Aggregation plays an integral role in multivalent protein-carbohydrate interactions, in Alzheimer's and other amyloid-related diseases, and in infection response. Efforts have been made to apply controlled-aggregation in toxin sensors. 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 one-second 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 are not associated with the initial lectin-sugar binding events. Rather, they appear to be caused by clustering and a subsequent conformational rearrangement of the lectins. Studies were conducted with mannose-functionalized PAMAM dendrimers of the second through the sixth generations with 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 suggest that all glycodendrimer generations studied reach a comparable state of aggregation. Although selfquenching 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.Aggregation plays an integral role in many cellular pathways, one of the most important being mediating the infection and proliferation potential of tumors and pathogens. 1 Protein aggregation has also been implicated in pathological conditions such as Alzheimer's and other amyloid-related diseases. 2 Owing to the importance of multivalently displayed carbohydrates on cell surfaces, sugar-induced aggregation has drawn considerable attention. 3,4 Sensor strategies based on controlled aggregation have been reported for the detection of toxins and other biologically relevant compounds. 5 Multivalent interactions often involve multiple weak monovalent binding events. An in-depth understanding of aggregation in complex systems requires studies that go beyond measuring the monovalent association constants. Particularly valuable would be methods capable of characterizing aggregation events in real time.We present here important new information about the aggregation of the mannose-specific lectin Concanavalin A (Con A) by glycodendrimers 1-4 (Figure 1, prepared as described in ref. 6 ). Glycodendrimers are very well suited for studying the formation and mediation of multivalent interactions. We have reported that binding and inhibition efficacies depend on gillispie@fluorescenceinnovations.com, mcloninger@chemistry.montana.edu. Supporting Information Available: Methods, description of instrumentation, and fluorescence data for methyl mannose, galactosefunctionalized dendrimers, and 1-4 with Con A. This material is ...

comparison to high-pressure crystal structures of L99A T4 lysozyme [Collins, et al. (2005), PNAS 102, 16668-16671], pressure denaturation of the structurally similar L99A and L99G/E108V mutants was studied at neutral pH. The pressure-denatured state at neutral pH is even more compact than at low pH, and the small volume changes associated with denaturation suggest that the preferential filling of large cavities results in a compact, pressure-denatured state. These results confirm that pressure denaturation is characteristically distinct from thermal or chemical denaturation.