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A5. Experimental Analyses of Binding - Biology

A5. Experimental Analyses of Binding - Biology


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It is often important to determine the Kd for a ML complex, since given that number and the concentrations of M and L in the system, we can predict if M is bound or not under physiological conditions. Again, this is important since whether M is bound or free will govern its activity. The trick in determining Kd is to determine ML and L at equilibrium. How can we differentiate free from bound ligand? The following techniques allow such a differentiation.

TECHNIQUES THAT REQUIRE SEPARATION OF BOUND FROM FREE LIGAND -

Care must be given to ensure that the equilibrium of (M + L ightleftharpoons ML) is not shifted during the separation technique.

  • gel filtration chromatography - Add M to a given concentration of L. Then elute the mixture on a gel filtration column, eluting with the free ligand at the same concentration. The ML complex will elute first and can be quantitated . If you measure the free ligand coming off the column, it will be constant after the ML elutes with the exception of a single dip near where the free L would elute if the column was eluted without free L in the buffer solution. This dip represents the amount of ligand bound by M.
  • membrane filtration - Add M to radiolableled L, equilibrate, and then filter through a filter which binds M and ML. For instance, a nitrocellulose membrane binds proteins irreversibly. Determine the amount of radiolabeled L on the membrane which equals [ML].
  • precipitation - Add a precipitating agent like ammonium sulfate, which precipitates proteins and hence both M and ML. Determine the amount of ML.

TECHNIQUES THAT DO NOT REQUIRE SEPARATION OF BOUND FROM FREE LIGAND

  • concentrations ligand gives ML. Repeat at many different stoichiometry, which for a 1:1 ligande, determine the amount of bound or spectroscopic techniques. At equilibrium, determine free L by sampling the solution surrounding the bag. By mass balancradioisotopic whose concentration can be determined using ligand- Place M in a dialysis bag and dialyze against a solution containing a equilibirum dialysis.
  • spectroscopy - Find a ligand whose absorbance or fluorescence spectra changes when bound to M. Alternatively, monitor a group on M whose absorbance or fluorescence spectra changes when bound to L.
  • isothermal titration calorimetry (ITC)- In ITC, a high concentration solution of an analyte (ligand) is injected into a cell containing a solution of a binding partner (typically a macromolecule like a protein, nucleic acid, vesicle).

Figure: Isothermal Titration Calorimeter Cells

On binding, heat is either released (exothermic reaction) or adsorbed, causing a small temperature changes in the sample cell compared to the reference cells containing just a buffer solution. Sensitive thermocouples measure the temperature difference (DT1) between the sample and reference cells and apply a current to maintain the difference at a constant value. Multiple injections are made until the macromolecules is saturated with ligand. The enthalpy change is directly proportional to the amount of ligand bound at each injection so the observed signal attenuates with time. The actual enthalpy change observed must be corrected for the change in enthalpy on simple dilution of the ligand into buffer solution alone, determined in a separate experiment. The enthalpy changes observed after the macromolecule is saturated with ligand should be the same as the enthalpy of dilution of the ligand. A binding curve showing enthalpy change as a function of the molar ratio of ligand to binding partner ((L_o/M_o) if (L_o gg M_o)) is then made and mathematically analyzed to determine Kd and the stoichiometry of binding.

Figure: Typical isothermal titration calorimetry data and analysis Reference: www.microcalorimetry.com/index.php?id=312

It should be clear in the example above, that the binding reaction is exothermic. But why is the graph of ΔH vs molar ratio of Lo/Mo sigmoidal (s-shaped) and not hyperbolic? One clue comes from the fact that the molar ratio of ligand (titrant) to macromolecule centers around 1 so, as explained above, when Lo is not >> Mo, the graph might not hyperbolic. The graphs below show a specific example of a Kd and ΔHo being calculated from the titration calorimetry data. They will shed light on why the graph of ΔH vs molar ratio of Lo/Mo is sigmoidal.

A specific example illustrates these ideas. Soluble versions of the HIV viral membrane protein, gp 120, 4 μM, was placed in the calorimetry cell, and a soluble form of its natural ligand, CD4, a membrane receptor protein from T helper cells, was placed a syringe and titrated into the cell (Myszka et al. 2000). Enthalpy changes/injection were determined and the data was transformed and fit to an equation which shows the ΔH "normalized to the number of moles of ligand (CH4) injected at each step". The line fit to the data in that panel is the best fit line assuming a 1:1 stoichiometry of CD4 (the "ligand") to gp 120 (the "macromolecule") and a Kd = 190 nM. Please note that the curve is sigmoidal, not hyperbolic.

Figure: Titration Calorimetry determination of Kd and DH for the interaction of gp120 and CD4

Note that the stoichiometry of binding (n), the KD, the ΔHo can be determined in a single experiment. From the value of ΔHo and KD, and the relationship

[ΔGo = -RTlnKeq = RTlnKD = ΔHo - TΔSo]

the ΔGo and ΔSo values can be calculated. No separation of bound from free is required. Enthalpy changes on binding were calculated to be -62 kcal/mol.

