Characterization of protein aggregation via intrinsic fluorescence lifetime.
Aggregation plays an integral role in multivalent protein-carbohydrate interactions, Alzheimer's and other amyloid-related diseases, and infection response. Efforts to apply controlled aggregation in toxin sensors have been made. We have developed a label-free intrinsic fluorescence lifetime assay that uniquely can monitor aggregation processes in real time without interference from precipitation. Fluorescence decay curves were measured with high precision at 1 s time intervals following addition of a glycodendrimer to a lectin-containing solution. Changes in the fluorescence intensity and lifetime signified formation of complexes. However, these changes were not associated with the initial lectin-sugar binding events. Rather, they appeared to be caused by clustering and subsequent conformational rearrangement of the lectins. Studies were conducted with mannose-functionalized polyamidoamine (PAMAM) dendrimers of the second through sixth generations and Concanavalin A. The apparent rate constant, when expressed on a per-mannose basis, increased with dendrimer generation, particularly in going from the fourth to the sixth generation. However, the identical fluorescence decay waveforms for saturating amounts of dendrimer suggested that all of the glycodendrimer generations studied reach a comparable state of aggregation. Although self-quenching of tryptophan resonances that was induced by clustering was monitored in this study, the reported method is not limited to such and is viable for numerous binding studies.
Schlick
Kristian H
KH
Department of Chemistry and Biochemistry, 103 Chemistry and Biochemistry Building, Montana State University, Bozeman, Montana 59717, USA.
Lange
Candace K
CK
Gillispie
Gregory D
GD
Cloninger
Mary J
MJ
D003208:Concanavalin A / Q000737:chemistry*; D050091:Dendrimers; D005453:Fluorescence; D008163:Luminescent Measurements; D011073:Polyamines / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D050091:Dendrimers; C104700:PAMAM Starburst; D011073:Polyamines; D011506:Proteins; D003208:Concanavalin A; D014364:Tryptophan