To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

Lectins are invaluable tools for chemical biology because they recognize carbohydrate arrays. Multivalent carbohydrate binding by lectins is important for processes such as bacterial and viral adhesion and cancer metastasis. A better understanding of mammalian lectin binding to carbohydrate arrays is critical for controlling these and other cellular recognition processes. Plant lectins are excellent model systems for the study of multivalent protein-carbohydrate interactions because of their robustness and ready availability. Here, we describe binding studies of mannose-functionalized poly(amidoamine) (PAMAM) dendrimers to a mitogenic lectin from Pisum sativum (pea lectin). Hemagglutination and precipitation assays were performed, and results were compared to those obtained from concanavalin A (Con A), a lectin that has been studied in more detail. Isothermal titration calorimetry (ITC) experiments are also described.

Understanding protein-carbohydrate interactions is essential for elucidating biological pathways and cellular mechanisms but is often difficult due to the prevalence of multivalent interactions. Here, we evaluate the multivalent glycodendrimer framework as a means to describe the inhibition potency of multivalent mannose-functionalized dendrimers using surface plasmon resonance (SPR). Using highly robust, mannose-functionalized dithiol self-assembled monolayers on gold surfaces, we found that glycodendrimers were efficient inhibitors of protein-carbohydrate interactions. IC50 values ranging from 260 nM to 13 nM were obtained for mannose-functionalized dendrimers with Concanavalin A.

The development of methodology that is designed to allow a significant increase in the patterning and in the functionalization of the dendrimer is the ultimate goal of the research described here. Glycoside clusters based on TRIS were formed using click chemistry and were attached to PAMAM dendrimers. A series of dendrimers bearing tris-mannoside and an ethoxyethanol group was synthesized, and the binding interactions of these dendrimers with Concanavalin A were evaluated using inhibition ELISAs. The results of the inhibition ELISAs suggest that tris-mannoside clusters can replace individual sugars on the dendrimer without loss of function. Since tris-mannoside clustering allows for a redistribution of the dendrimers' surface functionalities, from this chemistry one can envision patterned dendrimers that incorporate multiple groups to increase the function and utility of the dendrimer.

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 ...