Using the standard binding equations (5, 7, and 10 above) to calculate free L and ML at a vary of Lo concentrations and R = Lo/Mo ratios, a series of plots can be derived. Two were shown earlier in this Chapter section to illustrate differences in Y vs L and Y vs Lo when Lo is not >> Mo. They are shown again below:

Figure: Y vs L and Y vs Lo when Lo is not >> Mo

Next a plot of ML vs R (= [Lo]/[Mo] (below, panel A1 right) was made. This curve appears hyperbolic but it has the same shape as the Y vs Lo graph above (right). However if the amount of ligand bound at each injection (calculated by subtracting [ML] for injection i+1 from [ML] for injection i) is plotted vs R (= [Lo]/[Mo]), a sigmoidal curve (below, panel A2, left) is seen, which resemble that best fit graph for the experimentally determine enthalpies above. The relative enthalpy change for each injection is shown in red. Note the graph in A2 actually shows the negative of the amount of ligand bound per injection, to make the graph look the that in the graph showing the actual titration calorimetry trace and fit above.

Figure: Binding Curves that Explain Sigmoidal Titration Calorimetry Data for gp120 and CD4

Surface Plasmon Resonance

A newer technique to measure binding is called surface plasmon resonance (SPR) using a sensor chip consisting of a 50 nm layer of gold on a glass surface. A carbohydrate matrix is then added to the gold surface. To the CHO matrix is attached through covalent chemistry a macromolecle which contains a binding site of a ligand. The binding site on the macromolecule must not be perturbed to any significant extent. A liquid containing the ligand is flowed over the binding surface.

The detection system consists of a light beam that passes through a prism on top of the glass layer. The light is totally reflected but another component of the wave called an evanescent wave, passes into the gold layer, where it can excite the Au electrons. If the correct wavelength and angle is chosen, a resonant wave of excited electrons (plasmon resonance) is produced at the gold surface, decreasing the total intensity of the reflected wave. The angle of the SPR is sensitive to the layers attached to the gold. Binding and dissociation of ligand is sufficient to change the SPR angle, as seen in the figure below.

  • Fig: Surface Plasmon Resonance. (CC BY-SA 3.0; SariSabban)

animation: SPR evanaescent wave

This technique can distinguish fast and slow binding/dissociation of ligands (as reflected in on and off rates) and be used to determine Kd values (through measurement of the amount of ligand bond at a given total concentration of ligand or more indirectly through determination of both kon and koff.

Binding DB: a database of measured binding affinities, focusing chiefly on the interactions of protein considered to be drug-targets with small, drug-like molecules

PDBBind-CN: a comprehensive collection of the experimentally measured binding affinity data for all types of biomolecular complexes deposited in the Protein Data Bank (PDB).


Efficient, systematic genetic analysis helps dissect disease inheritance

Many genetic variants have been found to have a linkage with genetic diseases, but the understanding of their functional roles in causing diseases are still limited. An international research team, including a biomedical scientist from City University of Hong Kong (CityU), has developed a high-throughput biological assay technique which enabled them to conduct a systematic analysis on the impact of nearly 100,000 genetic variants on the binding of transcription factors to DNA. Their findings provided valuable data for finding key biomarkers of type 2 diabetes for diagnostics and treatments. And they believe that the new technique can be applied to studies of variants associated with other genetic diseases.

The study was co-led by Dr Yan Jian, Assistant Professor in the Department of Biomedical Sciences at CityU, Professor Bing Ren from the University of California San Diego and Professor Jussi Taipale from the University of Cambridge. Their findings were published in the scientific journal Nature.

"Based on our findings, we believe that our high-throughput experimental method can be applied in the study of different genetic diseases, including colorectal cancer and prostate cancer. It can help dissect the mechanism of the genetic inheritance of the disease and find the biomarkers for clinical diagnosis," said Dr Yan.

Unveiling the roles of noncoding variants in diseases

Genome-wide association studies (GWAS), which investigate the entire genome, has been the most important strategy in finding the genes associated with complex genetic diseases. Researchers have found hundreds of thousands of genetic variants in association with human diseases and traits. But studies on the functions of these variants are still limited.

"Understanding the molecular functions of the noncoding variants will help us find out why people carrying these mutations are more susceptible to genetic diseases. This will help us develop methods or strategies to prevent, to detect or to cure the diseases early," explained Dr Yan.

One of the variants' functions is to affect the binding of transcription factors to DNA. The transcription factors will then control the gene expression in cells, turning the specific genes "on" and "off," modulating the cellular functions.

To systematically characterise the effects of genetic variants on the transcription factor binding, the team modified their previously developed experimental method into an ultra-high-throughput multiplex protein-DNA binding assay, termed "single-nucleotide polymorphism evaluation by systematic evolution of ligands by exponential enrichment" (SNP-SELEX). Then they chose the genetic variants from the gene locations on the genome (called "gene loci") that are known to be associated with the risk of type 2 diabetes as the object of analysis.

Utilising the SNP-SELEX, they successfully analysed the impact of 95,886 genetic variants on the binding of 270 distinct human transcription factors to DNA. They demonstrated that noncoding genetic variant SNP rs7118999 that increases the risk of type 2 diabetes can affect the DNA binding with one of the transcription factors, and the resulting molecular mechanism regulates the blood lipid level.

