Ultrafast structural dynamics in BLUF domains: transient infrared spectroscopy of AppA and its mutants.
The structural dynamics following photoexcitation of a photosensing BLUF (blue light sensing using FAD) domain protein have been investigated by ultrafast transient infrared spectroscopy. Specifically, the transcriptional antirepressor AppA from Rhodobacter sphaeroides has been studied in the light and dark adapted forms and in photoactive and inactive mutants W104F and Q63L. A transient absorption has been observed at 1666 cm(-1) which is a marker mode for the photoactive state of the protein. This instantaneously formed transient is tentatively assigned to a vibrational mode of a protein residue modified through its interaction with the excited state of the chromophore. A plausible candidate consistent with the mutant studies is the carbonyl stretch of the Q63 amide side chain. These results suggest that modification of the strength of protein chromophore H-bonded interactions is the primary step in the BLUF domain photocycle. No new species were observed to be formed during the first nanosecond. Measurement of the ultrafast ground state recovery showed that the excited state of light adapted AppA is strongly quenched compared to the dark adapted state. It is proposed that the reorganization which occurs to form the signaling state is favorable to electron-transfer quenching.
Stelling
Allison L
AL
Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
Ronayne
Kate L
KL
Nappa
Jérôme
J
Tonge
Peter J
PJ
Meech
Stephen R
SR
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism*; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics*; D013055:Spectrophotometry, Infrared; D013997:Time Factors
C116785:AppA protein, Rhodobacter sphaeroides; D001426:Bacterial Proteins; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide