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On the mechanism of activation of the BLUF domain of AppA.

Abstract：

:AppA, a transcriptional antirepressor, regulates the steady expression of photosynthesis genes in Rhodobacter sphaeroides in response to high-intensity blue light and to redox signals. Its blue-light sensing is mediated by an N-terminal BLUF domain, a member of a novel flavin fold. The photocycle of this domain (AppA(5-125)) includes formation of a slightly red-shifted long-lived signaling state, which is formed directly from the singlet excited state of the flavin on a subnanosecond time scale [Gauden et al. (2005) Biochemistry 44, 3653-3662]. The red shift of the absorption spectrum of this signaling state has been attributed to a rearrangement of its hydrogen-bonding interactions with the surrounding apoprotein. In this study we have characterized an AppA mutant with an altered aromatic amino acid: W104F. This mutant exhibits an increased lifetime of the singlet excited state of the flavin chromophore. Most strikingly, however, it shows a 1.5-fold increase in its quantum yield of signaling state formation. In addition, it shows a slightly increased rate of ground-state recovery. On top of this, the presence of imidazole, both in this mutant protein and in the wild-type BLUF domain, significantly accelerates the rate of ground-state recovery, suggesting that this rate is limited by rearrangement of (a) hydrogen bond(s). In total, an approximately 700-fold increase in recovery rate has been obtained, which makes the W104F BLUF domain of AppA, for example, suitable for future analyses with step-scan FTIR. The rate of ground-state recovery of the BLUF domain of AppA follows Arrhenius kinetics. This suggests that this domain itself does not undergo large structural changes upon illumination and that the structural transitions in full-length AppA are dominated by interdomain rearrangements.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Laan W,Gauden M,Yeremenko S,van Grondelle R,Kennis JT,Hellingwerf KJ

doi

10.1021/bi051367p

subject

Has Abstract

pub_date

2006-01-10 00:00:00

pages

51-60

issue

1

eissn

0006-2960

issn

1520-4995

journal_volume

45

pub_type

杂志文章

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