Biphasic transition curve on denaturation of chicken cystatin by guanidinium chloride Evidence for an independently unfolding structural region

Björk, Ingemar; Pol, Ewa

doi:10.1016/0014-5793(92)80102-m

Cited by 14 publications

(4 citation statements)

References 19 publications

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“…( A ) Unfolding profile of dimeric (open circles) and monomeric (filled circles) cC incubated at different GdmCl concentrations for 24 h and monitored using tryptophan fluorescence. Data for monomeric cC match those reported previously (Bjork and Pol, 1992) and are superimposable on those of dimeric cC at GdmCl activity >2.5 M. Below this activity, conversion of monomer to dimer does not reach equilibrium within the incubation period. ( B ) The GdmCl dependence of the observed bimolecular rate constant for dimerization ( k obs , M −1 s −1 ), determined from the time dependencies of the unique NMR signals.…”

Section: Resultsmentioning

confidence: 99%

Three-dimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily

et al. 2001

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R.A.Staniforth and S.Giannini contributed equally to this workCystatins, an amyloid-forming structural superfamily, form highly stable, domain-swapped dimers at physiological protein concentrations. In chicken cystatin, the active monomer is a kinetic trap en route to dimerization, and any changes in solution conditions or mutations that destabilize the folded state shorten the lifetime of the monomeric form. In such circumstances, amyloidogenesis will start from conditions where a domain-swapped dimer is the most prevalent species. Domain swapping occurs by a rearrangement of loop I, generating the new intermonomer interface between strands 2 and 3. The transition state for dimerization has a high level of hydrophobic group exposure, indicating that gross conformational perturbation is required for domain swapping to occur. Dimerization also occurs when chicken cystatin is in its reduced, molten-globule state, implying that the organization of secondary structure in this state mirrors that in the folded state and that domain swapping is not limited to the folded states of proteins. Although the interface between cystatin-fold units is poorly de®ned for cystatin A, the dimers are the appropriate size to account for the electron-dense regions in amyloid proto®laments.

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Section: Resultsmentioning

confidence: 99%

Three-dimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily

et al. 2001

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“…Several cases showing biphasic unfolding characteristics were reported, such as high-affinity ligand binding [29], an increase of the free ligand during the unfolding of the protein caused by the release of the ligand from the denatured protein [30], and the presence of multiple structural regions with lower and higher stabilities [31]. While the former two cases unlikely occurred on T1r2a/T1r3LBD, because the biphasic features in those cases were observed at low concentrations of the ligands, the last case might conform with this protein.…”

Section: Discussionmentioning

confidence: 99%

Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3

et al. 2019

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Taste receptor type 1 (T1r) is responsible for the perception of essential nutrients, such as sugars and amino acids, and evoking sweet and umami (savory) taste sensations. T1r receptors recognize many of the taste substances at their extracellular ligand-binding domains (LBDs). In order to detect a wide array of taste substances in the environment, T1r receptors often possess broad ligand specificities. However, the entire ranges of chemical spaces and their binding characteristics to any T1rLBDs have not been extensively analyzed. In this study, we exploited the differential scanning fluorimetry (DSF) to medaka T1r2a/T1r3LBD, a current sole T1rLBD heterodimer amenable for recombinant preparation, and analyzed their thermal stabilization by adding various amino acids. The assay showed that the agonist amino acids induced thermal stabilization and shifted the melting temperatures (Tm) of the protein. An agreement between the DSF results and the previous biophysical assay was observed, suggesting that DSF can detect ligand binding at the orthosteric-binding site in T1r2a/T1r3LBD. The assay further demonstrated that most of the tested l-amino acids, but no d-amino acid, induced Tm shifts of T1r2a/T1r3LBD, indicating the broad l-amino acid specificities of the proteins probably with several different manners of recognition. The Tm shifts by each amino acid also showed a fair correlation with the responses exhibited by the full-length receptor, verifying the broad amino-acid binding profiles at the orthosteric site in LBD observed by DSF.

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“…From each spectrum, the CD signal (∆A 248 -∆A 224 ) was extracted. 27 In Figure 1, this quantity is plotted on a relative scale against the Gua concentration. A similar series of experiments was conducted with urea as the denaturant, using a maximum concentration of 10 M.…”

Section: Methodsmentioning

confidence: 99%

“…The circular dichroism (CD) spectra (using Jobin-Yvon circular dichrograph V) were recorded immediately after mixing in a range of 208−250 nm using a 1-mm cuvette. From each spectrum, the CD signal (Δ A 248 − Δ A 224 ) was extracted . In Figure , this quantity is plotted on a relative scale against the Gua concentration.…”

Section: Methodsmentioning

confidence: 99%

Spectral Diffusion Experiment with a Denatured Protein

Ponkratov¹,

Friedrich²,

Marković³

et al. 2003

J. Phys. Chem. B

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Spectral diffusion broadening of cytochrome c carrying the free-base analogue of heme is investigated in its unfolded state and compared with the corresponding broadening in the native state. Spectral diffusion is much larger in the unfolded state, in comparison to the native state. Interestingly, the time law that governs spectral diffusion changes as the aging time increases, from a power-law behavior in the native state to an apparent logarithmic behavior in the unfolded state.

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Biphasic transition curve on denaturation of chicken cystatin by guanidinium chloride Evidence for an independently unfolding structural region

Cited by 14 publications

References 19 publications

Three-dimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily

Three-dimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily

Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3

Spectral Diffusion Experiment with a Denatured Protein

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