Stephen J. Williams, Ph.D. Writer, Curator
Rational Design of Allosteric Inhibitors and Activators Using the Population-Shift Model: In Vitro Validation and Application to an Artificial Biosensor.(1)
The population-shift mechanism allows for the re-engineering of biosensors utilizing the concept of allosterism to allow for a structure-based switching on/off capacity into biosensors, “smart-biomaterials, and other artificial biotechnologies. A fundamental problem in the design of valuable biosensors has been limited number of biomolecules that produce enough signal (for example emission of light, etc.) upon binding to its target. However this issue has been resolved with the development of biosensors in which target binding is transduced into a quantifiable optical or electrochemical signal after coupling with conformational changes in the receptor (for review see (2)).
There are a few advantages to this biosensor design:
- Works well in complex samples, such as blood, serum; Low background noise from nonspecific adsorption from interfering biomolecules
- Supports real-time monitoring- allosteric biosensors do not rely on additional reagents and are rapidly reversible
- Binding of the receptors is dependent on an unfavorable conformational change, so there is possibility to fine tune this conformational switch.
Concept of allosterism
Allosterism is generally defined as a change in the activity and conformation of an enzyme/protein resulting from the binding of a compound at a site on the enzyme other than the active binding site. Allosterism plays a critical role in the control and integration of molecular events in biological systems. Frequently, allosterism is seen with multisubunit proteins/enzymes, where subunit interaction is necessary for allosteric effects, and is distal to the binding site. Examples of allosteric systems include hemoglobin, phosphofructose kinase and many
NAD+ -dependent dehydrogenases. For example, the binding of O2 to hemoglobin is enhanced the binding of addition O2, the Bohr effect (the affinity of hemoglobin to O2 depends on H+), and the metabolic product diphosphoglycerate regulates O2 binding.
Types of DNA Biosensors
DNA-based biosensors rely on the hybridization of complementary DNA. Many optical biosensors based on the phenomenon of surface plasmon resonance (SPR) utilize a property of and other materials; specifically that a thin layer of gold on a high refractive index glass surface can absorb laser light, producing electron waves (surface plasmons) on the gold surface. This occurs only at a specific angle and wavelength of incident light and is highly dependent on the surface of the gold, such that binding of a target analyte to a receptor on the gold surface produces a measurable signal.
Electrochemical biosensors are normally based on enzymatic catalysis of a reaction that produces or consumes electrons (such enzymes are rightly called redox enzymes). The sensor substrate usually contains three electrodes; a reference electrode, a working electrode and a counter electrode. The target analyte is involved in the reaction that takes place on the active electrode surface, and the reaction may cause either electron transfer across the double layer (producing a current) or can contribute to the double layer potential (producing a voltage). We can either measure the current (rate of flow of electrons is now proportional to the analyte concentration) at a fixed potential or the potential can be measured at zero current (this gives a logarithmic response). The label-free and direct electrical detection of small peptides and proteins is possible by their intrinsic charges using bio-functional ion-sensitive field-effect transistors.
Piezoelectric sensors utilize crystals which undergo an elastic deformation when an electrical potential is applied to them. An alternating potential produces a standing wave in the crystal at a characteristic frequency. This frequency is highly dependent on the elastic properties of the crystal, such that if a crystal is coated with a biological recognition element the binding of a (large) target analyte to a receptor will produce a change in the resonance frequency, which gives a binding signal. In a mode that uses surface acoustic waves (SAW), the sensitivity is greatly increased.
Type | Biological Element | Transducer |
OpticalFiber Optics
Surface plasmon resonance Biomolecular interactionAnalysis |
DNA | Optical fiberResonant mirror
BIAcore SERG probe |
Electrochemical | DNA | Carbon paste electrodes |
Piezoelectric FrequencyAcoustics | DNA | CrystalsCrystals |
The most popular of optical DNA biosensors is molecular beacons, DNA probes containing a fluorescent moiety and a quencher of on the same DNA strand. This probe has an internal complementary sequence so as the DNA folds into a secondary structure, most likely a stem-loop or hairpin structure, so the fluor and quencher are held in close proximity, quenching the fluorescent signal. Target hybridization opens up the stem-loop structure, thereby emitting the fluorescent signal. A typical molecular beacon probe is 25 nucleotides long. A typical molecular beacon structure can be divided in 4 parts:
- Loop: This is the 18–30 base pair region of the molecular beacon which is complementary to the target sequence.
