Unfolding and Conformational Studies on Bovine Adrenodoxin Probed by Engineered Intrinsic Tryptophan Fluorescence†

Biochemistry ◽  
2002 ◽  
Vol 41 (36) ◽  
pp. 11008-11016 ◽  
Author(s):  
Frank Hannemann ◽  
Aloke Kumar Bera ◽  
Birgitta Fischer ◽  
Michael Lisurek ◽  
Klaus Teuchner ◽  
...  
Keyword(s):  
Tryptophan Fluorescence ◽  
Conformational Studies ◽  
Intrinsic Tryptophan Fluorescence

scholarly journals Inactivation of sarcoplasmic-reticulum Ca2+-ATPase in low-frequency-stimulated muscle results from a modification of the active site

1992 ◽  
Vol 285 (1) ◽  
pp. 303-309 ◽  
Author(s):  
S Matsushita ◽  
D Pette
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Active Site ◽  
Low Frequency ◽  
Fluorescein Isothiocyanate ◽  
Tryptophan Fluorescence ◽  
Atp Binding ◽  
Binding Characteristics ◽  
Intrinsic Tryptophan Fluorescence ◽  
Inactive Form ◽  
Induced Changes

Molecular changes underlying the partial inactivation of the sarcoplasmic-reticulum (SR) Ca(2+-) ATPase in low-frequency-stimulated fast-twitch muscle were investigated in the present study. The specific Ca(2+)-ATPase activity, as well as the ATP- and acetyl phosphate-driven Ca2+ uptakes by the SR, were reduced by approx. 30% in 4-day-stimulated muscle. Phosphoprotein formation of the enzyme in the presence of ATP or Pi was also decreased to the same extent. Measurements of ATP binding revealed a 30% decrease in binding to the enzyme. These changes were accompanied by similar decreases in the ligand-induced (ATP, ADP, Pi) intrinsic tryptophan fluorescence. A decreased binding of fluorescein isothiocyanate (FITC) corresponded to the lower ATP binding and phosphorylation of the enzyme. Moreover, Pi-induced changes in fluorescence of the FITC-labelled enzyme did not differ between SR from stimulated and contralateral muscles, indicating that Ca(2+)- ATPase molecules which did not bind FITC were responsible for the decreased Pi-dependent phosphorylation, and therefore represented the inactive form of the enzyme. No differences existed between the Ca(2+)-induced changes in the intrinsic fluorescence of SR from stimulated and contralateral muscles which fit their similar Ca(2+)-binding characteristics. Taking the proposed architecture of the Ca2(+)-ATPase into consideration, our results suggest that the inactivation relates to a circumscribed structural alteration of the enzyme in sections of the active site consisting of the nucleotide-binding and phosphorylation domains.

scholarly journals Substrate-induced conformational transition in human phenylalanine hydroxylase as studied by surface plasmon resonance analyses: the effect of terminal deletions, substrate analogues and phosphorylation

2003 ◽  
Vol 369 (3) ◽  
pp. 509-518 ◽  
Author(s):  
Anne J. STOKKA ◽  
Torgeir FLATMARK
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Phenylalanine Hydroxylase ◽  
Conformational Transition ◽  
Tryptophan Fluorescence ◽  
Catalytic Core ◽  
Intrinsic Tryptophan Fluorescence ◽  
Human Phenylalanine Hydroxylase ◽  
Activity State

The optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, was used for real-time measurements of the slow conformational transition (isomerization) which occurs in human phenylalanine hydroxylase (hPAH) on the binding/dissociation of l-phenylalanine (l-Phe). The binding to immobilized tetrameric wt-hPAH resulted in a time-dependent increase in the refractive index (up to approx. 3min at 25°C) with an end point of approx. 75RU (resonance units)/(pmolsubunit/mm2). By contrast, the contribution of binding the substrate (165Da) to its catalytic core enzyme [ΔN(1—102)/ΔC(428—452)-hPAH] was only approx. 2RU/(pmolsubunit/mm2). The binding isotherm for tetrameric and dimeric wt-hPAH revealed a [S]0.5-value of 98±7μM (h = 1.0) and 158±11μM, respectively, i.e. for the tetramer it is slightly lower than the value (145±5μM) obtained for the co-operative binding (h = 1.6±0.4) of l-Phe as measured by the change in intrinsic tryptophan fluorescence. The responses obtained by SPR and intrinsic tryptophan fluorescence are both considered to be related to the slow reversible conformational transition which occurs in the enzyme upon l-Phe binding, i.e. by the transition from a low-activity state ('T-state') to a relaxed high-activity state ('R-state') characteristic of this hysteretic enzyme, however, the two methods reflect different elements of the transition. Studies on the N- and C-terminal truncated forms revealed that the N-terminal regulatory domain (residues 1—117) plus catalytic domain (residues 118—411) were required for the full signal amplitude of the SPR response. Both the on- and off-rates for the conformational transition were biphasic, which is interpreted in terms of a difference in the energy barrier and the rate by which the two domains (catalytic and regulatory) undergo a conformational change. The substrate analogue 3-(2-thienyl)-l-alanine revealed an SPR response comparable with that of l-Phe on binding to wild-type hPAH.

