Enhancing the fluorescence of tyr-59 in ubiquitin by blocking proton transfer.
Two highly fluorescent mutants of ubiquitin (E51Q and E51A) were produced by mutation of a single amino acid, demonstrating that excited-state proton transfer from the tyrosine residue to the carboxylate group of Glu-51 in ubiquitin is responsible for the reduced fluorescence of wild-type ubiquitin (wt-UBQ) at pH 5. E51A shows a Tm=59 degrees C at pH 1.5 and a Tm>80 degrees C at pH 5 similar to wt-UBQ, which shows that the mutation has not altered the protein structure significantly. The high and constant fluorescence from pH 1.5 to pH 7 allows for the study of the folding/unfolding over a wide range of pHs which would otherwise be impossible with wt-UBQ.
Departamento de Química, Instituto Superior Técnico, Universidade Técnica de Lisboa, 1049-001, Lisboa, Portugal. mnoronha@ist.utl.pt
Melinda
M
Noronha
Hana
H
Gerbelová
Tiago Q
TQ
Faria
Maria Manuel
MM
Sampaio
Rainer
R
Rudolph
António L
AL
Maçanita
Helena
H
Santos
D002478:Cells, Cultured; D005453:Fluorescence; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D017354:Point Mutation; D017510:Protein Folding; D011522:Protons; D013053:Spectrophotometry; D013696:Temperature; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics*
D011522:Protons; D025801:Ubiquitin; D018698:Glutamic Acid; D014443:Tyrosine