- Stem: The beacon stem is formed by the attachment, to both termini of the loop, of two short (5 to 7 nucleotide residues) oligonucleotides that are complementary to each other.
- 5′ fluorophore: At the 5′ end of the molecular beacon, a fluorescent dye is covalently attached.
- 3′ quencher (non fluorescent): The quencher dye is covalently attached to the 3′ end of the molecular beacon. When the beacon is in closed loop shape, the quencher resides in proximity to the fluorophore, which results in quenching the fluorescent emission of the latter.
Structure of a molecular beacon. Description and figure from Wikipedia (5).
Common applications of DNA biosensors include cDNA microarray and Affymetrix GeneChip™ technology.
Ricci et al. provide a proof-of –principle paper to demonstrate how allosteric switching can be introduced into biosensors(1). The authors engineered allosteric inhibition into a molecular beacon by the addition of two single-stranded tails that serve as an allosteric site where binding of an inhibitor sequence would bridge the two tails and prevent target binding (holding the probe in the inactivated state). Using this approach the authors demonstrated over a three-fold increase in the dynamic range of the beacon.
The authors also demonstrated this effect, with an allosterically activated biosensor in which “allosteric activation was engineered into a molecular beacon using one single-stranded tail as an allosteric binding site. The activator sequence binding to this tail partially invades the stem, destabilizing the nonbinding state and thus improving the target affinity.” Thus this population-shift mechanism allows for the design of sensors that can be allosterically activated using activators that destabilize the beacon’s nonbinding conformation, increasing the beacon’s dynamic range without compromising target specificity. Finally the authors suggest that population-shift mechanisms can be engineered into many different types of “switching” biosensors including aptamer-based and protein-based sensors (3,4).
1. Ricci, F., Vallee-Belisle, A., Porchetta, A., and Plaxco, K. W. (2012) Journal of the American Chemical Society 134, 15177-15180
2. Vallee-Belisle, A., and Plaxco, K. W. (2010) Current opinion in structural biology 20, 518-526
3. White, R. J., Rowe, A. A., and Plaxco, K. W. (2010) The Analyst 135, 589-594
4. Kohn, J. E., and Plaxco, K. W. (2005) Proceedings of the National Academy of Sciences of the United States of America 102, 10841-10845
5. http://en.wikipedia.org/wiki/Molecular_beacon
Other research papers on Biosensors were published on this Scientific Web site as follows:
Measuring glucose without needle pricks: nano-sized biosensors made the test easy
New Definition of MI Unveiled, Fractional Flow Reserve (FFR)CT for Tagging Ischemia
New Drug-Eluting Stent Works Well in STEMI
Sensor detects glucose in saliva, tears for diabetes testing
Dr. Williams,
This is a very important post.
Do you suggest which of the following:
1. This post is a first in a series for a new investigator initiated research category called Enzymology and/or biosensors in DNA research
2. This post belongs to the following research categories: nano… And / or Proteomics And /or bio instrumentation, and/or medical devices
Please advise, let’s decide and thereafter, I’ll make changes in the Research Category System and you will check the appropriate ones, as appropriate.
Aviva I think this should stay in bioinstrumentation and not in its own separate category such as Enzymology which might be too broad a subject.and is not relevant to a Proteomics category. I feel it may belong to nanotechnology. Biosensors in DNA researdch may be too restrictive and might want to combine all nano related biotechnology in one category such as proteomic methods, DNA methodology, and applications of each…
I tend to agree with Dr. Williams on this, it should stay in this category and not as it’s own. Nice post, Dr. Williams, I really liked it
Allostericity goes back to Jacob and Monod, the French Nobelists. This is an application in enzymology that uses a molecular tag. It is far more fine tuned than was the case in clinical chemistry in developing enzyme probes 15 years ago (Elisa). The field progressed from coupled reactions to fluorescence quenching, and so forth. Now the target is many orders smaller.