scholarly journals Intrinsic tryptophan fluorescence spectroscopy reliably determines galectin-ligand interactions

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Paulina Sindrewicz ◽  
Xiaoxin Li ◽  
Edwin A. Yates ◽  
Jeremy E. Turnbull ◽  
Lu-Yun Lian ◽  
...  
Keyword(s):  
Fluorescence Spectroscopy ◽  
Tryptophan Fluorescence ◽  
Intrinsic Tryptophan Fluorescence ◽  
Ligand Interactions

scholarly journals An intrinsic-tryptophan-fluorescence study of phage phi29 connector/nucleic acid interactions

1994 ◽  
Vol 225 (2) ◽  
pp. 747-753 ◽  
Author(s):  
Maria Angeles Urbaneja ◽  
Susana Rivas ◽  
Jose L. Carrascosa ◽  
Jose Maria Valpuesta
Keyword(s):  
Nucleic Acid ◽  
Tryptophan Fluorescence ◽  
Fluorescence Study ◽  
Intrinsic Tryptophan Fluorescence ◽  
Nucleic Acid Interactions

Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

Biologia ◽  
2014 ◽  
Vol 69 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Ping-Lin Ong ◽  
Tzu-Ting Chuang ◽  
Tzu-Fan Wang ◽  
Long-Liu Lin
Keyword(s):  
Bacillus Licheniformis ◽  
Specific Activity ◽  
Chloride Ion ◽  
Tryptophan Fluorescence ◽  
Host Cells ◽  
Glycoside Hydrolase Family ◽  
Chloride Binding ◽  
Mutant Proteins ◽  
Nickel Affinity Chromatography ◽  
Intrinsic Tryptophan Fluorescence

AbstractBased on sequence alignment of selected Cl− dependent and independent glycoside hydrolase family 13 enzymes, two invariant residues (Arg201 and Asn347) and one tyrosine (Tyr365) that might be responsible for the binding of Bacillus licheniformis trehalose-6-phosphate hydrolase (BlTreA) to chloride ion were identified. The role of these three residues was further explored by mutational and biophysical analyses. The mutant enzymes (R201Q/E/K, N327Q/D/K, and Y365A/R) and BlTreA were individually overexpressed in Escherichia coli M15 host cells and purified by one-step nickel affinity chromatography on Ni-NTA resin. The purified BlTreA and Y365A had a specific activity of 236.9 and 47.6 U/mg protein, respectively. The remaining enzymes lost their hydrolase activity completely even in the presence of high salt. With the exception of Y365A, all mutant enzymes did not have the ability to bind fluoride, chloride and nitrate anions. Structural analyses showed that the circular dichroism spectra of the mutant proteins were consistent with those of BlTreA. However, wild-type and mutant enzymes displayed a slight difference in the profiles of intrinsic tryptophan fluorescence. Collectively, these results clearly indicate that Arg201 and Agr327 residues might play an essential role in chloride binding of BlTreA.

Intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1-ATPase. A powerful probe for phosphate and nucleotide interactions

Biochemistry ◽  
1991 ◽  
Vol 30 (13) ◽  
pp. 3256-3262 ◽  
Author(s):  
Gilles Divita ◽  
Attilio Di Pietro ◽  
Gilbert Deleage ◽  
Bernard Roux ◽  
Daniele C. Gautheron
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Tryptophan Fluorescence ◽  
F1 Atpase ◽  
Intrinsic Tryptophan Fluorescence ◽  
Nucleotide Interactions

scholarly journals Glycolipid Acquisition by Human Glycolipid Transfer Protein Dramatically Alters Intrinsic Tryptophan Fluorescence

2009 ◽  
Vol 284 (20) ◽  
pp. 13620-13628 ◽  
Author(s):  
Xiuhong Zhai ◽  
Margarita L. Malakhova ◽  
Helen M. Pike ◽  
Linda M. Benson ◽  
H. Robert Bergen ◽  
...  
Keyword(s):  
Tryptophan Fluorescence ◽  
Transfer Protein ◽  
Intrinsic Tryptophan Fluorescence
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