Instrumentation Development for Characterization of Multivalent Interactions: Fluorescence Lifetime Measurements
This project is supported by NSF CHE 0821802, a major research instrumentation (MRI) development grant.
Many assays are used for the study of multivalent binding interactions. Hemagglutination inhibition assays, precipitation assays, and turbidity assays are straightforward but provide only qualitative assessments of binding. Isothermal titration calorimetry (ITC) provides valuable quantitative energetic values for binding, but precipitation often complicates these studies. In addition, ITC requires significant quantities of material. Surface plasmon resonance is difficult to analyze for heterogeneous (multivalent) binding events. ELLAs provide binding information but are most useful for studying interactions at surfaces.
Although many assays are available, in most circumstances data analysis is hindered by the multivalent nature of the targeted interactions. Moreover, each assay is likely to provide information only about one aspect of multivalent interactions (for example, an equilibrium constant or a value for inhibition of aggregation). Since many factors including kinetic on and off rates, enthalpy/entropy compensations, equilibrium constants, and formations of aggregates are likely all contributing to the sensitivity and selectivity of biologically relevant interactions, new assays with which multivalent interactions can be reliably studied must be developed.
We have been developing a fluorescent lifetime microplate reader to enable the characterization of lectin aggregates that are formed when lectins interact with their glycosylated binding partners.
Since fluorescence lifetime (FL) measurements contain the added dimension of time relative to steady state fluorescence, measurements using FL allow for more reproducible detection of small changes in fluorescence.1 The binding of lectins to glycodendrimers relies on a series of individually weak interactions, so the change in the environment occupied by the tryptophan residues of the lectins is often small and causes only small changes in fluorescence. However, conventional methods of data collection for FL measurements such as time-correlated single-photon counting (TCSPC) require long collection times, and we have been studying binding processes with half-lives as short as just a few seconds. With a prototype instrument from Fluorescence Innovations, Inc. (Bozeman, MT), we were able to collect data that is equally accurate to that obtained from TCSPC but which can be collected about 100 times faster. A proprietary digitizer that is capable of recording the entire decay curve for each pulse was developed by Fluorescence Innovations. Their prototype instrument uses a cuvette; a microplate reader platform for this fluorescence lifetime instrument is being developed with funding from an NSF MRI development grant. The new microplate reader will allow for significantly higher throughput using much less protein, making the measurement of fluorescence lifetimes a practical and efficient method for evaluating glycodendrimer/lectin interactions.
Figure 1. Fluorescence assay data for additions of mannose-functionalized G(4)-PAMAM into 100 mg/mL Con A.
Assay data for complex formation upon addition of mannose functionalized G(4)-PAMAM to Con A is shown in Figure 1. Control experiments in which a large excess of methyl mannose was added to Con A did not reveal any change in fluorescence and suggest that the primary sugar-Con A binding events are not the source of the observed fluorescence changes. Instead, fluorescence changes are most likely associated with a Con A/Con A protein-protein interaction orchestrated by binding to the glycodendrimer framework. The primary effect of the dendrimers is most likely to hold the Con A lectins in close proximity, thereby increasing their effective concentration (Figure 2). A protein-protein interaction, presumably a conformational change affecting the environment of one of more Con A tryptophans, can then occur.
Figure 2. Glycodendrimer-mediated lectin aggregation. Left: uncomplexed Con A. Right: cross-linked state.
Although our research is focused on the study of glycodendrimer/lectin aggregates, the fluorescence lifetime microplate reader that we are developing will also be useful for the study of a wide variety of interactions. For example, intrinsic fluorescence can be used for the study of protein-protein interactions, protein-small molecule interactions, vesicle and micelle formation, oligomerization events, and protein folding. Labeled compounds can be studied as well using the methods described here.
- Berezin, M. Y.; Achilefu, S., Fluorescence Lifetime Measurements and Biological Imaging. Chemical Reviews 2010, 110(5), 2641-2684.