There is room for several additional categories, and this is one of them.
PUT IT IN CONTEXT OF CANCER CELL MOVEMENT
The contraction of skeletal muscle is triggered by nerve impulses, which stimulate the release of Ca2+ from the sarcoplasmic reticuluma specialized network of internal membranes, similar to the endoplasmic reticulum, that stores high concentrations of Ca2+ ions. The release of Ca2+ from the sarcoplasmic reticulum increases the concentration of Ca2+ in the cytosol from approximately 10-7 to 10-5 M. The increased Ca2+ concentration signals muscle contraction via the action of two accessory proteins bound to the actin filaments: tropomyosin and troponin (Figure 11.25). Tropomyosin is a fibrous protein that binds lengthwise along the groove of actin filaments. In striated muscle, each tropomyosin molecule is bound to troponin, which is a complex of three polypeptides: troponin C (Ca2+-binding), troponin I (inhibitory), and troponin T (tropomyosin-binding). When the concentration of Ca2+ is low, the complex of the troponins with tropomyosin blocks the interaction of actin and myosin, so the muscle does not contract. At high concentrations, Ca2+ binding to troponin C shifts the position of the complex, relieving this inhibition and allowing contraction to proceed.
Figure 11.25
Association of tropomyosin and troponins with actin filaments. (A) Tropomyosin binds lengthwise along actin filaments and, in striated muscle, is associated with a complex of three troponins: troponin I (TnI), troponin C (TnC), and troponin T (TnT). In (more ) Contractile Assemblies of Actin and Myosin in Nonmuscle Cells
Contractile assemblies of actin and myosin, resembling small-scale versions of muscle fibers, are present also in nonmuscle cells. As in muscle, the actin filaments in these contractile assemblies are interdigitated with bipolar filaments of myosin II, consisting of 15 to 20 myosin II molecules, which produce contraction by sliding the actin filaments relative to one another (Figure 11.26). The actin filaments in contractile bundles in nonmuscle cells are also associated with tropomyosin, which facilitates their interaction with myosin II, probably by competing with filamin for binding sites on actin.
Figure 11.26
Contractile assemblies in nonmuscle cells. Bipolar filaments of myosin II produce contraction by sliding actin filaments in opposite directions. Two examples of contractile assemblies in nonmuscle cells, stress fibers and adhesion belts, were discussed earlier with respect to attachment of the actin cytoskeleton to regions of cell-substrate and cell-cell contacts (see Figures 11.13 and 11.14). The contraction of stress fibers produces tension across the cell, allowing the cell to pull on a substrate (e.g., the extracellular matrix) to which it is anchored. The contraction of adhesion belts alters the shape of epithelial cell sheets: a process that is particularly important during embryonic development, when sheets of epithelial cells fold into structures such as tubes.
The most dramatic example of actin-myosin contraction in nonmuscle cells, however, is provided by cytokinesisthe division of a cell into two following mitosis (Figure 11.27). Toward the end of mitosis in animal cells, a contractile ring consisting of actin filaments and myosin II assembles just underneath the plasma membrane. Its contraction pulls the plasma membrane progressively inward, constricting the center of the cell and pinching it in two. Interestingly, the thickness of the contractile ring remains constant as it contracts, implying that actin filaments disassemble as contraction proceeds. The ring then disperses completely following cell division.
Figure 11.27
Cytokinesis. Following completion of mitosis (nuclear division), a contractile ring consisting of actin filaments and myosin II divides the cell in two.
http://www.ncbi.nlm.nih.gov/books/NBK9961/
This is good. I don’t recall seeing it in the original comment. I am very aware of the actin myosin troponin connection in heart and in skeletal muscle, and I did know about the nonmuscle work. I won’t deal with it now, and I have been working with Aviral now online for 2 hours.
I have had a considerable background from way back in atomic orbital theory, physical chemistry, organic chemistry, and the equilibrium necessary for cations and anions. Despite the calcium role in contraction, I would not discount hypomagnesemia in having a disease role because of the intracellular-extracellular connection. The description you pasted reminds me also of a lecture given a few years ago by the Nobel Laureate that year on the mechanism of cell division.