"This is a clear example of applying the data generated by SNP-SELEX that it can help identify the genetic variants which play key roles in the inheritance of type 2 diabetes. This would help the subsequent investigation in finding diagnostic biomarkers and therapeutic targets," said Dr Yan.

Speeding Up Analysis Significantly

Moreover, previous studies could only single out one or a few variants to find out its molecular mechanism. Each study took around 2-3 years. "So it was impossible to completely understand the complex genetic diseases like type 2 diabetes which are associated with hundreds of genetic variants within a short period. But with the SNP-SELEX, we could systematically analyse approximately 100,000 variants within a much shorter timeframe," said Dr Yan.

"In this study, we only covered a relatively small portion of variants and transcription factors. So we will expand our study. By utilising the SNP-SELEX, hopefully it will help us uncover the underlying mechanisms of more and more of these noncoding variants very soon," said Professor Ren.


Contents

Antibodies directed against annexin A5 are found in patients with a disease called the antiphospholipid syndrome (APS), a thrombophilic disease associated with autoantibodies against phospholipid compounds.

Annexin A5 forms a shield around negatively charged phospholipid molecules. The formation of an annexin A5 shield blocks the entry of phospholipids into coagulation (clotting) reactions. In the antiphospholipid antibody syndrome, the formation of the shield is disrupted by antibodies. Without the shield, there is an increased quantity of phospholipid molecules on cell membranes, speeding up coagulation reactions and causing the blood-clotting characteristic of the antiphospholipid antibody syndrome.
Annexin A5 showed upregulation in papillary thyroid carcinoma. [5]

Annexin A5 is used as a non-quantitative probe to detect cells that have expressed phosphatidylserine (PS) on the cell surface, an event found in apoptosis as well as other forms of cell death. [6] [7] [8] Platelets also expose PS and PE on their surface when activated, which serves as binding site for various coagulation factors.

The annexin A5 affinity assay typically uses a conjugate of annexin V and a fluorescent or enzymatic label, biotin or other tags, or a radioelement, in a suitable buffer (annexin V binding to aminophospholipids is Ca 2+ dependent). The assay combines annexin V staining of PS and PE membrane events with the staining of DNA in the cell nucleus with propidium iodide (PI) or 7-Aminoactinomycin D (AAD-7), distinguishing viable cells from apoptotic cells and necrotic cells. [9] Detection occurs by flow cytometry or a fluorescence microscope.


Volume I

Conformationally Selective Parathyroid Hormone Ligands and Prolonged Signaling

Kinetic binding and signaling assays performed using various PTH and PTHrP ligand analogues provide data to suggest that the PTHR-1 can indeed adopt different protein conformations that display differential affinities for structurally diverse ligands, and can thereby mediate different types of signaling responses. 373-376 In particular, these studies show that certain PTH analogues can form complexes with the PTHR-1 that maintain a high affinity state, even upon the addition of GTPγS, a reagent that promotes the dissociation of G proteins from the receptor and thus typically is used to shift a GPCR into a low-affinity state. These PTH ligand analogues are thought to bind to a novel PTHR-1 conformation, called R 0 , that can maintain high affinity even independently of G protein coupling. In contrast, other ligands, such as the shorter-length M-PTH(1-14) analogues, form complexes with the PTHR-1 that rapidly dissociate upon addition of GTPγS. 373 Accordingly, these PTH ligands are thought to bind primarily to a G protein–coupled conformation called RG.

The biological consequences of such ligand-directed conformational selectivity at the PTHR-1 are not evidenced by a change in signaling pathway type (e.g., from the Gαs/cAMP/PKA pathway toward a non–Gαs-mediated pathway), but rather by the duration of the cAMP response that is induced by the different ligands. Thus, R 0 -selective ligands are seen to induce prolonged cAMP responses in PTHR-1-expressing cells, whereas RG-selective PTH ligands induce more transient cAMP responses that diminish soon after initial ligand exposure. While the complete mechanisms underlying the prolonged signaling responses observed for the R 0 -selective ligands are far from clear, a potential explanation is that they arise from the capacity of the ligand to remain bound to the receptor through multiple and repeated rounds of coupling to Gαs. In any event, the effects are robust and observable not only in cell-culture systems, but also in animals, in which R 0 -selective ligands have been shown to induce increases in blood calcium levels and decreases in blood phosphate levels that persist for at least several hours longer than those observed for PTH(1-34), even when the R 0 analogue is injected at a dose severalfold lower than the dose used for PTH(1-34). 374,376

