Photounbinding of calmodulin from a family of CaM binding peptides.
BACKGROUND
Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/antigen binding.


RESULTS
We present studies of the photounbinding of labeled calmodulin (CaM) from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked.


CONCLUSIONS
The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformational change of the CaM which lowers their binding affinity with the peptide or its respective binding partner.
Department of Optical Engineering, Research Institute of Molecular Pathology, Vienna, Austria.
Klaus G
KG
Neumüller
Kareem
K
Elsayad
Johannes M
JM
Reisecker
M Neal
MN
Waxham
Katrin G
KG
Heinze
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects / Q000528:radiation effects; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004735:Energy Transfer; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D016166:Free Radical Scavengers / Q000494:pharmacology; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D038761:Photobleaching; D011485:Protein Binding / Q000187:drug effects / Q000528:radiation effects
D002147:Calmodulin; D005456:Fluorescent Dyes; D016166:Free Radical Scavengers; D005609:Free Radicals; D010455:Peptides; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2