Spectral perturbations and oligomer/monomer formation in 124-kilodalton Avena phytochrome

Abstract

We have studied the effects of pH, ionic strength, and hydrophobic fluorescence probes, 8-anilinonaphthalene-1-sulfonate (ANS) and bis-ANS, on the structure of intact (124-kDa) Avena phytochrome. The Pfr form of phytochrome forms oligomers in solution to a greater extent than the Pr form. Hydrophobic forces play a major role in the oligomerization of phytochrome, as suggested by fluorescence and monomerization by bis-ANS. However, electrostatic charges also take part in the phytochrome oligomerization. The partial proteolytic digestion patterns for the Pr and Pfr species are different, but binding of bis-ANS to the phytochrome abolishes this difference and yields an identical proteolytic peptide mapping for both spectral forms of phytochrome. This appears to result from bis-ANS binding at the carboxy-terminal domain, which induces monomerization of phytochrome oligomers. A second bis-ANS binding at an amino-terminal site blocks cleavage sites of trypsin and alpha-chymotrypsin. Bis-ANS especially blocks access of the proteases to the amino-terminal cleavage site that produces an early proteolytic product (114/118 kDa) on SDS gels. The bis-ANS binding does not, however, affect the proteolytic cleavage site that occurs in the hinge region between the two structural domains of phytochrome, the chromophore domain and the C-terminal non-chromophore domain. A chromophore binding site in the Pfr form is apparently exposed for preferential binding of bis-ANS, causing cyclization of the chromophore and bleaching of its absorbance at 730 nm. These observations have been discussed in terms of a photoreversible topographic change of the chromophore/apoprotein during the phototransformation of phytochrome.

Purification and Characterization of Recombinant Affinity Peptide‐Tagged Oat Phytochrome A

“…The SEC-HPLC studies on ASPHYA-ST and its bilin adducts, summarized in Table 3, are in good agreement with the dimeric quaternary structure reported for native phytochrome A (3942). The Pfr forms of the photoreversible adducts (PcPB and PCB) have a larger apparent size than the P, forms, which is consistent with results for native oat phyA presented in this study and in previously published work (40,41,43). Interestingly, the PCB adduct is significantly larger in the Pfr form such that its size surpasses that of the largest molecular weight standard.…”

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity

“…Allosteric effects were investigated by hydrogen/deuterium exchange mass spectrometry on the full length sGC, and the results revealed that PAS and CC domains play a critical role in the activation by NO . The structure of the human isoform of sGC was investigated using far-UV circular dichroism (CD) spectroscopy, intrinsic tryptophan fluorescence, fluorescence of the hydrophobic dye bis-8-anilino-1-naphtalene­sulfonic acid (bis-ANS), size exclusion chromatography, and small angle X-ray scattering (SAXS), which allowed for a three-dimensional model of the enzyme to be constructed. ,− …”

Improved conditions for fluorescent staining of proteins with 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid in SDS‐PAGE

“…0 -Dianilino-1,1 0 -binaphthyl-5,5 0 -disulfonic acid (BisANS), has been frequently used to probe hydrophobic sites in proteins and to study protein-substrate interaction and protein conformational changes, because it binds to hydrophobic regions of protein surrounded by positively charged residues with high affinity [1][2][3][4][5][6][7][8]. Furthermore, BisANS was applied to visualize the protein in SDS-PAGE with the enhancement of KCl or BaCl 2 , which gives much less nonspecific background fluorescence.…”