One long-acting analogue of particular interest, called LA-PTH, is a hybrid peptide composed of the M-PTH(1-14) sequence joined to the PTHrP(15-36) sequence. 376 LA-PTH binds to the R 0 PTHR-1 conformation with severalfold higher affinity than does PTH(1-34), and while the cAMP potency measured for LA-PTH is the same as that measured for PTH(1-34), consistent with their equivalent RG-binding affinities, the cAMP response induced by LA-PTH persists for many hours longer than that induced by PTH(1-34). When injected into mice, LA-PTH induces elevations of serum calcium that can last for 24 hours or longer, whereas PTH(1-34) injection at the same dose results in responses that last only a few hours ( Fig. 56-10 ). Importantly, pharmacokinetic studies have indicated that the prolonged responses to such R 0 -selective PTH analogues are not due to prolonged durations of the peptides in the circulation, 88 and so they are more likely to be due to a persistent binding of the ligands to the PTHR-1 in bone and kidney target cells. Because of their prolonged actions in vivo, the class of such R 0 -selective PTH analogues would appear to hold promise as a potential new line of therapy for patients with hypoparathyroidism. 135


Contents

The basic apparatus comprises an optical microscope, a light source and some fluorescent probe. Fluorescent emission is contingent upon absorption of a specific optical wavelength or color which restricts the choice of lamps. Most commonly, a broad spectrum mercury or xenon source is used in conjunction with a color filter. The technique begins by saving a background image of the sample before photobleaching. Next, the light source is focused onto a small patch of the viewable area either by switching to a higher magnification microscope objective or with laser light of the appropriate wavelength. The fluorophores in this region receive high intensity illumination which causes their fluorescence lifetime to quickly elapse (limited to roughly 10 5 photons before extinction). Now the image in the microscope is that of a uniformly fluorescent field with a noticeable dark spot. As Brownian motion proceeds, the still-fluorescing probes will diffuse throughout the sample and replace the non-fluorescent probes in the bleached region. This diffusion proceeds in an ordered fashion, analytically determinable from the diffusion equation. Assuming a Gaussian profile for the bleaching beam, the diffusion constant D can be simply calculated from:

where w is the radius of the beam and tD is the "Characteristic" diffusion time. [1] [2]

Supported lipid bilayers Edit

Originally, the FRAP technique was intended for use as a means to characterize the mobility of individual lipid molecules within a cell membrane. [1] While providing great utility in this role, current research leans more toward investigation of artificial lipid membranes. Supported by hydrophilic or hydrophobic substrates (to produce lipid bilayers or monolayers respectively) and incorporating membrane proteins, these biomimetic structures are potentially useful as analytical devices for determining the identity of unknown substances, understanding cellular transduction, and identifying ligand binding sites.

Protein binding Edit

This technique is commonly used in conjunction with green fluorescent protein (GFP) fusion proteins, where the studied protein is fused to a GFP. When excited by a specific wavelength of light, the protein will fluoresce. [3] When the protein that is being studied is produced with the GFP, then the fluorescence can be tracked. Photodestroying the GFP, and then watching the repopulation into the bleached area can reveal information about protein interaction partners, organelle continuity and protein trafficking. [4]

If after some time the fluorescence doesn't reach the initial level anymore, then some part of the fluorescence is caused by an immobile fraction (that cannot be replenished by diffusion). Similarly, if the fluorescent proteins bind to static cell receptors, the rate of recovery will be retarded by a factor related to the association and disassociation coefficients of binding. This observation has most recently been exploited to investigate protein binding. [3] [5] [6] Similarly, if the GFP labeled protein is constitutively incorporated into a larger complex, the dynamics of fluorescence recovery will be characterized by the diffusion of the larger complex. [7]

FRAP can also be used to monitor proteins outside the membrane. After the protein of interest is made fluorescent, generally by expression as a GFP fusion protein, a confocal microscope is used to photobleach and monitor a region of the cytoplasm, [3] mitotic spindle, nucleus, or another cellular structure. [8] The mean fluorescence in the region can then be plotted versus time since the photobleaching, and the resulting curve can yield kinetic coefficients, such as those for the protein's binding reactions and/or the protein's diffusion coefficient in the medium where it is being monitored. [9] Often the only dynamics considered are diffusion and binding/unbinding interactions, however, in principle proteins can also move via flow, i.e., undergo directed motion, and this was recognized very early by Axelrod et al. [1] This could be due to flow of the cytoplasm or nucleoplasm, or transport along filaments in the cell such as microtubules by molecular motors.

The analysis is most simple when the fluorescence recovery is limited by either the rate of diffusion into the bleached area or by rate at which bleached proteins unbind from their binding sites within the bleached area, and are replaced by fluorescent protein. Let us look at these two limits, for the common case of bleaching a GFP fusion protein in a living cell.

Diffusion-limited fluorescence recovery Edit

In practice, in a cell none of these assumptions will be strictly true.

