Mechanistic Interpretation of Tryptophan Fluorescence Quenching in the Time Courses of Glutamate Dehydrogenase Catalyzed Reactions†

Biochemistry ◽  
1996 ◽  
Vol 35 (51) ◽  
pp. 16483-16488 ◽  
Author(s):  
Swapan K. Saha ◽  
Steven J. Maniscalco ◽  
Harvey F. Fisher
Keyword(s):  
Fluorescence Quenching ◽  
Glutamate Dehydrogenase ◽  
Tryptophan Fluorescence ◽  
Time Courses ◽  
Catalyzed Reactions ◽  
Tryptophan Fluorescence Quenching

scholarly journals Biochemical Study of Multiple CheY Response Regulators of the Chemotactic Pathway of Rhodobacter sphaeroides

2004 ◽  
Vol 186 (15) ◽  
pp. 5172-5177 ◽  
Author(s):  
Axelle Ferré ◽  
Javier de la Mora ◽  
Teresa Ballado ◽  
Laura Camarena ◽  
Georges Dreyfus
Keyword(s):  
Fluorescence Quenching ◽  
Rhodobacter Sphaeroides ◽  
Biochemical Study ◽  
Response Regulator ◽  
Tryptophan Fluorescence ◽  
Flagellar Motor ◽  
Acetyl Phosphate ◽  
Response Regulators ◽  
Tryptophan Fluorescence Quenching

ABSTRACT The six copies of the response regulator CheY from Rhodobacter sphaeroides bind to the switch protein FliM. Phosphorylation by acetyl phosphate (AcP) was detected by tryptophan fluorescence quenching in three of the four CheYs that contain this residue. Autophosphorylation with Ac32P was observed in five CheY proteins. We also show that all of the cheY genes are expressed simultaneously; therefore, in vivo all of the CheY proteins could bind to FliM to control the chemotactic response. Consequently, we hypothesize that in this complex chemotactic system, the binding of some CheY proteins to FliM, does not necessarily imply switching of the flagellar motor.

scholarly journals Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching

2013 ◽  
Vol 42 (4) ◽  
pp. 2736-2749 ◽  
Author(s):  
Kirsten E. Robinson ◽  
Jillian Orans ◽  
Alexander R. Kovach ◽  
Todd M. Link ◽  
Richard G. Brennan
Keyword(s):  
Fluorescence Quenching ◽  
Rna Binding ◽  
Tryptophan Fluorescence ◽  
Binding Motif ◽  
Gram Positive ◽  
C Terminus ◽  
Specific Regulation ◽  
Rna Mapping ◽  
Rna Interaction ◽  
Tryptophan Fluorescence Quenching

Abstract Hfq is a posttranscriptional riboregulator and RNA chaperone that binds small RNAs and target mRNAs to effect their annealing and message-specific regulation in response to environmental stressors. Structures of Hfq-RNA complexes indicate that U-rich sequences prefer the proximal face and A-rich sequences the distal face; however, the Hfq-binding sites of most RNAs are unknown. Here, we present an Hfq-RNA mapping approach that uses single tryptophan-substituted Hfq proteins, all of which retain the wild-type Hfq structure, and tryptophan fluorescence quenching (TFQ) by proximal RNA binding. TFQ properly identified the respective distal and proximal binding of A15 and U6 RNA to Gram-negative Escherichia coli (Ec) Hfq and the distal face binding of (AA)3A, (AU)3A and (AC)3A to Gram-positive Staphylococcus aureus (Sa) Hfq. The inability of (GU)3G to bind the distal face of Sa Hfq reveals the (R-L)n binding motif is a more restrictive (A-L)n binding motif. Remarkably Hfq from Gram-positive Listeria monocytogenes (Lm) binds (GU)3G on its proximal face. TFQ experiments also revealed the Ec Hfq (A-R-N)n distal face-binding motif should be redefined as an (A-A-N)n binding motif. TFQ data also demonstrated that the 5′-untranslated region of hfq mRNA binds both the proximal and distal faces of Ec Hfq and the unstructured C-terminus.

scholarly journals Domain-domain associations in cystic fibrosis transmembrane conductance regulator

2002 ◽  
Vol 282 (5) ◽  
pp. C1170-C1180 ◽  
Author(s):  
Wenlan Wang ◽  
Zhaoping He ◽  
Thomas J. O'Shaughnessy ◽  
John Rux ◽  
William W. Reenstra
Keyword(s):  
Cystic Fibrosis ◽  
Fluorescence Quenching ◽  
Tryptophan Fluorescence ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Tryptophan Fluorescence Quenching ◽  
Overlay Assay ◽  
R Domain

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR is a chloride channel whose activity requires protein kinase A-dependent phosphorylation of an intracellular regulatory domain (R-domain) and ATP hydrolysis at the nucleotide-binding domains (NBDs). To identify potential sites of domain-domain interaction within CFTR, we expressed, purified, and refolded histidine (His)- and glutathione- S-transferase (GST)-tagged cytoplasmic domains of CFTR. ATP-binding to his-NBD1 and his-NBD2 was demonstrated by measuring tryptophan fluorescence quenching. Tryptic dig estion of in vitro phosphorylated his-NBD1-R and in situ phosphorylated CFTR generated the same phosphopeptides. An interaction between NBD1-R and NBD2 was assayed by tryptophan fluorescence quenching. Binding among all pairwise combinations of R-domain, NBD1, and NBD2 was demonstrated with an overlay assay. To identify specific sites of interaction between domains of CFTR, an overlay assay was used to probe an overlapping peptide library spanning all intracellular regions of CFTR with his-NBD1, his-NBD2, and GST-R-domain. By mapping peptides from NBD1 and NBD2 that bound to other intracellular domains onto crystal structures for HisP, MalK, and Rad50, probable sites of interaction between NBD1 and NBD2 were identified. Our data support a model where NBDs form dimers with the ATP-binding sites at the domain-domain interface.

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