The tryptophan residues of aspartate transcarbamylase: site-directed mutagenesis and time-resolved fluorescence spectroscopy

Biochemistry ◽  
1992 ◽  
Vol 31 (49) ◽  
pp. 12504-12513 ◽  
Author(s):  
Luc Fetler ◽  
Patrick Tauc ◽  
Guy Herve ◽  
Moncef M. Ladjimi ◽  
Jean Claude Brochon
Keyword(s):  
Fluorescence Spectroscopy ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Time Resolved Fluorescence ◽  
Aspartate Transcarbamylase ◽  
Time Resolved ◽  
Tryptophan Residues

scholarly journals Recombinant human O6-alkylguanine-DNA alkyltransferase (AGT), Cys145-alkylated AGT and Cys145→Met145 mutant AGT: comparison by isoelectric focusing, CD and time-resolved fluorescence spectroscopy

1997 ◽  
Vol 324 (1) ◽  
pp. 321-328 ◽  
Author(s):  
Matthias FEDERWISCH ◽  
Ulrich HASSIEPEN ◽  
Kirsten BENDER ◽  
Manfred DEWOR ◽  
Manfred F. RAJEWSKY ◽  
...  
Keyword(s):  
Fluorescence Spectroscopy ◽  
Isoelectric Focusing ◽  
Covalent Binding ◽  
Site Directed Mutagenesis ◽  
Time Resolved Fluorescence ◽  
Active Centre ◽  
Time Resolved ◽  
Helical Content ◽  
Dna Repair Protein ◽  
Dna Alkyltransferase

Isoelectric focusing, CD, steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA-repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with AGT derivatives methylated or benzylated on Cys145 or modified by site-directed mutagenesis at the active centre (Met145 mutant). The AGT protein is approximately spherical with highly constrained Trp residues, but is not stabilized by disulphide bridges. In contrast with native AGT, alkylated AGT precipitated at 25 °C but remained monomeric at 4 °C. As revealed by isoelectric focusing, pI changed from 8.2 (AGT) to 8.4 (Cys145-methylated AGT) and 8.6 (Cys145-benzylated AGT). The α-helical content of the Met145 mutant was decreased by approx. 5% and Trp residues were partially liberated. Although non-covalent binding of O6-benzylguanine did not alter the secondary structure of AGT, its α-helical content was increased by approx. 2% on methylation and by approx. 4% on benzylation, altogether indicating a small conformational change in AGT on undergoing alkylation. No signal sequences have been found in AGT that mark it for polyubiquitination. Therefore the signal for AGT degradation remains to be discovered.

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