  1. Bleaching will not be instantaneous. Particularly if strong bleaching of a large area is required, bleaching may take a significant fraction of the diffusion timescale τ D > . Then a significant fraction of the bleached protein will diffuse out of the bleached region actually during bleaching. Failing to take account of this will introduce a significant error into D. [11][12][13]
  2. The bleached profile will not be a radial step function. If the bleached spot is effectively a single pixel then the bleaching as a function of position will typically be diffraction limited and determined by the optics of the confocal laser scanning microscope used. This is not a radial step function and also varies along the axis perpendicular to the plane.
  3. Cells are of course three-dimensional not two-dimensional, as is the bleached volume. Neglecting diffusion out of the plane (we take this to be the xy plane) will be a reasonable approximation only if the fluorescence recovers predominantly via diffusion in this plane. This will be true, for example, if a cylindrical volume is bleached with the axis of the cylinder along the z axis and with this cylindrical volume going through the entire height of the cell. Then diffusion along the z axis does not cause fluorescence recovery as all protein is bleached uniformly along the z axis, and so neglecting it, as Soumpasis' equation does, is harmless. However, if diffusion along the z axis does contribute to fluorescence recovery then it must be accounted for.
  4. There is no reason to expect the cell cytoplasm or nucleoplasm to be completely spatially uniform or isotropic.

Thus, the equation of Soumpasis is just a useful approximation, that can be used when the assumptions listed above are good approximations to the true situation, and when the recovery of fluorescence is indeed limited by the timescale of diffusion τ D > . Note that just because the Soumpasis can be fitted adequately to data does not necessarily imply that the assumptions are true and that diffusion dominates recovery.

Reaction-limited recovery Edit

The equation describing the fluorescence as a function of time is particularly simple in another limit. If a large number of proteins bind to sites in a small volume such that there the fluorescence signal is dominated by the signal from bound proteins, and if this binding is all in a single state with an off rate koff, then the fluorescence as a function of time is given by [14]

Note that the recovery depends on the rate constant for unbinding, koff, only. It does not depend on the on rate for binding. Although it does depend on a number of assumptions [14]

  1. The on rate must be sufficiently large in order for the local concentration of bound protein to greatly exceed the local concentration of free protein, and so allow us to neglect the contribution to f of the free protein.
  2. The reaction is a simple bimolecular reaction, where the protein binds to localised sites that do not move significantly during recovery
  3. Exchange is much slower than diffusion (or whatever transport mechanism is responsible for mobility), as only then does the diffusing fraction recovery rapidly and then acts as the source of fluorescent protein that binds and replaces the bound bleached protein and so increases the fluorescence. With r the radius of the bleached spot, this means that the equation is only valid if the bound lifetime 1 / k off >> r 2 / D >>>r^<2>/D> .

If all these assumptions are satisfied, then fitting an exponential to the recovery curve will give the off rate constant, koff. However, other dynamics can give recovery curves similar to exponentials, so fitting an exponential does not necessarily imply that recovery is dominated by a simple bimolecular reaction. One way to distinguish between recovery with a rate determined by unbinding and recovery that is limited by diffusion, is to note that the recovery rate for unbinding-limited recovery is independent of the size of the bleached area r, while it scales as r − 2 > , for diffusion-limited recovery. Thus if a small and a large area are bleached, if recovery is limited by unbinding then the recovery rates will be the same for the two sizes of bleached area, whereas if recovery is limited by diffusion then it will be much slower for the larger bleached area.

Diffusion and reaction Edit

In general, the recovery of fluorescence will not be dominated by either simple isotropic diffusion, or by a single simple unbinding rate. There will be both diffusion and binding, and indeed the diffusion constant may not be uniform in space, and there may be more than one type of binding sites, and these sites may also have a non-uniform distribution in space. Flow processes may also be important. This more complex behavior implies that a model with several parameters is required to describe the data models with only either a single diffusion constant D or a single off rate constant, koff, are inadequate.

There are models with both diffusion and reaction. [2] Unfortunately, a single FRAP curve may provide insufficient evidence to reliably and uniquely fit (possibly noisy) experimental data. Sadegh Zadeh et al. [15] have shown that FRAP curves can be fitted by different pairs of values of the diffusion constant and the on-rate constant, or, in other words, that fits to the FRAP are not unique. This is in three-parameter (on-rate constant, off-rate constant and diffusion constant) fits. Fits that are not unique, are not generally useful.


An Analysis of the Effects of pH on Oxygen Binding by Squid (Illex Illecebrosus, Loligo Pealei) Haemocyanin

HANS-OTTO PÖRTNER An Analysis of the Effects of pH on Oxygen Binding by Squid (Illex Illecebrosus, Loligo Pealei) Haemocyanin. J Exp Biol 1 May 1990 150 (1): 407–424. doi: https://doi.org/10.1242/jeb.150.1.407

Address for reprint requests.

The in vitro oxygen-binding characteristics of haemocyanin were investigated in whole blood of two species of pelagic squid, Illex illecebrosus and Loligo pealei. pH-independent Haldane coefficients (ΔHCO3 − /ΔHcyO2) (where HcyO2 is haemocyanin-bound oxygen) slightly smaller than —1 were found in both species. Oxygen-linked CO2 binding was not present. Buffer values ranged between 5 and 5.8 m mol l −1 pH unit −1 . For further analyses a pH/saturation diagram was selected to show the effect of pH on oxygen binding at constant POO2 in a continuous plot. The slopes of the resulting oxygen isobars (ΔHcyO2/ΔpH or ΔSpH) (where S is oxygen saturation) depend on pH. The diagram allows evaluation of both the Bohr coefficients (ΔlogP50/ΔpH) and the Hill coefficients (n50) at specific pH values. It provides an integrated illustration of the importance of the Bohr effect and cooperativity for oxygen binding.

