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Single-molecule F?rster Resonance Energy Transfer Measurement Reveals Dynamic Partially Ordered Structure of the Epidermal Growth Factor Receptor C-tail Domain
摘要： Intrinsically disordered proteins (IDPs) or regions (IDRs) are thought to exhibit unique functionalities without forming ordered structures. However, these molecular mechanisms are not easily elucidated, partly because of the difficultly of measuring structural information. In this study, we applied the alternative laser excitation (ALEX) method and circular dichroism (CD) spectroscopy to investigate the structure of the C-terminal tail (CTT) domain of the human epidermal growth factor receptor (EGFR). The single-molecule distributions of F?rster resonance energy transfer (FRET) obtained by ALEX under solution conditions modified by the addition of potassium chloride (KCl), urea or guanidinium chloride (GdmCl) allowed us to separately examine the influences of charge interactions and secondary structure formation. The CD spectrum analyses indicated the types of included secondary structure. The results suggested that the structure of the CTT is influenced by secondary structure formation, which is principally antiparallel β-sheet, rather than by charge interactions, and that phosphorylation of the major Grb2-binding sites partially denature that secondary structure. Our findings suggest that the EGFR CTT might regulate ligand binding kinetics by local β-sheet formation or by the disruption associated with phosphorylation states.
关键词： Circular dichroism,Epidermal Growth Factor Receptor,F?rster resonance energy transfer,Secondary structure,Intrinsically disordered proteins
更新于2025-09-23 15:19:57
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Protective characterization of low dose sodium nitrite on yak meat myoglobin in a hydroxy radical oxidation environment: Fourier Transform Infrared spectroscopy and laser Micro-Raman spectroscopy
摘要： The effect of NaNO2 in hydroxyl radical-mediated oxidative damage of yak meat myoglobin was investigated. Laser micro-Raman spectroscopy and Fourier Transform Infrared spectroscopy were used to assess protein (carbonyls, total sulfhydryl and Disulfide bonds) oxidation, atomic (or molecular) interactions and secondary structural changes. The addition of NaNO2 during the oxidation of myoglobin significantly reduced the content of carbonyl and disulfide bonds and protected the sulfhydryl groups from hydroxyl radical oxidation (P < 0.05). There was no significant difference in the secondary structure of myoglobin between the control group and the SN treatment group(P > 0.05). At the same time, NaNO2 had an inhibitory effect on the expansion of the hemoglobin center size and the transition of Fe from a low spin state to a high spin state caused by radical-oxidized. These findings suggest that NaNO2 has potential for treatment effects in a hydroxyl radical-oxidized myoglobin.
关键词： Secondary structure,Hydroxyl radical,Raman spectroscopy,NaNO2,Myoglobin
更新于2025-09-11 14:15:04
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Encyclopedia of Biophysics || Protein Circular Dichroism Analysis
摘要： The far-ultraviolet circular dichroism spectra of proteins contain information about the electrostatic and magnetic environment of the chromophores and may be analyzed to give a close approximation of the content of secondary structure types. Different protein secondary structure types produce characteristic spectral shapes, and, due to the additive nature of the spectra, the shapes contribute to the total spectrum in the proportions that their secondary structures are found within the sample. Typically, radiation of wavelengths 240–190 nm and shorter is used for this type of analysis. More electronic transitions are included by collecting data at shorter wavelengths, so more information is available.
关键词： Deconvolution,Protein Circular Dichroism,Secondary structure analyses
更新于2025-09-10 09:29:36
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Photoactivation mechanism, timing of protein secondary structure dynamics and carotenoid translocation in the Orange Carotenoid Protein
摘要： The Orange Carotenoid Protein (OCP) is a two-domain photoactive protein that noncovalently binds an echinenone (ECN) carotenoid and mediates photoprotection in cyanobacteria. In the dark, OCP assumes an orange, inactive state known as OCPO; blue light illumination results in the red active state, known as OCPR. The OCPR state is characterized by large-scale structural changes that involve dissociation and separation of C-terminal and N-terminal domains accompanied by carotenoid translocation into the N-terminal domain. The mechanistic and dynamic-structural relations between photon absorption and formation of the OCPR state have remained largely unknown. Here, we employ a combination of time-resolved UV-visible and (polarized) mid-infrared spectroscopy to assess the electronic and structural dynamics of the carotenoid and the protein secondary structure, from femtoseconds to 0.5 milliseconds. We identify a hereto unidentified carotenoid excited state in OCP, the so-called S* state, which we propose to play a key role in breaking conserved hydrogen-bond interactions between carotenoid and aromatic amino acids in the binding pocket. We arrive at a comprehensive reaction model where the hydrogen-bond rupture with conserved aromatic side chains at the carotenoid β1-ring in picoseconds occurs at a low yield of <1 %, whereby the β1-ring retains a trans configuration with respect to the conjugated π-electron chain. This event initiates structural changes at the N-terminal domain in 1 μs, which allow the carotenoid to translocate into the N-terminal domain in 10 μs. We identified infrared signatures of helical elements that dock on the C-terminal domain β-sheet in the dark and unfold in the light to allow domain separation. These helical elements do not move within the experimental range of 0.5 ms, indicating that domain separation occurs on longer timescales, lagging carotenoid translocation by at least 2 decades of time.
关键词： protein secondary structure dynamics,photoactivation mechanism,Orange Carotenoid Protein,time-resolved spectroscopy,carotenoid translocation
更新于2025-09-09 09:28:46