Two-photon fluorescence spectroscopy and imaging of 4-dimethylaminonaphthalimide peptide and protein conjugates.
We report detailed photophysical studies on the two-photon fluorescence processes of the solvatochromic fluorophore 4-DMN as a conjugate of the calmodulin (CaM) and the associated CaM-binding peptide M13. Strong two-photon fluorescence enhancement has been observed which is associated with calcium binding. It is found that the two-photon absorption cross-section is strongly dependent on the local environment surrounding the 4-DMN fluorophore in the CaM conjugates, providing sensitivity between sites of fluorophore attachment. Utilizing time-resolved measurements, the emission dynamics of 4-DMN under various environmental (solvent) conditions are analyzed. In addition, anisotropy measurements reveal that the 4-DMN-S38C-CaM system has restricted rotation in the calcium-bound calmodulin. To establish the utility for cellular imaging, two-photon fluorescence microscopy studies were also carried out with the 4-DMN-modified M13 peptide in cells. Together, these studies provide strong evidence that 4-DMN is a useful probe in two-photon imaging, with advantageous properties for cellular experiments.
Department of Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.
Alan M
AM
McLean
Elke
E
Socher
Oleg
O
Varnavski
Travis B
TB
Clark
Barbara
B
Imperiali
Theodore
T
Goodson
D010455:Peptides / Q000737:chemistry*; D017785:Photons; D010797:Phthalimides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
C492935:4-(N,N-dimethylamino) phthalimide; D010455:Peptides; D010797:Phthalimides; D011506:Proteins