In accordance with Wyman's linkage equation, Bohr and Haldane coefficients are found to be identical. Both are pH-independent between pH7 and 8. The changing slopes of the oxygen isobars are likely to reflect changes in cooperativity with pH. Maximum values of n50 coincide with maximum steepness of the oxygen isobars in the physiological range of pH and POO2. Assuming that the haemocyanin acts as a buffer for venous POO2, this maximum in pH sensitivity and its decrease in the higher and lower pH ranges are discussed in the light of the maintenance of pigment function in vivo.


A5. Experimental Analyses of Binding - Biology

If it’s a virus, we study it – how they work and where they come from, to better understand new diseases before they happen. Please visit the Neuman Lab website for more information about the lab and our current projects.

Coronavirus, arenavirus and influenzavirus particles are quite variable in appearance, and the proteins that control virion shape and size are the same ones that guide the process of assembling new virions. Understanding these proteins is key to designing better vaccines and opens up new ways to potentially control infection by limiting not just what goes into a cell, but also what comes out. We have used cryo-electron microscopy and mass spectroscopy to probe the structure of virion proteins and the ways that changes in protein conformation are linked to the assembly process.

Viruses like SARS-CoV-2 that seem new to us are usually just recently arrived from another species. Finding and understanding viruses before they cause problems is an important component of pandemic preparedness. We use bioinformatics and molecular biology approaches to discover new RNA viruses, which are also a source of useful proteins that can potentially be exploited as molecular tools.

Part of understanding a virus is learning how each component contributes to the replication cycle. Taking away components that are important to the virus is a good strategy for designing and testing new antivirals. We believe that building up a good suite of antivirals as a complement to vaccines, can be a hedge against the rise of potentially vaccine-resistant viral strains.

We also look for the good in viruses. Phage therapy, where bacteria-killing viruses are deliberately used to control the growth of harmful bacteria and improve the growth of important food crops. We are exploring new methods like experimental evolution to create more effective phage cocktails for agricultural use


ChIP in series: Sequential ChIP

Amit Paul , Nicole C. Riddle , in Epigenetics Methods , 2020

2.2.1 DNA footprinting

To overcome the limitations of the EMSA, which provides no information regarding which DNA sequences a particular chromatin protein interacts with, DNA footprinting is used. The DNA footprinting method is based on the principle that the site where a protein binds to DNA is protected from nuclease digestion, which means that by isolating the “protected” pieces of DNA the precise protein binding site can be identified [38–40] . DNA footprinting is a very sensitive technique, robust protocols are available, and it is an excellent method of choice to identify binding motifs in a limited sequence context [41] . Initially, footprinting was developed as an in vitro technique, newer methods allow for the study the of DNA/protein interactions in vivo. Mueller and colleagues developed ligation mediated PCR (LMPCR) in 1989 to study the interaction between the developmentally regulated enhancer of the muscle creatine kinase (MCK) gene and the myogenic regulator MyoD1 (Myogenic Differentiation 1) [42] . The LMPCR footprinting approach demonstrated that MyoD1 was bound at several sites in the enhancer region of actively transcribed MCK in differentiated muscle cells but absent from these sites in non-differentiated myogenic cells [42] . Similarly, LMPCR footprinting was used to demonstrate that the mdm2 oncogene promoter contains a p53 response element that is bound by p53 during transcriptional activation of mdm2 [43] . Restriction endonuclease digestion followed by LMPCR was used to map the polymerase density at the heat shock responsive Hsp82 promoter [44] , and in vivo LMPCR was used to show that the transcription factor NFI acts as a powerful repressor of the p21 gene transcription, binding a target site between nucleotide position − 161 and − 149 relative to the transcription start site (TSS) [45] . These examples illustrate that DNA footprinting methods remain useful for the study of protein/DNA interactions in the chromatin context, specifically if very high resolution at a small number of loci is needed.

In recent years, efforts have been made to develop genome-wide methods for DNA footprinting. For example, DNase-seq (DNase I hypersensitive sites sequencing), developed by Boyle and colleagues in 2008, a technique in which nuclei are digested with DNase I (Deoxyribonuclease I), and after several processing steps including end repair, linker ligation, and digestion, the sequences adjacent to the DNase I cut sites are identified by next generation sequencing [46] (for a detailed protocol, see [47] ). Boyle and colleagues use this technique to map DNase I hypersensitive sites in human primary CD4 (cluster of differentiation 4) + T cells, allowing them to map regions of open chromatin across the genome [46] . Hesselberth and colleagues used a similar method involving DNase I digestion followed by next generation sequencing to carry out “digital genomic footprinting” [48] . Their analysis of yeast chromatin revealed large numbers of protected regions indicative of transcription factor binding or precisely positioned nucleosomes [48] . The genome-wide DNA footprinting approaches continue to evolve, and in 2019, XL (crosslink)-DNase-seq, which includes a light cross-linking step was introduced by Oh and colleagues to allow for the mapping to factors with shorter chromatin occupancy rates [49] . These examples illustrate that DNA footprinting continues to be utilized by researchers needing high resolution information on binding of chromatin components.


