenesis is a powerful technique that is widely used to identify the specific roles of target amino acid in proteins.
An example of such outcome is the G121V mutation of DHFR whereby the hydride transfer is severely reduced, despite the mutated residue being ~15 Å away from the active site.11 In chapter 3 we tested this mutant and showed that this mutated protein was unable to bind the cofactor NADPH. However, the binding of the transition-state analogue methotrexate induced the subsequent binding of NADPH. Importantly, under NADPH binding conditions, multiple crossing between conformers were observed. Although the G121V mutant is highly studied, the question still remains if the behaviour of the mutated enzyme is caused by altered structure, or by dynamics, or maybe even both.
Following this example, additional mutations in DHFR can be analysed at the single-molecule level to gain further insight in the role of certain residues of the enzyme during catalysis. Multiple point mutations in DHFR have already been described in literature and researched extensively, but were only studied in ensemble technique. The relationship between the mutants and the conformer distribution will be particularly interesting. For example, the M42W mutation has been shown to alters the dynamics within the closed conformation of the enzyme and is expected to have this results due to the disruption of a network of coordinated motion that promotes hydride transfer.12 However, how this mutation can affect correlated motions in the protein and what the role of these motions are in altering the hydride transfer is not known in detail. Therefore, experimental single-molecule data of this mutated protein could provide more insight into the catalytic mechanism and how this residue is participating in enzyme dynamics. Other point mutations might be interesting to study as well. For example, S148P, which is known to not form the occluded conformation and binds NADP+ more tightly than wild-type DHFR13, or the L28F mutation, which influences the pathway how tetrahydrofolate is dissociated from the enzyme.14,15 Results from these mutated enzymes might provide additional new insight into the mechanism of DHFR and may even give a better understanding of the role of these residues in the enzyme its structure.
6.3 Dihydrofolate reductase from other species
As pointed out in the discussion of chapter 4, the dynamic mechanism of Escherichia coli DHFR can be explained in light of divergent evolution. Although the structure of human DHFR is almost identical to that of E.coli DHFR, their sequence identity is highly divergent. This causes subtle differences in the catalytic cycle, and although the major cycle they perform is
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the same, human DHFR has a higher activity.16 The human DHFR remains in the closed conformation and the Met20 loop region does not undergo any conformational change, while the E.coli DHFR does. It has already been suggested in literature that the changes in the enzyme catalytic details between the two species arose from the different concentration of ligand available in the cells. In E. coli the concentration of NADP+ and NADPH is similar, while in humans, the concentration of NADPH is much higher than that of NADP+. Hence, it is possible that the enzymes evolved to diverge in their function to work more efficient in their native environment.17 Therefore, studying the human variant at the single-molecule level might provide additional informational about the role of conformers and dynamics in the catalytic cycle.
DHFR from many species have been studied, including from Moritella profunda13,18–20,
Thermotoga maritima21–23, Bacillius anthracis24 and Lactobacillus casei25,26 and Salmonella
enterica27. All these enzymes have unique conformational changes during catalysis despite performing the same chemical reaction. Therefore, single-molecule studies of these enzyme should also uncover the evolutionary aspects of dynamics and conformational changes in enzymatic reactions.
The study of human DHFR and its relation to the different conformers is additionally important because this enzyme is a major drug target in anticancer therapy.28,29 Several DHFR inhibitors have already been found clinical utility as antitumor agent, whereby methotrexate (MTX) is the most used.30 However, due to tumour resistance or ineffectiveness of this inhibitor in some cases, methotrexate has been chemically modified, although so far not with better inhibitory or antitumor activity than methotrexate.31 Due to its unbearable side effects, researchers are still trying to enhance the pharmaco-toxicological profile of methotrexate, for example by making it the activity of the drug for example photocontrolled.32 Therefore to understand the working mechanism of binding for inhibitors is also possible with a nanopore.
6.4 Test more enzymes in pores
Although the study presented in this thesis, together with many others, have progressed our knowledge on enzymes, it should be empathized that although DHFR is considered an archetype for enzyme dynamics, not all findings and conclusions on this enzyme can be extended to other enzymes. Therefore, more enzymes should be sampled using nanopores. In addition, by showing the single-molecule analysis of different enzymes, nanopores can become a more standardized approach for studying enzyme catalysis on the single-molecule level.
Attempts in our research group have been done on trapping the adenylate kinase (AdK) enzyme in the ClyA pore. Adenylate kinase catalyzes the reversible transfer of the terminal phosphate group between adenine nucleotides (ATP, ADP, and AMP) in the presence of magnesium and therefore plays an important role in cellular energy homeostasis.33 Just as DHFR, adenylate
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kinase is a highly studied enzyme in regards to catalysis-related conformational dynamics with techniques as NMR34,35 and single-molecule FRET35,36. The enzyme consists of three domains, and upon substrate binding the protein undergoes a major conformational change.37,38 Recently, it was proposed that instead that the closing of the enzyme is accompanied by a catalytic event34,36, the enzyme opens and closes multiple times before a single chemical step occurs39. We observed something similar with DHFR as described in chapter 4.
Other examples of enzymes from which the role of conformational dynamics in their catalytic cycles has been explored in literature are for example; glucose oxidase, 2-deoxyribose-5-phosphate aldolase, retro-aldolases, β-lactamases.40,41 This are enzymes that could be trapped inside the nanopore and studied at the single-molecule level.
6.5 Computational research
Computational modelling has become indispensable in the last years in the field of biochemistry. Although sampling the dynamics of DHFR on the microsecond-to-millisecond timescale is still out of reach for convention molecular dynamics simulations, the field is developing rapidly. However, to model an enzyme, input from the experimental data is necessary, thus it is important to emphasize that they are both needed to proof new mechanism concepts. Yet, by combining computer predictions and calculations with experimental nanopore data, the science on proteins can be further expanded.
As the results presented in chapter 3 contributes to an intriguing new property of enzymes, this could eventually be implemented with the engineering of enzymes with computational methods. Enzymes used to be described as having only one folded structure. Here we showed that DHFR folds into multiple ground-state conformers. This is likely to be a generic property of enzymes, which could be incorporated into computational modelling. Computational studies can give accurate energetic description of the protein, which can be used by the improvement of the catalytic function of enzymes for industrial purposes as well as for the design of de novo enzymes.
6.6 New biological pores
The ClyA nanopore can be isolated with higher oligomeric states42, extending the range of protein size that will fit into the lumen to up to 40 kDA. Currently, only a few nanopores with different size, shape and properties are available, and new (mostly bigger) nanopores are sought after. Recently, the association of both the YaxA and YaxB proteins has shown to form pores in membranes with pore sizes between 2.0 and 2.4 nm.43 Additionally, when the two component pleurotolysin nanopores (PlyAB)44 can be successfully reconstituted into a standard lipid bilayer, this pore can be the largest biological nanopore used for sensing with a diameter of 5.5 nm in its narrowest site, fitting proteins up to 80 kDa in its lumen. These nanopores can be used
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to study the subunit kinetics and cooperativity of oligomeric enzymes, and these studies can contribute to the separation and determination of the contribution from each subunit and study the kinetics of an individual subunit.
6.7 References
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