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SyncroPatch PE. Contact screener genedata. Raghavan M, Bjorkman PJ. BIAcore: a microchip-based system for analyzing the formation of macromolecular complexes. Structure ;—3. Nicotinic acetylcholine receptor from rat brain. Solubilization, partial purification, and characterization. J Biol Chem ;— Structure and function of alpha-bungarotoxin.
Journal of Toxicology-Toxin Reviews ;— Dutertre S, Lewis RJ. Toxin insights into nicotinic acetylcholine receptors. Biochem Pharmacol ;— Recent advances in Cys-loop receptor structure and function. Nature ;— Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors.
Anal Biochem ;— A glia-derived acetylcholine-binding protein that modulates synaptic transmission. Nature ;—8. Mimotopes of the nicotinic receptor binding site selected by a combinatorial peptide library. Biochemistry ;—9. When the kinetic parameters are known, then it is possible to add refractive index bulk effect or drift. Kinetic titration The kinetic titration single cycle kinetics is useful with interactions which are difficult to regenerate or when regeneration is detrimental to the ligand 1.
Reaction equation Reaction equation Differential equation Differential equation. Parameters L concentration of free ligand in RU A concentration of free analyte in M LA concentration of ligand-analyte complex in RU k a association rate constant in M -1 s -1 k d dissociation rate constant in s Differential equation.
References 1 Dougan, D. The model using a non-linear least squares method, with n, DHbin and Kbin degree of reversibility in the temperature-induced unfolding of as adjusted parameters. Binding of Alba to the DNA at high density Isothermal Titration Calorimetry was performed by subsequent docking of additional Alba The heat flow that resulted from the binding between Alba1 and proteins into the nearest chemically identical environment, Alba2 and the DNA oligonucleotides was measured using a high- which prevented steric clashes with the bound Alba protein.
Alba2 migrated in determined at the Tm. GC-DNA The data from the gel electrophoresis indicate that the Alba proteins probably appear as dimers in solution. Previously, the forma- respectively. Kbin values were agarose chromatography in which only one of two Aeropyrum calculated using Eq.
For the application of Eq. Buffer conditions: pH 7. Bovine serum albumin was used as a negative control, Characterisation of the Alba Proteins and it showed no DNA binding in all cases.
Alba1 showed There are two known DNA sequences for the Alba proteins in saturable binding under the conditions used, with fast association. The addition of the Under the reducing conditions dithiothreitol added , Alba1 Alba1 protein to AT-DNA had no significant effects on its thermal migrated as a protein of 13 kDa Figure 2, right side, lanes e-g , stability, although condensation of AT-DNA was seen at molar which corresponded to the migration of the weak 13 kDa band in ratios higher than Figure 5a.
CD spectra of Alba1. The measured shifts in the melting temper- the CT-DNA at the lower molar ratios and and ature, Tm, can be expressed as: stabilised it at ratios higher than The Tm at the limited molar ratio before condensation occurred was 91uC Figure 4b.
The thermograms revealed two phase transitions at molar ratios above Figure 5b. At molar ratios the helix-to-coil transition enthalpy of the ligand-free duplex, higher than , the Tm was shifted above uC.
GC-DNA which is 8. The reported for genomic DNA. Assuming that the range. CD spectra of Alba2. These data would suggest that the Alba2 protein of the spectra were measured at 25uC, 50uC, 70uC and 90uC. The does not change in secondary structure according to temperature, secondary structures of the Alba proteins in A.
In the presence of CT-DNA at the Alba proteins in other hyperthermophiles, with alternating a, b a molar ratio of Figure 8b , as with Alba1, Alba2 also adopted structures a1—b1—a2—b2—b3. The estimated proportion of a-helices in representing a less-ordered structure Figure 8c , although the Alba1 was ca.
In molar ellipticity increased with temperature. As the became more ordered, with a higher content of b-strands.
When temperature was raised, the Alba1 protein again adopted a more the temperature approached 90uC, the structure became less ordered structure, with the most ordered structure reached at ordered again, similar to that at 25uC Figure 8d.
Again, the 90uC Figure 7b. These changes in the structural organisation at explanation for these structural changes might be related to the higher temperatures might be the result of melting of the CT- equilibrium of dsDNA« 26 ssDNA Table 1.
Isothermal titration calorimetry profiles of Alba1 binding to various DNA. The Alba1 protein had approxi- mately the same level of structural organisation as at all of the temperatures in the absence of DNA.
Detailed higher temperatures with the most apparent increase seen in the inspection of the thermodynamic data listed in Table 2 reveals b-sheet content. The binding of Alba1 to conformation of the proteins at 25uC , with the amount of b-sheet A-DNA has an enthalpically favourable DHbin, while entropically, increasing with the increase in temperature Figure 9 c, d. The estimated secondary structure of Alba1 using studied here, due to the sensitivity limitations of the method. Molecular modelling of the Alba protein dimers.
Right: Alba2 dimers covering the DNA duplex with maximal binding density of one dimer per six base pairs. Sequence composition of the Alba proteins.
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