Tyrosine Phosphorylation

Scott T. Brady , . Scott T. Brady , in Basic Neurochemistry (Eighth Edition) , 2012

Receptor protein tyrosine phosphatases consist of an extracellular domain, a transmembrane domain and one or two intracellular catalytic domains

RPTPs can be divided into different classes by the structural features of the extracellular domain ( Fig. 26-10 ), which includes the immunoglobulin-like, fibronectin III-like, MAM and carbonic anhydrase domains ( Hunter, 1996 ). The immunoglobulin-like domains contain intramolecular disulfide bonds and a homophilic binding site for cell–cell adhesion molecules, such as neural cell adhesion molecule (NCAM). The fibronectin III-like domains originally were identified in the extracellular matrix protein fibronectin. They consist of conserved hydrophobic residues and may interact with integrins. The MAM domains are named because of their presence in meprins, A5 glycoprotein and PTPm. These domains contain four conserved cysteine residues. The carbonic anhydrase domains contain only one of the three histidine residues required for catalyzing the hydration of carbon dioxide and are unlikely to be catalytically active. It has been suggested that both the MAM and carbonic anhydrase domains play a role in cell adhesion (see Chapter 9).

The catalytic domains of RPTPs are in the intracellular region of the protein. Most RPTP families, except RPTPβ, contain two tandem catalytic domains. The proximal catalytic domain of most RPTPs contains all of the enzymatic activity. The distal catalytic domain appears to be inactive in some cases, critical catalytic residues are missing. Despite the lack of enzyme activity, the distal catalytic domain may be important for mediating intra- or intermolecular interactions and biological activity of RPTPs. It has been shown that a chimeric CD45 in which the distal catalytic domain is replaced with an equivalent region from LAR becomes deficient in the induction of interleukin-2 secretion and ZAP-70 phosphorylation.

It was originally believed that PTP activity was constitutive and that tyrosine phosphorylation was regulated solely by activating the PTKs. However, it is now clear that PTPs play an active role in the regulation of tyrosine phosphorylation ( Stoker, 2005 Tonks, 2006 ). This was suggested first by the discovery of RPTPs, such as CD45, that have a large extracellular domain reminiscent of that of RPTKs. Their activities are regulated by ligand binding to the extracellular domain. Chimeric studies fusing the intracellular domain of CD45 with the extracellular and transmembrane domains of the EGFR show that the CD45 intracellular catalytic domain is constitutively active. Addition of EGF suppresses the PTP activity of the chimera, suggesting that dimerization may negatively regulate RPTP activity ( Desai et al., 1993 ). The mechanism of dimerization-induced inhibition has been revealed by crystallographic studies of an RPTPα fragment consisting of membrane-proximal region and the proximal catalytic domain ( Tonks, 2006 ). These fragments form symmetrical dimers in which the active site of one molecule is blocked by an inhibitory wedge from the membrane-proximal region of the other. Based on this model, the inactive distal catalytic domain may promote tyrosine phosphatase activity of CD45 by competing with and inhibiting homodimerization of the proximal catalytic domain. In summary, activity of RPTPs may be diminished by ligand-induced dimerization, in contrast to activation by dimerization of RPTKs.


BD FACSymphony™ A5 Cell Analyzer

Contact your local representative to discuss the ideal instrument to meet your needs.

The BD FACSymphony™ A5 Cell Analyzer improves sensitivity to enable you to identify and analyze rare cell types and events

The instrument features an ultra-quiet VPX electronics system that supports up to 50 high-performance detectors.

The capabilities of this platform technology uniquely allow you to conduct deep and broad phenotyping and gain richer scientific insights by fully leveraging the broad portfolio of catalog and custom BD Horizon Brilliant™ Reagents.

The BD FACSymphony™ A5 Cell Analyzer allows you to select up to 9 lasers for maximum flexibility with new lasers, dyes and applications in the pipeline

Configure your instrument to meet your research needs using traditional or unique wavelengths and power ratings.

Optical arrays offer more flexibility than ever

  • Classic tube photomultiplier tubes (PMTs) in decagon arrays with up to 10 detectors
  • Small square form PMT and GaAs/GaAsP* detectors in high-parameter cascade (HPC) arrays
    • Detectors optimized for increased sensitivity and lower noise in ranges of the spectrum
    • Up to 20 detectors per laser
    • Discrete pre-amplifiers

    Filter sets are exchangeable for traditional fluorescent markers as well as specialized sets for unique dyes or proteins.

    * Gallium arsenide phosphide

    The BD ® High Throughput Sampler (HTS) and BD FACSFlow™ Supply System (FFSS) options are available for increased efficiency

    The HTS option:

    • Automates and accelerates sample acquisition
    • Compatible with 96- and 384-well plates
    • <0.5% sample carryover in high-throughput mode

    The FFSS option:

    • Increases capacity and ease of use while maintaining a stable fluidics pressure
    • Reduces daily maintenance by incorporating a 20-L BD FACSFlow™ Cubitainer

    As a Special Order Research Product (SORP), the BD FACSymphony™ A5 Cell Analyzer offers a choice of lasers from 26 different laser wavelengths to optimally configure your instrument for your specific research application.

