Thiol-reactive derivatives of the solvatochromic 4-N,N-dimethylamino-1,8-naphthalimide fluorophore: a highly sensitive toolset for the detection of biomolecular interactions.
The solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) possesses extremely sensitive emission properties due largely to the low intrinsic fluorescence it exhibits in polar protic solvents such as water. This makes it well suited as a probe for the detection of a wide range of biomolecular interactions. Herein we report the development and evaluation of a new series of thiol-reactive agents derived from this fluorophore. The members of this series vary according to linker type and the electrophilic group required for the labeling of proteins and other biologically relevant molecules. Using the calcium-binding protein calmodulin as a model system, we compare the performance of the 4-DMN derivatives to that of several commercially available solvatochromic fluorophores identifying many key factors important to the successful application of such tools. This study also demonstrates the power of this new series of labeling agents by yielding a fluorescent calmodulin construct capable of producing a greater than 100-fold increase in emission intensity upon binding to calcium.
Loving
Galen
G
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Imperiali
Barbara
B
D002118:Calcium; D002147:Calmodulin; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D015336:Molecular Probe Techniques; D053644:Naphthalimides / Q000138:chemical synthesis*; D012997:Solvents; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D002147:Calmodulin; D005456:Fluorescent Dyes; D053644:Naphthalimides; D012997:Solvents; D013438:Sulfhydryl Compounds; D002118:Calcium