Direct conversion of Cytochrome c spectral shifts to fluorescence using photochromic FRET

Abstract

Photochromic fluorescence resonance energy transfer (pcFRET) was used to monitor the redox activity of non-fluorescent heme protein. Venus fluorescent protein was used as a donor where its emission intensity was reversibly modulated by the absorption change of Cytochrome c.

Biological Stimuli-Induced Phase Transition of a Synthesized Block Copolymer: Preferential Interactions between PNIPAM-b-PNVCL and Heme Proteins

“…Figure a illustrates UV–visible absorption spectroscopy measurements of ANS-PNIPAM- b -PNVCL aqueous solution in the presence of Cyt c. Native Cyt c shows n−Π* transition of aromatic amino acids at 280 nm, an intense soret band due to Π–Π* transition at ∼410 nm, and a broad Q (α+β) band due to electronic transition in a heme group at 528 nm (Figure S2a). ,, The presence of diblock polymer changes the intensity and position of these bands, which is further affected by increasing the concentration of Cyt c. With the addition of 0.5 mg/mL concentration of Cyt c to the ANS-PNIPAM- b -PNVCL aqueous solution, the peak at 280 nm corresponding to Cyt c completely vanished due to n−Π* transition, which signifies the interaction of copolymer with aromatic amino acid residues of Cyt c. Peaks appearing at ∼410 and ∼528 nm for native Cyt c have shown a negligible effect on copolymer; however, less intense transition with a slight redshift was observed for copolymer (peak at ∼308 nm).…”

Comprehensive Insight into the Protein–Surface Biomolecular Interactions on a Smart Material: Complex Formation between Poly(N-vinyl Caprolactam) and Heme Protein

“…Native conformation of Cyt c (Figure a inset) shows bands at 280, ∼410, and 528 nm which are due to n−Π* transition of aromatic amino acids, an intense Soret band due to Π–Π* transition where His 18 and Met 80 stabilize low spin state by coordinating at axial positions, and a broad Q (α + β) band due to electronic transition in the heme group, respectively. , The intensity of these bands increases with change in the protein concentration, and the broad Q band splits into α (520 nm) and β (550 nm) bands in the polymer–protein complex, which was initially absent either in the pure protein in ANS (the red line shown in the inset of Figure a) or pure polymer in the case of ANS (the black line shown in Figure a). This clearly represents that the polymer interacts with the heme moiety of Cyt c , which is responsible for the splitting during absorbance.…”

“…Cyt c is in the shape of a prolate spheroid and the heme group is attached to it through two thioester bonds, that is, cysteine (Cys) 14 and cysteine (Cys) 17, and the “closed crevice” where the heme iron is held, with the axial ligands, histidine (His) 18 and the methionine (Met) 80 . Cyt c contains five α-helical segments which are N-terminal (residues 2–14), C-terminal (residues 87–103), and helical segments 49–55, 60–70, and 70–75. , Mb, which is a cytoplasmic heme protein, belongs to the globin superfamily of proteins containing 153 amino acids. Out of these 153 amino acids, 121 (79%) are present in the helical region while the remaining 32 amino acids are distributed over the nonhelical region. , The histidine group (His-93) is directly attached to iron and a distal histidine group (His-64) is hovered near the opposite face .…”

“…15 Cyt c contains five α-helical segments which are N-terminal (residues 2−14), C-terminal (residues 87−103), and helical segments 49−55, 60−70, and 70−75. 15,16 Mb, which is a cytoplasmic heme protein, belongs to the globin superfamily of proteins containing 153 amino acids. Out of these 153 amino acids, 121 (79%) are present in the helical region while the remaining 32 amino acids are distributed over the nonhelical region.…”

Development of heme protein based oxygen sensing indicators

“…Previously, Manioglu et al . measured redox states of the heme protein cytochrome c using YFP 45 . In this study, cytochrome c (Cyt c ) was attached to YFP via a short peptide linker (Ala-Ala-Ala) and redox dependent absorption changes of heme were successfully converted to changes in fluorescence intensity of YFP.…”