    For most lasers, multiple power ratings that can be adjusted, stored and recalled using the digital laser command and control functionality are available.

    Dependent on the laser, power settings from 20–1,000 mW may be available.

    Fluorochrome availability and excitation characteristics across various wavelengths should be discussed during the configuration process to identify the best optical options for your research.

    Laser Wavelength (nm) Power (mW)
    UV 355 20–100
    Violet 405 100–200
    Blue 488 100–200
    Yellow-Green 561 100–200
    Red 637 140

    The BD FACSymphony™ A5 Cell Analyzer works with conventional and BD Horizon Brilliant™ Fluorochromes to expand experimental design and help accelerate time to insight

    Enable on-site training and instrument characterization with CD4 fluorochrome evaluation kits

    Each BD fluorochrome optimized for flow cytometry has been conjugated to anti-human or mouse CD4 and is provided for PMT voltrations and instrument characterization. Higher-parameter configurations may also include custom fluorochromes suitable for additional channels. Mid- and low-expressed antigen specificities are available on a custom basis.

    Representative PMTV titration and measured stain index of PBMCs stained with clone SK3 mouse anti-human CD4 acquired at varying PMTVs in 25 V increments. Stain index and resolution for this detector is nearing maximal at a PMTV of 425 V and above as indicated by the square.

    Reduce cost and risk of optimizing new high-parameter panels

    Speak with your representative about including reagents as part of your instrument purchase to enable you to minimize the cost and risk of optimizing new high-parameter panels on your BD FACSymphony™ Cell Analyzer.

    Our high-parameter specialists will work with you to identify the optimal panel for your experimental design and instrument configuration and provide the reagents needed to get to the final rendition of your panel.


    References

    Schepers A, de Vries MR, van Leuven CJ, Grimbergen JM, Holers VM, Daha MR, van Bockel JH, Quax PH

    Leon C, Nandan D, Lopez M, Moeenrezakhanlou A, Reiner NE

    Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM

    Andree HA, Stuart MC, Hermens WT, Reutelingsperger CP, Hemker HC, Frederik PM, Willems GM

    Chen HH, Vicente CP, He L, Tollefsen DM, Wun TC

    Thiagarajan P, Benedict CR

    van Heerde WL, Sakariassen KS, Hemker HC, Sixma JJ, Reutelingsperger CP, De Groot PG

    Kenis H, Hofstra L, Reutelingsperger CP

    van Genderen HO, Kenis H, Hofstra L, Narula J, Reutelingsperger CP

    Boersma HH, Kietselaer BL, Stolk LM, Bennaghmouch A, Hofstra L, Narula J, Heidendal GA, Reutelingsperger CP

    Leroyer AS, Tedgui A, Boulanger CM

    van Tits LJ, van Heerde WL, van der Vleuten GM, de Graaf J, Grobbee DE, van de Vijver LP, Stalenhoef AF, Princen HM

    Ravassa S, Gonzalez A, Lopez B, Beaumont J, Querejeta R, Larman M, Diez J

    Peetz D, Hafner G, Blankenberg S, Peivandi AA, Schweigert R, Brunner K, Dahm M, Rupprecht HJ, Mockel M

    Landmesser U, Hornig B, Drexler H

    Frey B, Munoz LE, Pausch F, Sieber R, Franz S, Brachvogel B, Poschl E, Schneider H, Rodel F, Sauer R, Fietkau R, Herrmann M, Gaipl US

    Henson PM, Bratton DL, Fadok VA

    Whitman SC, Ravisankar P, Daugherty A

    Lardenoye JH, Delsing DJ, de Vries MR, Deckers MM, Princen HM, Havekes LM, van Hinsbergh van Weel , van Bockel JH, Quax PH

    Monraats PS, Pires NM, Schepers A, Agema WR, Boesten LS, de Vries MR, Zwinderman AH, de Maat MP, Doevendans PA, de Winter RJ, Tio RA, Waltenberger J, 't Hart LM, Frants RR, Quax PH, van Vlijmen BJ, Havekes LM, van der LA, van der Wall EE, Jukema JW

    Schepers A, Eefting D, Bonta PI, Grimbergen JM, de Vries MR, van W, V , de Vries CJ, Egashira K, van Bockel JH, Quax PH

    Heeneman S, Lutgens E, Schapira KB, Daemen MJ, Biessen EA

    Lardenoye JH, de Vries MR, Grimbergen JM, Havekes LM, Knaapen MW, Kockx MM, van Hinsbergh VW, van Bockel JH, Quax PH

    Sugawara J, Komine H, Hayashi K, Yoshizawa M, Yokoi T, Otsuki T, Shimojo N, Miyauchi T, Maeda S, Tanaka H

    Kemerink GJ, Liu X, Kieffer D, Ceyssens S, Mortelmans L, Verbruggen AM, Steinmetz ND, Vanderheyden JL, Green AM, Verbeke K



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